Polycaprolactone-b-poly(N-isopropylacrylamide) nanoparticles : synthesis and temperature induced coacervation behaviour

S. Vandewalle,a M. Van De Walle,a S. Chattopadhyay*,a, b F.E. Du Prez*a

aCentre of Macromolecular Chemistry (CMaC), Polymer Chemistry Research group, Department of Organic and Macromolecular Chemistry, Ghent University, Krijgslaan 281 S4bis, 9000 Ghent, Belgium

bDepartment of Chemistry, Indian Institute of Technology Patna, Bihta, Patna 801103, Bihar, India

E-mail addresses corresponding authors : Filip.DuPrez@UGent.be, sch@iitp.ac.in

Keywords : amphiphilic block copolymers, click chemistry, triazolinediones, self-assembly

Abstract

Poly(caprolactone)-b-poly(N-isopropylacrylamide) (PCL-b-PNIPAM) copolymers were prepared with different compositions of the individual blocks using a combination of enzyme catalyzed anionic ring opening (AROP) polymerization, reversible addition fragmentation chain transfer (RAFT) polymerization and triazolinedione-diene click chemistry. Study of the self-assembly of these block copolymers at different temperatures demonstrates the formation of nanoparticles, characterized by a thermo-responsive behaviour that is strongly dependent on the PNIPAM block length. At elevated temperature, self-aggregation (coacervation) of the nanoparticles could occur, leading to the formation of larger polymer aggregates.

Introduction

Amphiphilic block and graft copolymers have been widely studied in the last decades. The topology enables these polymers to self-assemble in various nanostructures such as nanoparticles, micelles, microgels and vesicles, which are applied for different (biomedical) applications, e.g. drug delivery[1-6]. In this respect, amphiphilic copolymers containing a biodegradable hydrophobic polyester core and a thermo-responsive hydrophilic corona are of great interest[7-10]. The biocompatibility and biodegradability[11-14] together with its great micellar stability[1], makes polyester-based copolymers interesting in a biomedical context. Next to poly(lactide) (PLA), poly(caprolactone) (PCL) is clearly emerging as one of the most used biodegradable and biocompatible polyesters and is approved by the Food and Drug Administration for specific biomedical applications. Besides the synthesis of the archetypical PCL-b-poly(ethyleneoxide) copolymers[15-20], recent efforts focus on the synthesis of stimuli-responsive copolymers of PCL, which are valuable in applications such as controlled and triggered drug delivery[21]. Poly(N-isopropylacrylamide) (PNIPAM) is a well-studied smart polymer[22-25], with a cloud point temperature in the range of 28-32°C, that is frequently applied as thermo-responsive block in amphiphilic polyester-based block copolymers. Up to now, these PCL-based copolymers were mainly synthesized using two different synthetic approaches: the grafting from and grafting onto method. In the first strategy, a macroinitiator that contains initiating functionalities, present as end group or in the side-chain, is used to polymerize a second functional monomer to obtain an amphiphilic block or graft copolymer respectively. Both, the use of PCL with thiocarbonlythio groups[26-28] or alkyl halides[29, 30], well-known controllable groups for reversible-deactivation radical polymerization (RDRP), and the use of hydroxyl containing PNIPAM macroinitiators[21, 31-33], are described in literature. In the grafting onto strategy, polymer blocks with clickable functionalities are synthesized individually. In a next step, these segments are conjugated together by means of efficient click reactions. This modular approach gives polymer chemists the possibility to design diversified polymer architectures starting from clickable prepolymers. Furthermore, it enables to select the appropriate polymerization technique for making distinct polymer blocks to obtain a copolymer with the desired polymer composition and functionality. With the advent of many versatile click platforms[34, 35], this grafting onto approach for polymer functionalization in general is gaining popularity in recent years[36]. The most widely used click reaction, also for the preparation of polyester based block and graft copolymers, is the copper catalyzed azide-alkyne cycloaddition reaction[37]. For example, starting from a PCL prepolymer with pendant azide groups Riva et al.[17] and Li et al.[38] synthesized poly(ethyleneoxide)-g-PCL and PNIPAM-g-PCL graft copolymers respectively. As triazolinedione (TAD) chemistry[35, 39, 40] is nowadays acknowledged as one of the most powerful click chemistry platforms in terms of ultrafast kinetics and efficiency, we aimed to apply it for the grafting onto synthesis of amphiphilic PCL-b-PNIPAM block copolymers (Figure 1). This chemistry was earlier employed for the functionalization and crosslinking of poly(caprolactone) polymers[41, 42].

