Introduction

Polymeric micelles, self-assembled from amphiphilic block copolymers in selective solvents, are of intensive interest in contemporary macromolecular science for both their diversiform morphologies and potential applications [1–3]. The so-obtained macromolecular assemblies demonstrate a series of attractive properties in drug delivery systems, such as good biocompatibility and high stability in vitro and in vivo, and have been described as promising materials in applications such as biosensors, tissue engineering, and selective drug delivery [4–6].

Micelles formed by stimuli-responsive block copolymers have received much attention in recent years [7–9]. Stimuli-responsive polymers that exhibit unique property changes in response to environmental stimuli, for example, temperature, pH, electric fields, and light, are promising for many biomedical applications, including smart drug/gene delivery systems, injectable tissue engineering scaffolds, cell culture, and separation sheets [10–12]. Among all intelligent polymers studied temperature-responsive polymeric systems have drawn more attention, because this is the important physiological factor in the body, and some disease states manifest themselves by a change in temperature [13, 14]. Thermo-responsive polymers are soluble in cold water, though precipitate with heating above a certain temperature, known as the lower critical solution temperature (LCST) [15]. This phenomenon is reversible; upon cooling the thermosensitive polymers become soluble again [16].

Poly(N-vinylcaprolactam) (PNVCL) is a well-known thermosensitive polymer with a LCST near body temperature, approximately at 32 °C [17–19]. These physiological temperatures open perspectives for biomedical applications such as micro-encapsulation of enzymes or cells and drug delivery systems [20–22]. PNVCL can be used as a hydrophilic shell segment at temperatures below its LCST together with various kinds of hydrophobic moieties to make core/shell micelles. The actual interest in PNVCL is connected with the combination of its thermo-responsive properties, complexation ability with different therapeutic agents and biocompatibility [23, 24].

Poly(ε-caprolactone) (PCL) and their copolymers have attracted increasing attention in the pharmaceutical and medical fields for potential uses as bioresorbable sutures, orthopedic fixation devices, artificial skin, tissue engineering scaffolds, and drug delivery systems because their biodegradation in physiological conditions yields nontoxic products that can be bioabsorbed or excreted by the human body [25–28]. But the application of PCL for drug delivery has a drawback of slow degradation rate in vivo due to its high crystallinity and hydrophobicity [29–31]. Introduction of hydrophilic units has been used to improve biodegradability and hydrophilicity [32].

In this study, new amphiphilic thermosensitive poly(N-vinylcaprolactam)/poly(ε-caprolactone) (PNVCL-b-PCL) block copolymers were synthesized by ring-opening polymerization of ε-caprolactone (ε-CL) using hydroxy-terminated poly(N-vinylcaprolactam) (PNVCL-OH) as macroinitiator. Their structures were characterized. Then, the PNVCL-b-PCL micelles were prepared by solvent evaporation method and their physicochemical characteristics and thermosensitivities were investigated.

Experimental

Materials

N-vinylcaprolactam (NVCL, analytical grade) was purchased from Sigma-Aldrich and recrystallized from dry n-hexane prior to use. ε-Caprolactone (ε-CL) was purchased from Alfa Aesar Co., Ltd. Stannous octoate (Sn(Oct)2) was purchased from Sigma-Aldrich Chemical Reagent Co., Ltd. 2-Mercaptoethanol (HSCH2CH2OH, analytical grade) was purchased from Amresco Co., Ltd. Pyrene was purchased from Alfa Aesar Co., Ltd. and recrystallized from dry ethanol prior to use. Azobisisobutyronitrile (AIBN, analytical grade) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China), and recrystallized from dry ethanol prior to use. 1,4-Dioxane (analytical grade) and dichloromethane (CH2Cl2, analytical grade) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China), dried and distilled prior to use. Other chemicals are all analytical reagents made in China and used without further purification.

