A straightforward synthesis of fluorescent and temperature‐responsive nanogels (2011)

Fluorescent nanogels from the biocompatible poly(N-vinylcaprolactam) are synthesized for the first time in direct miniemulsions. This straightforward method yields nanogels with a thermoresponsive behavior that is highly dependent on the degree of crosslinking. Poly(N-vinylcaprolactam) PVCL is a temperature-responsive polymer of great interest for biomedical applications, as it possesses a lower critical solution concentration in water in the physiological range,1-3 very similar to the well-known poly(N-isopropylacrylamide) PNIPAM (32 °C).4 Other groups already proposed interesting alternatives to PNIPAM such as copolymers of oligo(ethylene glycol) methacrylate and 2-(2-methoxyethoxy)ethyl methacrylate.5 The polymer PVCL has a number of key features that distinguish it from other temperature-responsive polymers: PVCL has a cytotoxicity much smaller than PNIPAM;6 moreover, PVCL is not hydrolyzed in pure water1 and therefore has considerable advantage for in vivo applications over the concurrent polymers that contain nonlactam amides4 and esters bonds5 in their chemical structure; finally, its monomer is easily obtained from the industrially relevant ε-caprolactam. Because PVCL has a low lower critical solution concentration in water, crosslinked gels of the same polymer display swelling/deswelling at the VPTT (volume phase transition temperature). PVCL nanogels are interesting candidates as biocompatible vehicles for drug-delivery activated by temperature.7 For this purpose, PVCL nanogels should be produced in a straightforward synthesis and possessed a controllable size. To preserve the well-defined lower critical solution concentration and the biocompatible character, the synthetic route should avoid the degradation of the monomer. It was demonstrated for instance that N-vinylcaprolactam was hydrolyzed to ε-caprolactam and acetaldehyde in the presence of KPS.8 Recent reports described the synthesis of PVCL nanogels by the precipitation/emulsion polymerization of N-vinylcaprolactam crosslinked with N-N′-methylenebisacrylamide, initiated by a persulfate salt in water.7-11 The dispersions obtained displayed low-solid content, that is, typically much less than 2 wt %. Miniemulsion polymerization is known to allow the fabrication of a large variety of functional polymers in dispersed medium with higher solid content.12 A parent monomer of N-vinylcaprolactam, ε-caprolactam, was already anionically polymerized in inverse miniemulsion.13 Recently, Medeiros et al.14 showed that N-vinylcaprolactam could be polymerized in inverse miniemulsion by emulsifying an aqueous solution of the monomer in hexadecane. Nanogels with size from about 200 to 600 nm could be successfully obtained. However, this procedure combines at least three drawbacks, which are the low-solid content of the dispersion due to the rather low solubility of N-vinylcaprolactam in water, the fact that the polymerization is again initiated by KPS, and finally more important the difficulty to remove the hexadecane continuous phase owing to its high-boiling point (287 °C at 760 mmHg15). The transfer of dispersions synthesized in inverse miniemulsion in an aqueous environment is difficult and involves the addition of surfactants with high HLB.16 Shah et al.17 proposed the copolymerization of N-vinylcaprolactam and methyl methacrylate in direct (oil-in-water) microemulsion in the presence of high concentration of surfactant (33 wt % compared to monomers). However, the polymerized materials were not crosslinked, and no physically crosslinked nanogels were formed. We propose here a straightforward synthesis of poly(N-vinylcaprolactam) nanogels dispersed in water, eliminating therefore the problems of hydrolysis of the monomer, low-solid content of the dispersions, and re-dispersion of the nanogel in water. Moreover, we describe the temperature-responsive properties of the obtained nanogels and demonstrate the possibility of functionalizing them with a fluorescent BODIPY comonomer. This is the first time that such temperature-responsive nanogel is synthesized in direct miniemulsion (oil-in-water). N-vinyl caprolactam (VCL, Aldrich 98%) was purified by recrystallization in hexane at 60°C and stored at 4°C. 2,2′-Azobis(2-methylpropionitrile) (AIBN, Fluka 98%+), sodium dodecyl sulfate (SDS, Fluka, 98.0%+), p-divinylbenzene (DVB, Sigma, 80% technical grade), toluene (Sigma-Aldrich, 99.5%+), and hexadecane (Sigma, 99%+) were used without further purification. The fluorescent monomer B504-MA was prepared according to a prior report.18 The chemical structures of the three monomers are displayed in Figure S1 (Supporting Information). Demineralized water was used throughout the work. 1.3 g of monomers (Table 1), 0.7 g of toluene, 80 mg of hexadecane, and 23 mg of the initiator