Figure 1: Followed approach for synthesis of PCL-b-PNIPAM and their self-assembly behaviour.

Initially, individual homopolymers were synthesized and functionalized with suitable end groups, being a TAD and a conjugated diene functionality. Enzyme catalyzed anionic ring opening polymerization (AROP) was used for the synthesis of poly(caprolactone) polymers with a conjugated diene end group, while reversible addition fragmentation chain transfer (RAFT) polymerization enabled the preparation of TAD-end functionalized PNIPAM polymers. In the next step, these homopolymers were coupled together to form amphiphilic block copolymers in minutes. The applied Diels-Alder reaction between TAD and conjugated diene components is characterized by an apparent rate constant in the order of 200 M-1s-1.[43] These high kinetics are, compared to other existing click reactions, beneficial for the ligation of polymers[39, 44-46], especially for segments with different polarity where fine mixing of both polymers becomes challenging. Three different block copolymers were prepared, varying the length of the individual PCL and PNIPAM homopolymer. Afterwards, the self-assembly behaviour of the synthesized polyester-based copolymers in aqueous environment were studied at different temperatures using dynamic light scattering (DLS) and scanning electron microscopy (SEM) analysis. These block copolymers were self-assembled in aqueous environment to form nanoparticles. Temperature responsive self-aggregation of the nanoparticles was observed, which was strongly dependent on the chain length of the PNIPAM block (Figure 1).

Experimental

Instrumentation

1H NMR spectra were recorded in CDCl3 or DMSO-d6 using a Bruker AM500 spectrometer at 500 MHz, Bruker AM400 spectrometer at 400 MHz or Bruker Avance 300 at 300 MHz at room temperature. Chemical shifts are presented in parts per million (δ) relative to the solvent CDCl3 (7.26 ppm for 1H NMR) or DMSO-d6 (2.50 ppm for 1H NMR) as an internal standard.

Size Exclusion Chromatography (SEC) with THF as solvent was performed using a Varian PLGPC50plus instrument, using a refractive index detector, equipped with two Plgel 5 µm MIXED-D columns 40 °C. Polystyrene standards were used for calibration and THF as eluent at a flow rate of 1 mL/min. Samples were injected using a PL AS RT autosampler. SEC with with DMA as solvent was performed on a Agilent 1260-series HPLC system equipped with a 1260 online degasser, a 1260 ISO-pump, a 1260 automatic liquid sampler (ALS), a thermostatted column compartment (TCC) at 50°C equipped with a PSS Gram30 column in series with a PSS Gram1000 column, a 1260 diode array detector (DAD) and a 1260 refractive index detector (RID). The used solvent was DMA containing 50mM of LiBr at a flow rate of 1 mL/min. The spectra were analyzed using the Agilent Chemstation software with the SEC add on. Molar mass and dispersity values were calculated against Varian PMMA standards.

Preparative SEC was done with a Shimadzu LC-2OAT pump, a Shimadzu SIL-IOAF autosampler, a RID-IOA Differential Refractive Index Detector, a FRC-1OA Fraction Collector and CMB-2OA PC Interface/System Controller. Software is LC solutions including LC solutions GPC software. Columns are Shodex: a K-LG guard column and a KF-2004 prep column. THF is used as eluent, flow rate 2.5 ml/min (room temperature).

Matrix Assisted Laser Desorption Ionisation – Time of Flight (MALDI-TOF) was performed on an Applied Biosystems Voyager De STR MALDI-TOF spectrometer equipped with 2 m linear and 3 m reflector flight tubes, a nitrogen laser operating at 337 nm, pulsed ion extraction source and reflectron. All mass spectra were obtained with an accelerating potential of 20kV in positive ion mode and in reflector mode. For measurement of PCL-diene and PNIPAM, trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene]malonitrile (DCTB) (20 mg/mL in THF) was used as a matrix, NaI (1 mg/mL in THF) was used as a cationizing agent, and polymer samples were dissolved in THF (2 mg/mL). Polymer solutions were prepared by mixing 10 μL of the matrix, 5 μL of the salt, and 5 μL of the polymer solution. Subsequently, 0.5 μL of this mixture was spotted on the sample plate, and the spots were dried in air at room temperature. A poly(ethylene oxide) standard (Mn = 2000 g/mol) was used for calibration. All data were processed using the Data Explorer 4.0.0.0 (Applied Biosystems) software package.