Synthesis of hydroxy-terminated poly(N-vinylcaprolactam) (PNVCL-OH) (Scheme 1)

The PNVCL-OH was synthesized by the radical polymerization of NVCL monomer initiated by AIBN with HSCH2CH2OH as chain transfer agents. In brief, 5.0425 g (0.0363 mol) of NVCL and 0.0597 g (0.3640 mmol) of AIBN were dissolved in 40 mL 1,4-dioxane and bubbling nitrogen for 30 min. Then, 0.0947 g (1.2141 mmol) of HSCH2CH2OH was added into the reactor. The polymerization was performed at 68 °C and terminated after 24 h under stirring. After being cooled to room temperature, 1,4-dioxane was removed under reduced pressure. The resulting polymer was dissolved in 30 mL anhydrous CH2Cl2 and precipitated in 300 mL petroleum ether three times. The precipitate was dried under reduced pressure at 40 °C for 48 h giving the PNVCL-OH as white solid, yield 48 %.

Scheme 1
scheme 1

The synthesis of PNVCL-b-PCL block copolymer

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Synthesis of poly(N-vinylcaprolactam)/poly(ε-caprolactone) (PNVCL-b-PCL) block copolymer (Scheme 1)

The PNVCL-b-PCL block copolymers were synthesized by ring-opening polymerization of ε-CL initiated by PNVCL-OH with Sn(Oct)2 as a catalyst. The necessary amounts of PNVCL-OH, ε-CL, Sn(Oct)2 and toluene were added to a 50-mL round flask and after several cycles of evacuation-purging with purified nitrogen, the polymerization was performed in an oil bath at 120 °C and terminated after 48 h under stirring. After being cooled to room temperature and removal of toluene under reduced pressure, the resulting polymer was dissolved in 15 mL anhydrous CH2Cl2 and precipitated in 150 mL ethyl ether several times. The precipitate was dried in vacuum at 30 °C for 48 h to give the desired PNVCL-b-PCL block copolymers as white solid. The yield was approximately 30 %. Different molar ratios of the feeding ε-CL to PNVCL-OH resulted in the corresponding copolymers with various compositions as listed in Table 1.

Table 1 Related data on polymers
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Preparation of micelles

The micelles were prepared applying a solvent displacement method with a tetrahydrofuran/water (THF/H2O) system [33]. A copolymer (50 mg) was first dissolved in 2.5 mL of THF; thereafter, the copolymer solution was slowly added into 10 mL of ultrapurified water (Aquaplus 18.2 MΩ). The THF was removed using a rotary evaporator at 25 °C for 2 h. The obtained solution was transferred into a 25-mL volumetric flask, followed by dilution to the calibration mark with ultrapurified water to obtain 2 mg mL−1 micelles.

Characterization

The IR spectra were collected by a Perkin-Elmer FT-IR spectrometer using KBr disks. 1H NMR spectra were measured on a Varian Mercury-300 NMR spectrometer at room temperature, using CDCl3 as solvent. Chemical shifts (δ) were given in ppm using tetramethylsilane (TMS) as an internal reference. The gel permeation chromatography (GPC) measurement was conducted with a Waters 1515 GPC instrument equipped with a HT4 and HT3 column (effective molecular-weight range 5,000–600,000 and 500–30,000) and a 2414 differential refractive index detector. THF was used as eluent at the flow rate of 1.0 mL/min at 30 °C, and the molecular weights were calibrated with polystyrene standards. The lower critical solution temperature (LCST) of the aqueous solutions of the polymers was investigated on a Perkin-Elmer Lambda Bio 20 UV–vis spectrophotometer together with a NESLAB RTE-111 temperature controller. In brief, the polymers were dispersed in ultrapurified water (Aquaplus 18.2 MΩ). The transmittance of aqueous solutions of polymer at λ = 500 nm was recorded in a 1.0-cm path-length quartz cell. The rate of heating was set at 1 °C/min with hold steps of 10 min at each temperature. Values for the LCST of aqueous solutions of the polymers were determined at a temperature with a half of the optical transmittance between blow and above transitions. The critical micelle concentrations (CMC) of the copolymers were determined by fluorescence measurements 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 6.0 × 10−7 mol/L; thereafter, the acetone was removed completely. The polymer solution was added into the volumetric flasks and diluted till the calibration mark using ultrapurified water to obtain the desired copolymer concentrations ranging from 1.0 × 10−4 to 0.9 mg/mL. The samples were stored at room temperature overnight to equilibrate the pyrene and micelles. Steady-state fluorescence excitation spectra were recorded on a Varian Cary Eclipse fluorescence spectrophotometer at 390-nm emission wavelength and 5.0-nm slit width. The scan rate was 120 nm/min. The size distribution of micelles was determined by dynamic light scattering (DLS) using a Malvern Nano ZS instrument. The morphology of the micelles was investigated by transmission electron microscopy (TEM), carried out on a Hitachi H-7650 electron microscope, operating at an accelerating voltage of 80 kV. Specimens were prepared by transferring a drop of the micelle solution onto a 200-mesh copper grid coated with carbon and allowing the sample to dry in air before measurements.

