Preparation of Thermo-Responsive and Cross-Linked Fluorinated Nanoparticles via RAFT-Mediated Aqueous Polymerization in Nanoreactors

In this work, a thermo-responsive and cross-linked fluoropolymer poly(2,2,2-Trifluoroethyl) methacrylate (PTFEMA) was successfully prepared by reversible addition-fragmentation chain transfer (RAFT) mediated aqueous polymerization with a thermo-responsive diblock poly(dimethylacrylamide-b-N-isopropylacrylamide) (PDMA-b-PNIPAM) that performed a dual function as both a nanoreactor and macro-RAFT agent. The cross-linked polymer particles proved to be in a spherical-like structure of about 50 nm in diameter and with a relatively narrow particle size distribution. 1H-NMR and 19F-NMR spectra showed that thermo-responsive diblock P(DMA-b-NIPAM) and cross-linked PTFEMA particles were successfully synthesized. Influence of the amount of ammonium persulfate (APS), the molar ratio of monomers to RAFT agent, influence of the amount of cross-linker on aqueous polymerization and thermo-responsive characterization of the particles are investigated. Monomer conversion increased from 44% to 94% with increasing the molar ratio of APS and P(DMA-b-NIPAM) from 1:9 to1:3. As the reaction proceeded, the particle size increased from 29 to 49 nm due to the consumption of TFEMA monomer. The size of cross-linked nanoparticles sharply decreased from 50.3 to 40.5 nm over the temperature range 14–44 °C, suggesting good temperature sensitivity for these nanoparticles.