Dynamic Light Scattering (DLS) measurements were performed on a Zetasizer Nano-ZS Malvern instrument using disposable cuvettes. A He-Ne laser at 633 nm was used as excitation light source. The intensity of the scattered light was measured at 173°.

Scanning electron microscope (SEM) analysis is performed on a JEOL JSM-5600 instrument. dispersion (concentration 0.5 mg/mL) was dried on a silicon wafer to analyze the nanoparticles by SEM.

Materials

Acetone (Sigma-Aldrich, > 99.8%), azobisisobutyronitrile (AIBN)(Sigma-Aldrich, 98%), benzyl alcohol (Sigma-Aldrich, 98.8%), bromine (Sigma-Aldrich, 99%), ε-caprolactone (Acros Organics, 99%), chloroform (Sigma-Aldrich, 99.8%), chloroform-d (Euriso-top, 99.8%), 1,4-diazabicyclo[2.2.2]octane (Sigma-Aldrich, 99%), dichloromethane (Acros Organics, 99.8%), N,N-dicyclohexyl-carbodiimide (Acros Organics, 99%), diethyl ether (Sigma-Aldrich, 99.8%), 4-dimethyl-aminopyridine (Aldrich, 99%), N,N-dimethylformamide (Acros Organics, > 99%), DMSO-d6 (Euriso-top, 99.8%), ethanol (Fisher Chemical, 99.99%), ethyl acetate (Sigma-Aldrich, > 99.7%), ethyl carbazate (Sigma-Aldrich, 97%), trans,trans-2,4-hexadien-1-ol (Sigma-Aldrich, > 97%), (2E,4E)-hexa-2,4-dien-1-yl acetate (Sigma-Aldrich, 97%), hexamethylene diisocyanate (Fluka, > 98%), n-hexane (Sigma-Aldrich, 97%), hydrochloric acid (Fluka, 1.25M in methanol), hydrochloric acid (Sigma-Aldrich, 4N in dioxane), N-hydroxysuccinimide (Sigma-Aldrich, 98%), N-isopropylacrylamide (TCI, > 98%), Lipase acrylic resin from Candida Antarctica (Novozyme 435) (Sigma-Aldrich), magnesium sulfate (Boom), methanol (Sigma-Aldrich, > 99.9%), molecular sieves, 4Å (Sigma-Aldrich), palladium on carbon (Sigma-Aldrich, 5%), potassium carbonate (Roth, > 99%), tin(II) 2-ethylhexanoate (Sigma-Aldrich, 92.5 - 100%), tetrahydrofuran (Sigma-Aldrich, > 99.9%), toluene (Sigma-Aldrich, 99.9%), triethylamine (Acros Organics, 99%).

The urazole functionalized RAFT agent (Ur-TTC) and DABCO-bromine were synthesized according to a literature procedure[44]. The molecular sieves were activated by heating under vacuum and were subsequently crushed and stored under argon atmosphere. The Lipase acrylic resin (Novozyme 435) was dried under vacuum before use.

SynthesisSynthesis of diene-functionalized poly(ε-caprolactone) by enzymatic AROP

A solution of ε-caprolactone (1 g, 8.76 mmol) in toluene (5 ml) and a solution of HDEO in toluene (for PCL3000 32.4 mg in 3mL, for PCL8000 9.80 mg in 1mL) were mixed in a dry 50 ml two-neck round-bottom flask. Afterwards, Novozyme 435 (100 mg, 10wt%) was added. The mixture was kept at 50°C and stirred under a nitrogen atmosphere. Aluminium foil was wrapped around the flask to prevent any influence from light. After reaction, the mixture was cooled and chloroform (+/- 10 ml) was added to stop the reaction. Filtration was done to remove the catalyst. Afterwards, the solvents (toluene and chloroform) were removed. A transparent oil was obtained. The PCL was purified by precipitation in cold methanol. The mixture was then filtered over a millipore filter. A white powder was obtained. The obtained product was analyzed by 1H-NMR, GPC and MALDI-TOF. PCL3000 : Recovered product: 0.567 g (55%). MW.: 3.5 kDa (1H-NMR), 3.8 kDa (Đ = 1.56) (DMA-GPC). PCL8000 : Recovered product: 0.452 g (45%). MW.: 8.9 kDa (1H-NMR), 8.6 kDa (Đ = 1.60) (DMA-GPC).