Results and discussion

Synthesis of poly(N-vinylcaprolactam)(PNVCL-OH)

The hydroxyl-terminated PNVCL-OH was synthesized by a radical polymerization of NVCL using 2-mercaptoethanol as a chain transfer agent and AlBN as an initiator (Scheme 1).

The IR spectrum of PNVCL-OH is depicted in Fig. 1B. The polymer gave a broad absorption in the 3,200–3,600 cm−1 region due to terminal hydroxyl groups. Furthermore, the absorption at 1,650 cm−1 was assigned to the stretch vibration ν C=O from the PNVCL-OH segment; the typical absorption of the NVCL monomer at 1,630, 2,980 and 3,100 cm−1 had completely disappeared.

Fig. 1
figure 1

IR spectra of NVCL (A), PNVCL-OH (B) and PNVCL-b-PCL3 (C)

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The 1H NMR spectrum of PNVCL-OH was shown in Fig. 2A. The peak at 4.25–4.68 ppm is assigned to proton a in PNVCL segment. The peak at 3.75 ppm is assigned to protons i in the –CH2CH 2 OH segment. The peak at 2.95–3.42 ppm is assigned to protons c in PNVCL segment. The peak at 2.80 ppm is assigned to protons h in the –CH 2 CH2OH segment. The peak at 2.45–2.82 ppm is assigned to protons g in the PNVCL segment. The peak at 2.15 ppm is assigned to proton j in the –CH2CH2OH segment. The peaks at 1.00–2.06 ppm are assigned to protons b, d, e, f in the PNVCL segments.

Fig. 2
figure 2

1H NMR spectra of PNVCL-OH (A) and PNVCL-b-PCL3 (B) block copolymer in CDCl3

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The GPC trace of the PNVCL-OH is shown in Fig. 3A. The sample showed unimodal molecular weight distribution. This further indicates that the polymerization is completed successfully and there is no other compound in the reaction product.

Fig. 3
figure 3

GPC traces of PNVCL-OH and PNVCL-b-PCL block copolymers

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Synthesis of poly(N-vinylcaprolactam)/poly(ε-caprolactone) (PNVCL-b-PCL) block copolymer

The amphiphilic block copolymers composed of PNVCL as the hydrophilic part and PCL as the hydrophobic one were synthesized through ring-opening polymerization. Stannous octoate, one of the most widely used initiators for cyclic esters polymerization, has been reported recently to induce polymerization of cyclic ε-caprolactone (ε-CL), lactide (LA), and phosphoester (PPE) by formation of stannous alcoholate active centers with ROH or RNH2 as co-initiator [34–36]. Because PNVCL-OH contains hydroxyl group, it can initiate ring-opening polymerization of cyclic ε-CL to generate PNVCL-b-PCL block copolymers. A series of the block copolymers with various molecular weights were synthesized and the results are summarized in Table 1.

The IR spectrum of PNVCL-b-PCL3 was shown in Fig. 1C. Compared to PNVCL-OH (Fig. 1B), the spectrum of the copolymer clearly showed the most important vibration bands, especially the vibrations of νC=O appearing at 1,723 cm−1 from the PCL segment.