Introduction
In recent years, the study of fluoropolymers has gained tremendous attention because of their versatility. Excellent chemical resistance, thermal stability and other special properties enable them to be widely used in the fields of leather, textiles, building, and coating. In addition, many fluorinated polymers and small molecules have been widely used in medicine and drug delivery because of their biocompatibility and non-toxicity in vivo [1][2][3]. For example, sitagliptin, a selective, potent dipeptidyl peptidase IV DPP-4 inhibitor, is the active ingredient in JANUVIA ® and JANUMET ® (a fixed dose combination with the antidiabetic agent metformin), which both recently received approval for the treatment of type 2 diabetes by the FDA [4]. The most widely used method for the preparation of polymer nanoparticles is heterogeneous polymerization, including emulsion polymerization and dispersion polymerization. Aqueous polymerization is a relatively economical and versatile tool to produce nanoparticles with a low sensitivity to impurities [5]. Usually emulsifiers are used to stabilize such type of emulsions. These emulsifiers are very toxic and also need to be removed from  Figure 2A shows the gel permeation chromatography (GPC) traces of the macro-RAFT agent of PDMA-S-(C=S)-S-C12H25 with a different ratio of monomer and RAFT agent, resulting in a different degree of polymerization. Based on the GPC traces eluted by THF, the different molecular weights ranging from 3900 to 10,800 g/mol with low dispersity (<1.1) for PDMA-S-(C=S)-S-C12H25 are obtained. Figure 2B shows the GPC traces of P(DMA-b-NIPAM)-S-(C=S)-S-C12H25 synthesized by the same macro-RAFT agent. Diblock copolymers ranging from 7200 to 22,000 g/mol with low dispersity were successfully obtained. These results confirm the well-controlled RAFT polymerization.  Figure 2A shows the gel permeation chromatography (GPC) traces of the macro-RAFT agent of PDMA-S-(C=S)-S-C 12 H 25 with a different ratio of monomer and RAFT agent, resulting in a different degree of polymerization. Based on the GPC traces eluted by THF, the different molecular weights ranging from 3900 to 10,800 g/mol with low dispersity (<1.1) for PDMA-S-(C=S)-S-C 12 H 25 are obtained. Figure 2B shows the GPC traces of P(DMA-b-NIPAM)-S-(C=S)-S-C 12 H 25 synthesized by the same macro-RAFT agent. Diblock copolymers ranging from 7200 to 22,000 g/mol with low dispersity were successfully obtained. These results confirm the well-controlled RAFT polymerization.    where Mn,PDMA is the molecular weight of the PDMA33 macro-CTA; d THF was used as eluent at a flow rate of 1.0 mL/min; e dispersity of PDMA-b-PNIPAM.
Optical photographs of the thermo-responsive block polymers that we synthesized are shown in Figure 3; the solution was transparent at 25 • C. When the temperature was raised to 40 • C, the P( Optical photographs of the thermo-responsive block polymers that we synthesized are shown in Figure 3; the solution was transparent at 25 °C. When the temperature was raised to 40 °C, the P can completely dissolve in water below the LCST of the PNIPAM block. The behavior of the PNIPAM block changes from soluble into insoluble once the temperature is above the LCST, resulting in the aggregation of PNIPAM segments to form nanoreactors, and hydrophilic segment PDMA acts as a stabilizer for the nanoreactors. The nanoreactors can encapsulate TFEMA monomers and cross-linker EGDMA for reaction. Intensity weighted distributions of the hydrodynamic diameter were obtained by dynamic light scattering (DLS). The micelle hydrodynamic diameter increased from 16 to 33 nm while the degree of polymerization of the PNIPAM segment increased from 63 to 163. The PNIPAM segments of P(DMA33-b-NIPAM32)-S-(C=S)-S-C12H25 are too short to stabilize the micelles. The aqueous solution containing P(DMA-b-NIPAM)-S-(C=S)-S-C12H25, APS, TFEMA and EGDMA will undergo polymerization in nanoreactors to generate cross-linked nanoparticles. Figure 4 shows the 19 F-NMR spectrum of lyophilized triblock copolymer P(DMA33-b-NIPAM163-b-TFEMA50)-S-(C=S)-S-C12H25. The characteristic signals of the PTFEMA segment (−73.8 ppm -CF3 in side chain PTFEMA) can be seen. The particle morphology, shown by the transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images in Figures 5 and 6, clearly suggests that stable and monodisperse spherical nanoparticles were formed. The particles' corona was formed by P(DMA-b-NIPAM) and the cross-linked PTFEMA block forms the hydrophobic core. The aqueous solution containing P(DMA-b-NIPAM)-S-(C=S)-S-C 12 H 25 , APS, TFEMA and EGDMA will undergo polymerization in nanoreactors to generate cross-linked nanoparticles. Figure 4 shows the 19 F-NMR spectrum of lyophilized triblock copolymer P(DMA 33 -b-NIPAM 163 -b-TFEMA 50 )-S-(C=S)-S-C 12 H 25 . The characteristic signals of the PTFEMA segment (−73.8 ppm -CF 3 in side chain PTFEMA) can be seen. The particle morphology, shown by the transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images in Figures 5 and 6, clearly suggests that stable and monodisperse spherical nanoparticles were formed. The particles' corona was formed by P(DMA-b-NIPAM) and the cross-linked PTFEMA block forms the hydrophobic core. the 19 F-NMR spectrum of lyophilized triblock copolymer P(DMA33-b-NIPAM163-b-TFEMA50)-S-(C=S)-S-C12H25. The characteristic signals of the PTFEMA segment (−73.8 ppm -CF3 in side chain PTFEMA) can be seen. The particle morphology, shown by the transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images in Figures 5 and 6, clearly suggests that stable and monodisperse spherical nanoparticles were formed. The particles' corona was formed by P(DMA-b-NIPAM) and the cross-linked PTFEMA block forms the hydrophobic core.

Influence of the Amount of APS on Aqueous Polymerization
Several sets of experiments using different amounts of APS were carried out. Samples were withdrawn at regular time intervals for determination of monomer conversion and particle size. The particle size and the distribution of highly diluted samples were measured by DLS. Figure 7 shows the evolution of TFEMA conversion versus time for RAFT-mediated aqueous polymerization using P(DMA77-b-NIPAM73)-S-(C=S)-S-C12H25 as macro-RAFT agent.

Influence of the Amount of APS on Aqueous Polymerization
Several sets of experiments using different amounts of APS were carried out. Samples were withdrawn at regular time intervals for determination of monomer conversion and particle size. The particle size and the distribution of highly diluted samples were measured by DLS. Figure 7 shows the evolution of TFEMA conversion versus time for RAFT-mediated aqueous polymerization using P(DMA77-b-NIPAM73)-S-(C=S)-S-C12H25 as macro-RAFT agent.