RAFT polymerization of N-isopropylacrylamide using Ur-TTC as CTA

For the preparation of PNIPAM3000: N-isopropylacrylamide (8 g, 70.70 mmol, 70 equivalents), Ur-TTC RAFT agent (424.79 mg, 1.01 mmol, 1 equivalent) and AIBN (16.58 mg, 0.10 mmol, 0.1 equivalent) were mixed and dissolved in DMF (23.56 ml) in a Schlenk tube. The oxygen in the tube was removed by three “freeze vacuum thaw” cycles. Afterwards, the mixture was kept at 65 °C and stirred for 95 minutes (conversion 34%). The polymerization was stopped by immersing the reaction mixtures in liquid nitrogen. Subsequently, the polymer was purified by three precipitation steps in cold diethyl ether. The obtained yellow powder was dried overnight under vacuum at 40°C. The product was analyzed by 1H-NMR, MALDI-TOF and GPC. The same procedure was followed for the preparation of PNIPAM8000 : N-isopropylacrylamide (8 g, 70.70 mmol, 150 equivalents), RAFT agent (197.67 mg, 0.47 mmol, 1 equivalent) and AIBN (7.79 mg, 0.047 mmol, 0.1 equivalent) were mixed and dissolved in DMF (23.56 ml). Reaction time of 125 minutes at temperature of 65°C (conversion of 40%).

PNIPAM3000 : MW.: 3.3 kDa (Đ = 1.15) (DMA-GPC), 3.5 kDa (MALDI-TOF). PNIPAM8000 : MW.: 6.1 kDa (Đ = 1.19) (DMA-GPC).

Oxidation of Urazole-functionalized poly(N-isopropylacrylamide)

The procedure for the oxidation is similar to the earlier reported procedure[44] and is given for the PNIPAM3000. Ur-PNIPAM (100 mg, 0.029 mmol, 1 equivalent) was dissolved in dry dichloromethane (3 ml) and was dried overnight using crushed molecular sieves. Next, this mixture was added to a solution of DABCO-Br (50.75 mg, 0.032 mmol, 1.1 equivalent) which was dissolved in dry dichloromethane (1 ml). After stirring of 5 hours at room temperature, the mixture was filtered (syringe filter) and an intense red coloured solution was obtained, which was directly used in further coupling experiments.

Synthesis of poly(caprolactone)-b- poly(N-isopropylacrylamide)

After oxidation, the purified TAD-PNIPAM was immediately added to the diene-functionalized PCL in 1 mL dry dichloromethane and was reacted for 45 minutes. After reaction, the dichloromethane was removed under reduced pressure. The residual product, dissolved in acetone, was brought in a dialysis membrane (with a cut off mass of 6 kDa) and dialyzed against acetone for 30 hours. The samples were dried afterwards and were analyzed by DMA-GPC and 1H-NMR. The equimolarity was optimized for the coupling of PNIPAM3000 and PCL30000. To calculate the amount of moles of the different polymer chains, molecular weights obtained by 1H-NMR and MALDI-TOF for PCL and PNIPAM respectively were considered. Four different ratios of PCL:PNIPAM were used for this optimization study (1.4, 0.9, 0.67 and 0.5). As seen from 1H-NMR analysis, the best coupling was achieved using a 0.9:1 ratio and was further used for all the coupling reactions. The quantitative amounts for all the couplings are given below. PCL3000-b-PNIPAM3000 (entry 3a) : 50.12 mg PCL3000 and 56.05 mg PNIPAM3000.

PCL8000-b-PNIPAM3000 (entry 3b) : 63.81 mg PCL8000 and 27.75 mg PNIPAM3000.

PCL3000-b-PNIPAM8000 (entry 3c) : 100 mg PCL3000 and 203 mg PNIPAM8000.

Results and discussion

Scheme 1 : General reaction procedure for the synthesis of amphiphilic PCL-b-PNIPAM block copolymers using TAD-diene click chemistry.