The 1H NMR spectrum of PNVCL-b-PCL3 block copolymer is shown in Fig. 2B. The peak at 4.25–4.68 ppm is assigned to proton a in PNVCL segment. The peak at 3.75 ppm is assigned to protons i in the –CH2CH 2 O– segment. The peak at 2.95–3.42 ppm is assigned to protons c in PNVCL segment. The peak at 2.80 ppm is assigned to protons h in the –CH 2 CH2O- segment. The peak at 4.11 ppm is assigned to protons p in the PCL segment. The peaks at 2.22–2.40 ppm are assigned to protons g, k in the PNVCL and PCL segments, respectively. The peaks at 1.32–1.78 ppm are assigned to protons b, d, e, f, l, o in the PNVCL and PCL segments, respectively. There are not additional peaks in the spectrum, which initially indicates that the block copolymer was prepared.

The GPC traces of the PNVCL-b-PCL copolymers were shown in Fig. 3. The three copolymers showed unimodal molecular weight distribution. This further indicates that the copolymerization is completed successfully and there is no homo-polymer in the reaction product. GPC data of the copolymers are listed in Table 1.

Formation of micelles

The micellar structures of PNVCL-b-PCL are confirmed by fluorescence technique using pyrene as a probe. The fluorescence excitation spectra of pyrene in the presence of PNVCL-b-PCL1 at various concentrations are shown in Fig. 4. A red shift from 333 to 337 nm is observed with increasing concentration of PNVCL-b-PCL1, indicating that micellization takes place for the PNVCL-b-PCL1 copolymer. Such results can be attributed to the transfer of pyrene molecules from water to a hydrophobic environment within the micelles core.

Fig. 4
figure 4

Excitation spectra of pyrene as a function of PNVCL-b-PCL1 concentration in water

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The onset of micellization and the critical micelle concentrations (CMC) can also be obtained from the studies of excitation spectra [37]. For the copolymer PNVCL-b-PCL1, 333 and 337 nm are chosen as the peak wavelength of the (0, 0) band in the pyrene excitation spectra in the aqueous phase and in the entirely hydrophobic core of polymeric micelle, respectively. The pyrene fluorescence intensity ratios (I 337/I 333) are plotted against the logarithm of copolymer concentration. The plots are shown in Fig. 5. Below a certain concentration, I 337/I 333 is constant, above this concentration, I 337/I 333 increases with increasing lg C and finally reaches a plateau. From this plot, the critical micelle concentration (CMC) of 2.7 × 10−2 mg mL−1 was obtained from the intersection of two straight lines: the base line and the rapidly rising I 337/I 333 line. The CMC of PNVCL-b-PCL2 and PNVCL-b-PCL3 were also obtained from the same methods and listed in Table 1. The CMC values were reduced with the increasing of the proportion of PCL segment. It is reasonable since higher content of the hydrophobic segments will result in stronger interactions between hydrophobic chains, leading to a more stable structure and therefore to lower CMC value. This trend is in agreement with the reported result by the literature [38].

Fig. 5
figure 5

Plots of I 337/I 333 versus logarithm of PNVCL-b-PCL block copolymers’ concentrations

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Size and size distribution of PNVCL-b-PCL micelles

The size and size distribution of micelles were measured by DLS. As shown in Fig. 6, the mean diameter of micelles formed by PNVCL-b-PCL1, PNVCL-b-PCL2, and PNVCL-b-PCL3 were about 106, 142, and 167 nm, respectively. The size of micelle was increased with the increasing of the proportion of PCL segment, so the size of the copolymer micelle could be adjusted by changed the proportion of PCL segment of the copolymer. The increment of micelle size with increasing PCL block length is mainly originated from the increase of hydrophobic property by the longer hydrophobic PCL chain in aqueous milieu.