Influence of the Amount of APS on Aqueous Polymerization
Several sets of experiments using different amounts of APS were carried out. Samples were withdrawn at regular time intervals for determination of monomer conversion and particle size. The particle size and the distribution of highly diluted samples were measured by DLS. Figure 7 shows the evolution of TFEMA conversion versus time for RAFT-mediated aqueous polymerization using P(DMA 77 -b-NIPAM 73 )-S-(C=S)-S-C 12 H 25 as macro-RAFT agent.

Influence of the Amount of APS on Aqueous Polymerization
Several sets of experiments using different amounts of APS were carried out. Samples were withdrawn at regular time intervals for determination of monomer conversion and particle size. The particle size and the distribution of highly diluted samples were measured by DLS. Figure 7 shows the evolution of TFEMA conversion versus time for RAFT-mediated aqueous polymerization using P(DMA77-b-NIPAM73)-S-(C=S)-S-C12H25 as macro-RAFT agent. As shown in Figure 7A the final monomer conversion was 44.3% when the molar ratio of APS and chain transfer agent was 1:9. The final monomer conversion reached 94.2% upon changing the molar ratio of APS and chain transfer agent from 1:9 to 1:3. The low APS concentration process could not generate enough radicals to initiate the reaction, resulting in low monomer conversion. By increasing the amount of APS, the polymerization rate increased due to the increase of the number of radicals. Figure 7B shows the evolution of TFEMA conversion versus time, keeping the same amount of initiator. In the two experiments, the molar ratio of TFEMA and macro-RAFT agent was varied, but the amount of initiator was held constant. In the later stages of the reaction, there was not enough initiator to initiate the reaction. As a result, the conversion of the reaction with more monomer was low [28].

Influence of Amount of Cross-Linker on Nanoparticles
Several experiments were conducted with adding different amounts of cross-linker EGDMA. During this process, P(DMA46-b-NIPAM31)-S-(C=S)-S-C12H25 was used as macro-RAFT agent. From the results we can conclude that the amount of the EGDMA has a significant influence on particle size. As shown in Figure 8, particle size increased linearly with increasing amount of EGDMA. This is because the increase of amount of cross-linker leads to high cross-linking density. As a result, the number of block copolymers connected on each nanoreactor is also increased, leading to larger particle size. As shown in Figure 7A the final monomer conversion was 44.3% when the molar ratio of APS and chain transfer agent was 1:9. The final monomer conversion reached 94.2% upon changing the molar ratio of APS and chain transfer agent from 1:9 to 1:3. The low APS concentration process could not generate enough radicals to initiate the reaction, resulting in low monomer conversion. By increasing the amount of APS, the polymerization rate increased due to the increase of the number of radicals. Figure 7B shows the evolution of TFEMA conversion versus time, keeping the same amount of initiator. In the two experiments, the molar ratio of TFEMA and macro-RAFT agent was varied, but the amount of initiator was held constant. In the later stages of the reaction, there was not enough initiator to initiate the reaction. As a result, the conversion of the reaction with more monomer was low [28].

Influence of Amount of Cross-Linker on Nanoparticles
Several experiments were conducted with adding different amounts of cross-linker EGDMA. During this process, P(DMA 46 -b-NIPAM 31 )-S-(C=S)-S-C 12 H 25 was used as macro-RAFT agent. From the results we can conclude that the amount of the EGDMA has a significant influence on particle size. As shown in Figure 8, particle size increased linearly with increasing amount of EGDMA. This is because the increase of amount of cross-linker leads to high cross-linking density. As a result, the number of block copolymers connected on each nanoreactor is also increased, leading to larger particle size. ratio of APS and chain transfer agent from 1:9 to 1:3. The low APS concentration process could not generate enough radicals to initiate the reaction, resulting in low monomer conversion. By increasing the amount of APS, the polymerization rate increased due to the increase of the number of radicals. Figure 7B shows the evolution of TFEMA conversion versus time, keeping the same amount of initiator. In the two experiments, the molar ratio of TFEMA and macro-RAFT agent was varied, but the amount of initiator was held constant. In the later stages of the reaction, there was not enough initiator to initiate the reaction. As a result, the conversion of the reaction with more monomer was low [28].