Amphiphilic block copolymers with a hydrophobic PCL segment and a thermo-responsive hydrophilic PNIPAM segment were synthesized using TAD-diene click chemistry (Scheme 1). For this, PCL was functionalized with a conjugated diene, while the PNIPAM polymer was functionalized by a urazole end group. Next, this end group can be oxidized towards a TAD moiety, which can undergo an ultrafast Diels-Alder reaction with the conjugated diene of the PCL block.

Table 1 : Molecular weights and conversion for all polymeric structures.

Entry

Polymer

DPa

Mn [kDa]b

Đb

Mp

[kDa]b

Mn

[kDa]

Conv.c

(%)

1a

PCL3000

26a1

2.5

1.56

3.6

3.5c

-

1b

PCL8000

88a1

5.6

1.57

8.1

8.9c

-

2a

PNIPAM3000

24

3.3

1.15

3.8

3.5d

34

2b

PNIPAM8000

60

6.1

1.19

7.9

n.d.d

40

3a

PCL3000-b-

PNIPAM3000

-

5.4

1.33

6.9

-

-

3b

PCL8000-b-

PNIPAM3000

-

7.0

1.38

8.9

-

-

3c

PCL3000-b-

PNIPAM8000

-

8.7

1.4

10.3

-

-

aCalculated based on monomer conversion, a1assuming full monomer conversion. bCalculated by SEC (DMA with 5g/L LiBr, PMMA standards). cCalculated by 1H NMR. dDetermined by MALDI-TOF.

The electron rich conjugated diene end groups were introduced in poly(ε-caprolactone) (diene-PCL) using a functional initiator, trans,trans-2,4-hexadien-1-ol (HDEO), via lipase catalyzed ring opening polymerization of ε-caprolactone (CL). In comparison with metal catalyzed AROP, this enzymatic polymerization can be performed under milder conditions[47], i.e. temperature, which guarantee the stability of the diene functionalities of the initiator, preserving the polymer end group during polymerization. This was evidenced in a model experiment where the thermal stability of a HDEO-like compound ((2E,4E)-hexa-2,4-dien-1-yl acetate) was investigated under both metal and enzyme catalyzed AROP reaction conditions (Figure S1 – S2). At first, diene-PCL’s with an average molecular weight of 3 and 8 kDa were targeted by varying the initial [ε-CL]0/[HDEO]0 ratio during polymerization. The 1H-NMR spectra of the poly(caprolactone) polymers show the presence of characteristic signals corresponding to the vinylic end groups and the polymer backbone (Figure S3). Based on the integration intensity of these peaks, the average molecular weights of the diene-PCL’s could be estimated and were in close agreement with the theoretical molecular weights (Table 1). Likewise, the molecular weights determined by size exclusion chromatography (SEC) were in the same range, with a peak molecular weight of 3.5 kDa and 8.9 kDa respectively and dispersity indices around 1.5. However, the shape of the SEC traces of the diene-PCL’s (Figure 2, 1a) is characterized by the presence of a small shoulder at low molecular weight, corresponding to non-functionalized cyclic PCL polymers originating from backbiting reactions during polymerization. This was confirmed by MALDI-TOF analysis of fractionated samples (using preparative SEC) of the PCL3000 (Figure S4), displaying the presence of cyclic PCL oligomers at low molecular weights (< 3000 Da) and diene functionalized PCL at higher molecular weights (Figure S5-S6). As the cyclic PCL’s have no functional diene end group, this minor fraction did not interfere in the coupling reactions and could be removed by dialysis after the block copolymer formation.

Figure 2 : SEC traces for PCL3000 (1a), PNIPAM3000 (2a) and the PCL3000-b-PNIPAM3000 block copolymer (3a).