Fig. 6
figure 6

The size distributions of PNVCL-b-PCL1 (A), PNVCL-b-PCL2 (B) and PNVCL-b-PCL3 (C) block copolymer micelles in aqueous solution measured by DLS at 25 °C

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Thermosensitivity of polymers

Figure 7 is a typical photograph of aqueous solutions of PNVCL-b-PCL2. Below the LCST, PNVCL-b-PCL copolymers are amphiphilic, consisting of a hydrophilic block (PNVCL) and a hydrophobic block (PCL). The solution was transparent and colorless (Fig. 7A). However, when heated closed to the LCST, the solution gradually turned into a semitransparent emulsion (Fig. 7B), then above the LCST, the copolymers are hydrophobic and the solution became a white opaque suspension (Fig. 7C), and finally the polymers precipitated from water if the solution was kept at a high temperature for enough time. When cooled, the semitransparent emulsion and transparent colorless solution were gotten again. Evidently, PNVCL-b-PCL2 showed a revisable LCST phase transition in water. This phenomenon takes place due to the different solvation of poly(N-vinylcaprolactam) chains by water molecules at the temperatures below and above the phase transition temperature [39].

Fig. 7
figure 7

LCST transitions of PNVCL-b-PCL2 at different temperatures: 25.6 °C (A), 38.9 °C (B) and 43.3 °C (C) (c = 0.5 mg/mL)

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Effect of molecular weight on the LCST of PNVCL-OH

LCSTs are expected to decrease with increasing polymer molecular weight based on the changes in the polymer–solvent interaction [19, 40, 41]. This phenomenon is ascribed to the fact that the solution behavior of the PNVCL-OH/water system corresponds to a typical Flory-Huggins demixing behavior with LCST, also called Type I behavior [42]. Figure 8A shows a definite effect of the molecular weight on the LCST of PNVCL-OH in aqueous solution. The LCST decreased as the molecular weight of the polymer increased. The variation of the number-average molecular weight of PNVCL-OH from 2572 to 5383 g mol−1 leaded to the variation of the LCST from 47.8 to 38.6 °C (Fig. 8B). This result is explained by the fact that the decrease of the molecular weight leads to an increase of the polymer hydrophilicity. Moreover, the increase of the hydrophilicity would also be explained by the contribution of the hydroxyl end group from the chain transfer agent as was evidenced by Santos for PNVCL with molecular weights between 2.497 × 104 and 5.903 × 104 g/mol [43]. As the molecular weights obtained in this work were in general much lower than those obtained by Santos et al., the influence of the hydroxyl end group was more evident. Therefore, the increase of molecular weight seems to have a more effective influence on the LCST. The difference on the LCST values in the range of molecular weights studied observed in this work was much higher than that in the literature [41, 43].

Fig. 8
figure 8

Effects of molecular weight on the LCST of PNVCL-OH (A) and LCST as a function of the molecular weight (B) (c = 5 mg/mL)

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Effect of hydrophobic block length on the LCST of copolymers

It is reported that the LCST of thermo-responsive polymers can be controlled by compositions of hydrophobic and hydrophilic units [18]. In this study, the LCST transition behavior of the PNVCL-b-PCL block copolymers was tuned by changing the length of the hydrophobic PCL block: the larger the hydrophobic block content of the copolymer, the lower the LCST at fixed PNVCL molecular weight. Figure 9A shows the temperature dependence of optical transmittance of micellar solutions of the copolymers with different hydrophobic PCL block lengths. The transmittance decreased significantly at a specific temperature on heating solutions of all of the copolymers. The LCST were evaluated as 39.3, 37.5, 35.3, and 33.7 °C for PNVCL-OH, PNVCL-b-PCL1, PNVCL-b-PCL2, and PNVCL-b-PCL3, respectively (Fig. 9B), thus showing a decreasing trend with increasing PCL block length. The LCSTs of PNVCL-b-PCL polymers were found to be lower than PNVCL-OH homopolymers and to depend on their molecular weights. This is considered to result from hydrating contributions from polar terminal hydrophilic hydroxyl groups in the copolymers [44]. In general, the LCST decreases with decreasing hydrophilicity of the polymer. Considering the fact that PCL block is more hydrophobic than PNVCL block, it is reasonable that the LCST decreases with the increasing molar fraction of PCL.15 These results clearly show that the phase transition of PNVCL-b-PCL can be controlled within a temperature range (33.7–39.3 °C) by varying the block length of the PCL block. Similar observations have been reported for several thermosensitive polymers [19, 45]. Thermoresponsivity under physiological conditions is effective for drug delivery or tissue engineering applications. In fact, the LCST of thermo-responsive polymers can be controlled by compositions of hydrophobic and hydrophilic units [14]. Thus, LCST can also be further tuned by adjusting the composition of the PNVCL block or adding new hydrophobic blocks to find its application under physiological temperatures.