AIBN were mixed and added to a solution of 24 mg SDS in 8 g of water. After stirring for 1 h at 800 rpm, the miniemulsion was prepared by ultrasonicating the emulsion for 120 s with a Branson sonifier 450-D (70% intensity and 0.25 in. tip) under ice cooling. The polymerization was carried out at 72 °C and a stirring speed of 500 rpm for 17 h. Table 1 gives an overview of the ratios of the reactants in the reaction mixture. After ultrasonication, 100 μL of each miniemulsion was removed for measuring the droplet diameter by DLS. The fluorescent nanogels were prepared by using 0.2 wt % B504-MA and 5 wt % DVB compared to the total monomer. A salmon-pink milky dispersion was obtained after polymerization. The nanogels were subsequently purified by dialysis. The reaction mixtures were placed in semipermeable SpectraPor membranes with MWCO of 2,000 g mol−1, which are immersed in distilled water (∼100-fold of sample volume). The samples were dialyzed for 4–7 days;9 during this time, the entire dialysate volume was changed for fresh dialysate solution repeatedly to wash away the unreacted monomers. Dialysis was allowed to run until the conductivity of the dialysate was close to that of the distilled water (2 μS cm−1).6 Elementary analysis performed on the nanogels after dialysis revealed that the materials still contained 0.2–0.5% of sulfur from SDS, bearing in mind that it was reported that elementary analysis is not a suitable method for exact measurements on poly(N-vinyl lactams).10 Between 1.24 and 1.42 g of dried product could be isolated after dialysis, and the values were used to calculate the yield after purification (Table 1). 1H NMR spectra were recorded on a Bruker 400.13 MHz (at 298 K) in deuterated DMSO. This measurement allowed the determination of the respective molar ratios of VCL and crosslinker in the swollen part of the nanogels. The droplet sizes before and after the polymerization were measured using a Nicomp particle sizer model 380 at a fixed scattering angle of 90° and at 23 °C and are reported as the average of two independent measurements. The logarithm of the field-field time autocorrelation function was expanded in terms of the cumulants of the distribution (method of cumulants). The second-order cumulant was converted to intensity weighted hydrodynamic diameter using instrumental constants and the dynamic viscosity of the continuous phase. The VPTT of the nanogels were determined by optical absorption measurements on a BioTek SynergyMx multidetection microplate reader at 490 nm. These measurements were carried out in triplicate at a concentration of 3 mg mL−1 (200 μL in each well) at temperatures from 28 to 45 °C with 1 °C increments. The solutions in the microplate were equilibrated for 5 min at each temperature. The samples were placed in a 96-well microplate from Nunc. The sizes of the dried nanogels were determined by TEM using a Hitachi S-4800 microscope. TEM grids were prepared at room temperature (RT). The microgels were swollen in water (5 or 10 mg/mL), one droplet was placed on a carbon-coated copper grid (QUANTIFOIL® 200mesh), and the excess of liquid was removed by placing the grid on a filter paper. Conventional CLSM (confocal laser scanning microscopy) and STED (stimulated emission depletion) microscopy were performed with the TCS SP5X (Leica Mikrosysteme Vertrieb GmbH, Wetzlar) microscope. The details of the experiment set-up and conditions were described previously.18 Steady-state fluorescence spectra of single particles were measured with the TCS SP5X microscope. Fluorescence spectra of diluted dispersions were recorded with A Spex FluoroLog 3 spectrofluorometer. Decays of fluorescence were recorded with time correlation single photon-counting technique (FluoTime 200, PicoQuant GmbH). A cuvette (thickness 10 mm) with a solution was excited by supercontinuum pulsed laser SC450-2-PP (10 MHz, ∼1 mW nm−1, and pulse duration ∼10 ps; Fianium Ltd, UK). Output radiation of the laser was passed through 4F-monochromator19 to select the needed wavelength. Typically, the radiation with a bandwidth 10–15 nm was used. Right angle geometry of sample was chosen for fluorescence collection. Glan–Thompson polarizers (for excitation and detection) were arranged under magic angle condition. Additional longpass filter Brightline 519/LP (Semrock Inc., USA) was placed in front of a Sciencetech Model 9030 monochromator for better eliminating scattered light. A counting photomultiplier PMA 165 was used as a detector. On the contrary to previous miniemulsion polymerizations of N-vinylcaprolactam,14 we dissolved the monomer in a hydrophobic solvent (toluene) and not in water. Because the solubility of the monomer is higher in toluene than in water, this procedure allows the formation of dispersions with much higher solid content (>10 wt %) than those obtained in inverse miniemulsion. For