As second building block for the synthesis of the amphiphilic polyester-based block copolymers, poly(N-isopropylacrylamide) (PNIPAM) was selected. Following a robust method developed earlier by our group, urazole functionalities - a stable precursor of TAD – were introduced as end groups in the PNIPAM segments using a functional urazole thiocarbonylthio (Ur-TTC) as chain transfer agent in a RAFT polymerization[44]. Orthogonal oxidation of these urazole precursor moieties introduces clickable TAD end groups on the PNIPAM polymers, which favour ultrafast Diels-Alder reaction with the electron rich diene end groups of the PCL polymers (Scheme 1). Initially, the RAFT polymerization of N-isopropylacrylamide (NIPAM) using the Ur-TTC was examined in full detail. The data of the kinetic analysis using typical RAFT conditions ([NIPAM]/[CTA]/[AIBN] = 100/1/0.1, 3M DMF, 65°C) are given in figure S7. For all polymerizations, a short inhibition period with no polymerization activity, which is a common phenomenon in RAFT polymerization[48-50], was observed at the start of the reaction. After this inhibition step, the molecular weight increases linearly with monomer conversion, revealing that the radical polymerization proceeds under controlled conditions. By varying the initial [NIPAM]0/[CTA]0 ratio, it was possible to synthesize low disperse PNIPAM polymers with a pre-determined theoretical molecular weight of respectively 3 and 8 kDa. Indeed, the SEC traces show unimodal peaks with narrow dispersities (Đ < 1.2) and molecular weights that are in close agreement with the theoretical values (Table 1 and Figure 2). The 1H-NMR spectra of the PNIPAM polymers are characterized by signals corresponding to the urazole end group and the polymer backbone, showing the presence of the urazole end groups in the PNIPAM polymers (Figure S8). In order to conjugate the PNIPAM segments to the diene-PCL polymers, the urazole moieties were oxidized to their corresponding TAD functionalities using a tetrameric bromine complex of DABCO (1,4-diazobicyclo[2.2.2]octane) in dry dichloromethane. This heterogeneous oxidation method was already successfully applied in earlier studies for the oxidation of urazole end groups on hydrophobic polymer backbones[44]. Herein, the procedure was further optimized for the oxidation of poly(N-isopropylacrylamide) using PNIPAM3000. For this, it was essential to dry the hydrophilic polymers prior to oxidation in order to prevent the loss of functional end groups due to the hydrolysis of TAD functionalities during the oxidation step. This drying step was performed using activated and crushed molecular sieves. The discoloration of the polymer solution, from yellow to red, during oxidation was a first indication for the successful generation of the TAD-chromophore that has a characteristic absorbance at 540 nm. To monitor this oxidation in a quantitative way, the generated TAD end group was functionalized with a complementary reaction partner (trans,trans-2,4-hexadien-1-ol) and this modified PNIPAM polymer was examined in full detail using 1H NMR with DMSO-d6 as solvent (Figure S8). From this, it is clear that the oxidation of the urazole towards the clickable TAD end group and its corresponding modification with HDEO was complete. Indeed, the singlet at 10 ppm that corresponds to the urazole protons disappeared entirely after modification, while new signals with comparable integration intensity appeared in the range of 3 – 6 ppm that correspond to the formed HDEO adduct.

The coupling reaction between the TAD-PNIPAM and diene-PCL polymers using the TAD-diene Diels Alder reaction was optimized using the polymers 1a and 2a. Ideally, the reaction was performed in dry dichloromethane at room temperature with a molar [PCL]/[PNIPAM] ratio of 0.9/1 (Figure S9), which was determined based on the molecular weights obtained by 1H-NMR for PCL and MALDI-TOF for PNIPAM (Table 1). As evidenced from the gradual disappearance of the distinctive red color, corresponding to the TAD chromosphere, a complete conversion was monitored in 45 minutes. Figure 3 shows the SEC traces of the PCL-b-PNIPAM 3a, which are characterized by an increased molar mass (Mn = 5.4 kDa, Mp = 6.9 kDa) in comparison with the molar masses measured for the PCL 1a (Mn = 2.5 kDa, Mp = 3.6 kDa) and PNIPAM 2a (Mn = 3.3 kDa, Mp = 3.8 kDa) starting materials. After coupling, a shoulder at low molecular weight was observed corresponding to a minor fraction of non- reacted cyclic PCL (vide supra), which could significantly be reduced using dialysis in acetone.

Figure 3 : 1H NMR spectra of the PCL3000 showing the characteristic diene signals* in the region 5-6 ppm (top, in CDCl3), the PNIPAM3000 showing the characteristic urazole* singlet at 10 ppm (middle, in DMSO-d6), the PCL3000-b-PNIPAM3000 showing the appearance of the characteristic signal for the adduct at 6 ppm (bottom, in DMSO-d6).