Fig. 9
figure 9

Effect of hydrophobic block length on the LCST of copolymers (A) (c = 5 mg/mL) and LCST as a function of the PCL block in the copolymer (B)

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Effect of concentration on the LCST of copolymers

Figure 10 shows the effect of concentration on the LCST of PNVCL-b-PCL2 aqueous solution. For the lowest concentration (2.0 × 10−3 mg/mL, which was below the CMC), nearly 100 % transmittance was maintained in the tested temperature range. As the polymer concentration became higher than the CMC and micelle formation occurred, the transmittance decreased sharply at about 43.9 °C on heating, indicating that a LCST transition readily occurred only in micellar solution. The polymer concentration had an effect on the LCST of the micellar solution: increasing the concentration from 0.1 to 0.9 mg/mL shifted the LCST 6.4 °C toward lower temperature. When the concentration is 0.9 mg/mL, LCST of the micellar solution was 37.5 °C. Similar observations have been reported for other thermosensitive polymers [46]. In addition, it should be noted that the sharpness of the thermally induced phase transition was dependent on the polymer concentration. A fairly sharp LCST transition was observed at concentrations of 0.9 and 0.5 mg/mL. At concentrations of 0.1 and 0.2 mg/mL, very limited variation of transmittance was displayed at the LCST. For concentrations >0.9 mg/mL, the transition became broader. This finding is consistent with the generally accepted LCST principle for dilute solutions, which states that higher water content enhances the hydrogen-bonding interactions between water and the polymer chain, which requires more thermal energy to break the water structure, thereby resulting in an increase of the LCST [21, 47].

Fig. 10
figure 10

Effect of concentration on the LCST of PNVCL-b-PCL2 (A) and LCST as a function of the concentration of PNVCL-b-PCL2 (B)

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Variable temperature TEM measurements

Figure 11 shows TEM images of the micelles obtained by the self-assembly of PNVCL-b-PCL2 block copolymer in aqueous solution at 25.6, 38.9, and 43.3 °C, respectively. All solution concentrations were kept at 0.5 mg/mL, which was above the CMC of PNVCL-b-PCL2 solution. When the solution temperature was below the LCST [<38.9 °C, Scheme 2(Stage 1)], spherical polymer micelles existed individually (Fig. 11A) and the micellar solution was clear (Fig. 7A). At temperatures close to the LCST [Scheme 2(Stage 2)], intermicelle aggregation gave rise to formation of larger aggregates with multicore structure that was detectable in their TEM images (Fig. 11B) and associated with an abrupt increase in aggregate radius. Moreover, the solution became cloudy (Fig. 7B). When the temperature was 43.3 °C, the micelles aggregated [Fig. 11C, Scheme 2(Stage 3)]. The micelle dehydration and aggregation will certainly increase the solution turbidity due to the scattering (Fig. 7C).

Fig. 11
figure 11

TEM images of PNVCL-b-PCL2 block copolymer micelles: (A) 25.6 °C, (B) 38.9 °C and (C) 43.3 °C (c = 0.5 mg/mL)

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Scheme 2
scheme 2

Schematic illustration of thermally induced aggregation and phase transition of PNVCL-b-PCL copolymers in water

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Conclusions

In the present study, the PNVCL-OH and PNVCL-b-PCL block copolymers with different PCL block lengths have been synthesized. The structures and compositions of the polymers characterized by FT-IR, 1H NMR and GPC. The nanosized micelles with a core–shell (corona) structure and regularly spherical shape were obtained by self-assembly from the block copolymer in aqueous solution. The property of micelles such as CMC and size was investigated. It has been revealed that the phase transition of PNVCL-b-PCL micelles is reversible. The LCST of PNVCL-OH is affected by the molecular weight. The LCST of PNVCL-b-PCL is affected by the composition and the concentration of the copolymer, which allows convenient adjustment of their thermosensitivity.