Influence of Amount of Cross-Linker on Nanoparticles
Several experiments were conducted with adding different amounts of cross-linker EGDMA. During this process, P(DMA46-b-NIPAM31)-S-(C=S)-S-C12H25 was used as macro-RAFT agent. From the results we can conclude that the amount of the EGDMA has a significant influence on particle size. As shown in Figure 8, particle size increased linearly with increasing amount of EGDMA. This is because the increase of amount of cross-linker leads to high cross-linking density. As a result, the number of block copolymers connected on each nanoreactor is also increased, leading to larger particle size.

Influence of Amount of TFEMA on the Nanoparticles
The effect of amount of TFEMA on particle size was investigated using P(DMA 46 -b-NIPAM 31 )-S-(C=S)-S-C 12 H 25 as macro-RAFT agent. Intensity weighted distributions of the hydrodynamic diameter were obtained by DLS. Figure 9 shows the evolution of particle size versus time or TFEMA conversion. As the reaction proceeded, the particle size in diameter gradually increased from 28.55 to 49.04 nm. This is because the monomer conversion gradually increased as the reaction proceeded, and the core of spherical particle formed by cross-linking PTFEMA will expand with the increase of monomer conversion. Higher monomer conversion means more monomer entered into nanoreactors for polymerization resulting in larger particle size.

Influence of Amount of TFEMA on the Nanoparticles
The effect of amount of TFEMA on particle size was investigated using P(DMA46-b-NIPAM31)-S-(C=S)-S-C12H25 as macro-RAFT agent. Intensity weighted distributions of the hydrodynamic diameter were obtained by DLS. Figure 9 shows the evolution of particle size versus time or TFEMA conversion. As the reaction proceeded, the particle size in diameter gradually increased from 28.55 to 49.04 nm. This is because the monomer conversion gradually increased as the reaction proceeded, and the core of spherical particle formed by cross-linking PTFEMA will expand with the increase of monomer conversion. Higher monomer conversion means more monomer entered into nanoreactors for polymerization resulting in larger particle size.  Figure 10A shows the effect of temperature on the particle size in the range of 14-44 °C. From 14 to 32 °C the particle diameter underwent small changes, but in the 32-37 °C range, the particle size was dramatically reduced from 48 to 41 nm. In the range of 35-44 °C, the particle size remained stable, indicating that the cross-linked nanospheres are temperature sensitive.

Thermo-Responsive Characterization of Cross-Linked Nanoparticles
Meanwhile, it suggests that the LCST of the nanospheres is 32 °C. PNIPAM segments are in a state of complete dissolution and stretching when the temperature is below 32 °C. As a result, the DLS-based size of the nanospheres is large. When the temperature is raised to the LCST of PNIPAM segments, PNIPAM segments begin to shrink rapidly leading to smaller particle size. After the complete shrinkage of PNIPAM segments at 37 °C, particle size will not change with the increase of temperature. This result proves that PNIPAM is connected with the cross-linked nanospheres, and it can freely stretch or shrink. To further prove this result, we synthesized the nanospheres with the same degree of polymerization of PDMA and PTFEMA and only changed the degree of polymerization of PNIPAM. As shown in (C)  Figure 10A shows the effect of temperature on the particle size in the range of 14-44 • C. From 14 to 32 • C the particle diameter underwent small changes, but in the 32-37 • C range, the particle size was dramatically reduced from 48 to 41 nm. In the range of 35-44 • C, the particle size remained stable, indicating that the cross-linked nanospheres are temperature sensitive.

Thermo-Responsive Characterization of Cross-Linked Nanoparticles
Meanwhile, it suggests that the LCST of the nanospheres is 32 • C. PNIPAM segments are in a state of complete dissolution and stretching when the temperature is below 32 • C. As a result, the DLS-based size of the nanospheres is large. When the temperature is raised to the LCST of PNIPAM segments, PNIPAM segments begin to shrink rapidly leading to smaller particle size. After the complete shrinkage of PNIPAM segments at 37 • C, particle size will not change with the increase of temperature. This result proves that PNIPAM is connected with the cross-linked nanospheres, and it can freely stretch or shrink. To further prove this result, we synthesized the nanospheres with the same degree of polymerization of PDMA and PTFEMA and only changed the degree of polymerization of PNIPAM. As shown in Figure 10B, the particle size increased with the increase of degree of polymerization of PNIPAM, indicating that PNIPAM is connected with the cross-linked nanospheres outside.