the evaluation of the partition of VCL in the aqueous and organic phase, both phases were isolated, and the aqueous phase was evaporated under vacuum. Both samples displayed no measurable amount of VCL in the aqueous phase. Different amounts of crosslinker (1, 5, and 16 mol %) were used in the synthesis to provide different degree of crosslinking, which leads to a different swelling for the nanogels. The yields after purification were between 62 and 71% (Table 1) as measured gravimetrically, that is, relatively low for a miniemulsion polymerization, but far above other poly(N-vinylcaprolactam) nanogel synthesis (<20 wt %).9 These relatively low yields can be explained by the presence of water-soluble oligomers that were dialyzed and by the low reactivity of the monomer N-vinylcaprolactam. Generally, the polymerizations with higher amounts of divinylbenzene resulted in a higher yield. The product was verified to be a copolymer of N-vinylcaprolactam by 1H NMR (Supporting Information Fig. S2). Moreover, 1H NMR analysis was used for determining the amounts of DVB incorporated in the nanogels. Interestingly enough, the calculated molar percentages of DVB are well correlated with the ones in the feed, but the absolute values are much smaller (Table 1). This effect is particularly pronounced for the dispersions synthesized with 15% DVB in the feed. This is due to the fact that upon incorporating more DVB in the nanoparticles, a microphase-separated structure is formed, with a DVB-rich core and a VCL-rich shell. The latter can be swollen by DMSO and thus reliably analyzed by solution 1H NMR, whereas the strongly crosslinked core does not contribute to the observed chemical signature. This observation was already reported for crosslinked poly(N-isopropylacrylamide).20 It was already shown that divinylbenzene can be homopolymerized by free-radical polymerization in the absence21 or presence of toluene in miniemulsion.22 The VCL/DVB/toluene miniemulsions were stable, displaying hydrodynamic diameters less than 200 nm (Table 1). The hydrodynamic diameters measured for the dispersions (after polymerization) were found to be larger than the original droplets. This is due to the swelling of the poly(N-vinylcaprolactam) nanogels in water at room temperature and probably owing to coalescence in the case of the highly crosslinked nanogels (VCL84). The dispersions were further dialyzed for removing toluene, the unreacted monomer, and the surfactant. Indeed, it is known that SDS has a strong influence on the VPTT of PVCL microgels.23 The nanogels with low and intermediate crosslinking could be redispersed (402 and 315 nm at 23 °C, respectively) in water as shown by DLS measurements, whereas the highly crosslinked formed micron-sized aggregates (>2 μm). The primary nanoparticles that build these aggregates are still visible in the aggregates themselves as identified by SEM by comparing nondialyzed [Fig. S3(a,b)] with dialyzed samples [Fig. S3(c)]. This is due to the higher hydrophobicity of the latter copolymer and the absence of surfactant to stabilize its interface with water. The less-crosslinked nanogels could be observed as singular objects by electron microscopy [Figs. 1(A) and S4]. The sizes of the dried and purified nanogels are in the range of ∼100 nm, that is, display much lower size than the nanogels swollen in water. TEM observations indicate that the core appears much darker in contrast than the surrounding part, thus confirming the 1H NMR measurements that revealed the presence of a core-shell structure for the nanogels. Such a behavior was documented in transmission electron micrographs of crosslinked poly(N-ethylmethacrylamide).24, 25 TEM micrographs of the microgels (a) VCL99, (b) VCL95, and (c) VCL84. The temperature-responsive character of the nanogels was evidenced by absorbance measurements for different copolymer concentrations (Fig. 2 and Supporting Information Fig. S5). The loss of transmittance upon increase of temperature was more pronounced for lower concentrations (1 mg mL−1) than for higher concentrations, and this is in a continuous manner from 2, 3, to 5 mg mL−1 [Supporting Information Fig. S5(a–c)]. However, at room temperature, a correlation between the DVB amount incorporated in the nanogels and the transmittance of the respective solutions can be observed regardless of the concentration. As such, nanogels with lower crosslinking degree appear to be well swollen in water, in contrast with the ones having higher crosslinker contents. The VPTT was defined as the temperature corresponding to a 10% reduction in the original transmittance of the solution as proposed by other authors for the determination of the lower critical solution concentration (method M1).26 Although the VPTT of the highly crosslinked sample could not be determined in the temperature range of measurement (28–45 °C), it was detected to be ∼33 °C for both nanogels that were less crosslinked (Table 1, M1). The VPTT was found to be higher by calculating the VPTT as abscissa of the inflection point: 37 and 38 °C for the samples with low and intermediate crosslinking, respectively. Both methods were used for concentration of polymer of 3 mg mL−1. The lower value calculated by method 1 is closer to expected values, because hydrophobic comonomers are known to reduce the lower critical solution concentration of temperature-responsive copolymers. Transmittance (%) of the dispersions monitored in function of the temperature (°C) for ccopolymer = 3 mg mL−1. Samples (a) VCL99, (b) VCL95, and (c) VCL84. The slightly crosslinked nanogel could be successfully fluorescently labeled by copolymerizing the VCL and DVB with a low amount of fluorescent comonomer [Fig. S1(c)]. The method affords an opportunity to incorporate a hydrophobic fluorescent material into polar environment keeping the unique photochemical properties of the dye. Figure 3(C) shows the CLSM image of single nanoparticles arranged on the top of a microscope slide. The observed individual features have various brightness due to differences of particles size. It is important to compare photophysical properties of the dye in polar and apolar environments. The nanogels showed strong and stable fluorescence independently on the polarity of the surrounding environment. The emission spectra (laser excitation at λ = 488 nm) measured for single particle pointed with red arrow [Fig. 3(C)] are presented in panel A of Figure 3. The spectra are compared to fluorescence spectra of the same dye chemically incorporated in hydrophobic polystyrene particles. The spectrum is clearly wider for fluorophores surrounded by strongly polar media (water). Therefore, intermolecular interaction between dyes can occur. The contrast between media got more evidenced by time resolved fluorescence measurements [Fig. 3(B)]. The decay of fluorescence is monoexponential with lifetime τ = 5.8 ns when the surrounding media are polystyrene. In contrast, the decay could be only fitted with a multiexponential model (average lifetime τav = 1.81 ns) when the fluorophore was located inside the nanogel. This fact emphasizes the complexity of intra- and intermolecular interaction inside the nanogels. Dye aggregation is an effect that can be responsible for shorter decay. In general, this is a negative effect leading to the reduction of brightness and enhancement of photodegradability. Remarkably, in the case of the nanogels, we did not observe any evidence of declining photochemical properties. We therefore tested the particles as potential contrast agent for STED microscopy.27 Indeed, STED microscopy is particularly interesting, because its resolution is not limited by diffraction and hence offers higher resolution than the standard CLSM. However, overall fluorescence decreases and photobleaching can occur when using STED microscopy. Thus, only very effective and stable photostable fluorophores can be used as contrast agent for this method. Panels D and F of Figure 3 present images obtained with conventional CLSM [Fig. 3(D)] and STED [Fig. 3(F)] microscopy. The obtained images clearly evidenced an improvement of optical resolution. Additionally, the nanogels showed high brightness and excellent photostability. The fluorescent nanogels are therefore suitable markers for STED microscopy. Fluorescent properties of obtained nanogel particles. A: Steady-state fluorescence of a dispersion of polystyrene nanoparicles (D = 92 nm) loaded with B504MA—black solid line; steady-state fluorescence of a single hydrogel particle loaded with B504MA (the particle is marked by red arrow on panel C; detection: Leica SP5 confocal microscope) —red circles. B: Decays of fluorescence measured by time correlation single photon counting. Dispersion of the polystyrene nanoparicles loaded with B504MA—blue line; dialyzed nanogel loaded with B504MA—red line. Instrument response—black line. C: CLSM image of single nanogels loaded with B504MA. D: Conventional CLSM image and (F) STED microscopy image show increasing resolution when the STED effect is used. N-Vinylcaprolactam was successfully copolymerized with a hydrophobic crosslinker in direct miniemulsion to yield nanogels of different composition. Unprecedented high-solid content of poly(N-vinylcaprolactam) nanogels could be obtained (>10 wt %), which could be redispersed when the nanogels contained less than 0.5 mol % DVB units in the corona. The nanogels with low-DVB content were found to be temperature responsive, exhibiting a VPTT around 33 °C. The nanogels could be labeled with a fluorescent comonomer, and the dialyzed nanogels displayed fluorescence making them suitable candidates for cell-nanogel interaction studies. 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