Furthermore, new signals at 6 ppm appeared in the 1H-NMR spectrum (Figure 3) of the PCL-b-PNIPAM block copolymer that could be assigned to the coupled Diels-Alder adduct between the two polymer segments. Moreover, the signals corresponding to the diene and the urazole end groups of the PCL and PNIPAM polymers at 5.5-6.5 and 10 ppm respectively, were not observed in the 1H-NMR spectrum of the block copolymer. These findings show that the PCL-b-PNIPAM conjugate 3a was successfully synthesized by efficient and equimolar clicking of the functionalized PCL and PNIPAM segments, with a minor impurity originating from unreacted cyclic PCL that could be reduced by dialysis. This optimized coupling reaction was subsequently used for the synthesis of the conjugates 3b and 3c, in which the lengths of both the PCL and PNIPAM segment were varied using suitable functionalized starting materials. The results of these coupling reactions can be found in table 1.

As expected, the prepared amphiphilic block copolymers self-assembled in water, forming spherical nanoparticles (Nps) which are characterized with a hydrophobic PCL core and a hydrophilic PNIPAM corona. In order to prepare these aggregates, the block copolymers were dissolved in THF (1 mg/ml) and were subsequently added dropwise to water. Afterwards, the remaining organic solvent traces were removed by evaporation under reduced pressure. The prepared Nps were characterized by dynamic light scattering (DLS) and scanning electron microscopy (SEM). The size of the different nanoparticles were observed in the range of 145 – 160 nm (Table 2). As expected, the size of the nanoparticles slightly increases with increasing chain length of the PCL block. All of these Nps were found to be very stable, also in very dilute solution. At room temperature, these micellar nanoparticles were stabilized by a hydrophilic PNIPAM shell.

Table 2: Analysis of block copolymer based nanoparticles using DLS.

Entry

Polymer

Diameter at

20 °C (nm)

(±10 nm)

Diameter at 50 °C (nm)

(±10 nm)

Volume (50 °C)/

Volume (25 °C)

3a

PCL3000-b-PNIPAM3000

145

145

1

3b

PCL8000-b-PNIPAM3000

170

170

1

3c

PCL3000-b-PNIPAM8000

160

1000

244

As PNIPAM is characterized by an LCST behaviour, temperature-responsive coacervation behaviour of the synthesized nanoparticles was expected. Thus, the colloidal dispersion was heated to 50 °C, a temperature above the cloud point temperature of the individaul PNIPAM blocks, as was determined by turbidimetry measurements (Figure S10). At this elevated temperature, the colloidal Nps were analyzed by SEM and DLS. From DLS experiments, it was clear that the size of the Nps, characterized by the shortest PNIPAM chains, (PCL3000-b-PNIPAM3000 and PCL8000-b-PNIPAM3000) did not change with temperature. However, the size of the PCL3000-b-PNIPAM8000 Nps, characterized by longer PNIPAM chains, increases at elevated temperature. The volume of these aggregates at 50 °C was approximately 250 times higher than the volume at 20 °C (Table 2 and Figure 4).

Figure 4: Dynamic light scattering (DLS) characterization of nanoparticles (entry 3c) and their self-assembly at lower (20 °C) and higher temperature (50 °C).

Thus, only when the chain length of PNIPAM was high enough, enhanced hydrophobicity of PNIPAM chain at a temperature above LCST enabled further coacervation of the nanoparticles through hydrophobic interaction, leading to the formation of larger aggregates. This phenomenon was further evidenced from SEM analysis, where aggregation of smaller nanoparticles was visualized at higher temperature (Figure S11). Such coacervation behaviour can be advantageous in different biomedical applications[51].

Conclusion

Three different PCL-b-PNIPAM block copolymers, featuring different composition of both blocks, have been synthesized using a combination of enzyme catalyzed AROP, RAFT and TAD-diene click reaction. First, the PCL-synthesis resulted in polyester prepolymers with different predefined molecular weights with functional conjugated diene end groups. During this polymerization mechanism, a minor fraction of side product was identified as unfunctionalized cyclic PCL which could be separated after polymer-polymer ligation by dialysis. As second block, PNIPAM polymers with good end group fidelity was prepared via RAFT polymerization using a functional urazole functionalized thiocarbonylthio agent. In a last step, both polymer segments were clicked together by the efficient TAD-diene Diels-Alder reaction, yielding amphiphilic PCL-b-PNIPAM block copolymers with thermo-responsive behaviour. Subsequently, the purified block copolymers were self-assembled in water to form nanoparticles. A further self-aggregation of those nanoparticles was possible upon heating, only if the chain length of PNIPAM is comparatively larger than the PCL segment of the amphiphilic block copolymer.

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