Materials and Reagents
All reagents and solvents were of analytical grade and used as received unless otherwise stated. Ethyleneglycol dimethacrylate (EGDMA, 98%, Aladdin, Shanghai, China) and N,N-dimethylacrylamide

NMR Analysis
The 1 H-NMR and 19 F-NMR analysis was performed on an AVANCE III 400 MHz digital NMR spectrometer (Bruker BioSpin, Karlsruhe, Germany) using deuterated acetone as solvent with tetramethylsilane as the internal standard at room temperature.

GPC Analysis
The molecular weights and dispersity (Ð) were determined by gel permeation chromatography (GPC) at 35 • C with tetrahydrofuran (THF) as the eluent at a flow rate of 1.0 mL/min. Narrow-polydispersity polystyrene was used as calibration standard. The system was equipped with a Model 1525 HPLC pump (Waters, Milford, MA, USA) and a Waters Model 2414 refractive index (RI) detector.

TEM Characterization
Transmission electron microscopy (TEM) observation was performed using a JEOL-1400 electron microscope (Jeol, Tokyo, Japan). A typical TEM grid preparation was as follows: A particle solution was diluted with MilliQ water to approximately 0.10 wt %. A formvar precoated copper TEM grid was covered with a drop of the solution for 60 s, and counterstained with 3% uranyl acetate (5 µL) for 20 s.

SEM Characterization
Morphology of lyophilized particle powder analysis was conducted by scanning electron microscopy (SEM, S-2500, Hitachi Seiki, Tokyo, Japan).

DLS Characterization
A Zetasizer Nano-ZS90 (Malvern Instruments, Malvern, England) was used for dynamic light scattering (DLS) characterization to measure particle size (hydrodynamic diameter Z-Ave) and particle size distribution. The sample refractive index (RI) was set at 1.59 for polystyrene.

Synthesis of Macro-RAFT Agent of PDMA 77 -S-(C=S)-S-C 12 H 25
In a typical synthesis, a round-bottomed flask was charged with DMA (15.00 g; 1.51 × 10 −1 mol), DOPAT (0.5289 g; 1.51 × 10 −3 mol), AIBN (0.0502 g, 3.06 × 10 −4 mol; CTA/initiator molar ratio = 4.9) and dioxane (23.00 g). The sealed reaction vessel was purged with nitrogen and placed in a pre-heated oil bath at 65 • C for 7 h. The solution was cooled in an ice bath, diluted with dioxane. The polymer was recovered by precipitation in diethyl ether, filtration and drying under vacuum for 48 h at 35 • C. 1 H-NMR indicated the actual degree of polymerization of 77 for the PDMA macro-CTA. M n = 7900 g·mol −1 and M w /M n = 1.12, as determined by GPC.

Conclusions
In summary, thermo-responsive and cross-linked PTFEMA nanospheres were successfully prepared. Firstly, RAFT solution polymerization was used to synthesize a thermo-responsive diblock copolymer P(DMA-b-NIPAM)-S-(C=S)-S-C 12 H 25 with low dispersity and well-controlled molecular weight. The 1 H-NMR spectral analysis confirmed that copolymers were successfully synthesized. Gel permeation chromatography was conducted to demonstrate the narrow molecular weight dispersity. Then we gave evidence that the thermo-responsive diblock copolymer can turn into nanoreactors when the temperature reaches 32 • C. We proceeded a successful RAFT-mediated aqueous polymerization of TFEMA using P(DMA-b-NIPAM)-S-(C=S)-S-C 12 H 25 trithiocarbonate as both a stabilizer and a macro-RAFT agent. During the process of aqueous polymerization, the monomer conversion increased with increasing the molar ratio of initiator to chain transfer agent. The particle size increased with increasing the monomer conversion. The particle size of the cross-linked nanospheres quickly decreased when the temperature reached 32 • C showing temperature sensitivity.