Self-assembly of upconversion nanoclusters with an amphiphilic copolymer for near-infrared- and temperature-triggered drug release

Abstract

Multifunctional triggered drug release systems have been one of the most intriguing and challenging topics. In this study, we synthesize amphiphilic copolymers composed of hydrophilic oligo ethylene glycol methyl ether methacrylate and hydrophobic azobenzene-containing methacrylate. These amphiphilic copolymers can easily self-assemble into micelles in aqueous media and show smart responses to temperatures, UV/vis lights and ions. Interestingly, the obtained copolymers can self-assemble onto hydrophobic upconversion nanoparticles in aqueous media via hydrophobic interaction to form hybrid colloidal clusters with the hydrophobic nanocrystal core and hydrophilic polymeric shell. The hybrid colloidal clusters exhibit a thermo-responsive and photosensitive performance. When served as a drug carrier, both the near-infrared light and temperature triggered drug release can be realized, which have potential applications in intelligent drug delivery.

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Introduction

Self-assembly of amphiphilic copolymers has been increasingly utilized as drug nanocarriers due to the high loading capacity, sustained or triggered release, extended blood circulation, and site-specific accumulation within tumor tissues.^1–3 As for practical clinical applications, the behaviors of biological tissues are often a result of adaptation to multiple environmental changes.⁴ Therefore, a variety of pathologically relevant stimuli, such as pH, temperature, enzymes, hypoxia, and redox milieu, have been exploited to design multiple responsive drug nanocarriers.^5–10 Among these drug delivery systems, light-controllable drug delivery systems (LDDs) have been investigated extensively because photo as an external stimulus offers controllable drug release both spatially and temporally, thereby affording better control of drug administration and improved therapeutic efficacy.^11,12 To date, although several types of LDDs have been developed, including those based on scaffolds such as coumarin,¹³ 2-nitrobenzyl,¹⁴ and 7-nitroindoline¹⁵ or their derivatives, traditional LDDs require UV light for excitation, which has a poor penetration depth and is harmful to living tissues, thus limiting their clinical applications.

In recent years, lanthanide-doped upconversion nanoparticles (UCNPs) have aroused tremendous interest in bioimaging. In particular, they have the ability to generate UV or visible emissions under continuous-wave near-infrared (NIR) excitation.¹⁶ Nonetheless, UCNPs are usually synthesized using a hydrophobic ligand (oleic acid) and cannot be directly used for biological applications. To address this issue, several strategies have been developed to transfer the hydrophobic nanocrystals into an aqueous phase, including the addition of extra chemicals (silica),¹⁷ replacement of the hydrophobic ligand with other ones,¹⁸ or self-assembly with amphiphilic polymers or molecules.¹⁹ For example, considering UCNPs co-doped with Yb³⁺ and Tm³⁺ are used to convert CW NIR light at 980 nm into UV emission in an anti-Stokes process,²⁰ Liu et al. reported NIR-triggered anticancer drug delivery system by the upconversion of nanoparticles with integrated azobenzene-modified mesoporous silica.²¹ Yan and co-workers successfully used the NIR laser to dissociate a block copolymer by encapsulating UCNPs inside the micelles. Nevertheless, the majority of the NIR drug delivery systems are based on silica and single stimuli-responsive polymers.²²

In this study, we successfully synthesized multi-responsive amphiphilic copolymer through the reversible addition–fragmentation chain transfer polymerization (RAFT) of azobenzene-containing methacrylate (AZO) and oligo ethylene glycol methyl ether methacrylate (OEGMA). The amphiphilic copolymer obtained not only exhibits excellent responses to UV/vis light, temperature and ions, but also is easily used to convert hydrophobic nanocrystals (e.g., QDs, SPIOs, and UCNPs) to hydrophilic colloidal nanoclusters by self-assembly. To assess the suitability of the hydrophilic UCNP clusters as a carrier for controlled drug release, drug loading and in vitro release studies were performed using doxorubicin (DOX) as a hydrophobic model drug. The results showed that drug release can be controlled by altering the temperature and NIR stimuli. These properties make hydrophilic UCNPs clusters potential candidates for novel intelligent delivery systems and smart biomaterials fields.

Experimental section

Materials

Oligo ethylene glycol methyl ether methacrylate (OEGMA, M_n = 475) was received from Aladdin Regent Co., Ltd (China) and passed through a basic alumina column to remove inhibitors prior to use. 2,2′-Azobis(2-methylpropionitrile) (AIBN) was obtained from J&K Chemical Ltd (USA) and recrystallized three times using ethanol. Iron(III) chloride hexahydrate (FeCl₃·6H₂O), iron(II) chloride tetrahydrate (FeCl₂·4H₂O), oleic acid (OA, 90%), NH₄F (98%), ammonium hydroxide (NH₃·H₂O, 25–28%), sodium hydroxide (NaOH, 98%), 1-octadecene (ODE, 90%), Rhodamine B (Rh-B), fluorescein isothiocyanate (FITC), ethanol, cyclohexane, hexane, and tetrahydrofuran (THF, 99.5%) were purchased from Sinopharm Chemical Reagent Co., Ltd (China). THF was refluxed and distilled over sodium (Na) before use. Propargyl alcohol (99%) was received from Xiya Chemicals (China). 4-(Dimethylamino)pyridine (99%), GdCl₃·6H₂O (99.99%), YCl₃·6H₂O (99.99%), YbCl₃·6H₂O (99.9%), TmCl₃·6H₂O (99.9%), N-(3-dimethylaminopropyl)-N′-ethyl-carbodiimide hydrochloride (EDC, 99%), doxorubicin hydrochloride (DOX), and dichloromethane (anhydrous grade) were purchased from Sigma-Aldrich. CdSe/ZnS nanocrystals (QDs, emission at 620 nm) were purchased from Xingzi Company (China). All these chemicals were used as received unless otherwise stated.

Synthesis of alkyne-terminated CTA (alkyne-CTA)

The RAFT agent 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid (CTA) was synthesized according to a previously reported procedure as follows:²³ 5 mmol CTA, 8 mmol 4-(dimethylamino)pyridine and 8 mmol EDC were added to 35 mL dichloromethane in a 50 mL round bottom flask. The solution was then kept in an ice bath for 30 min under nitrogen flow, followed by the dropwise injection of 20 mmol propargyl alcohol. This reaction system was maintained at 0 °C for 1 h and then at room temperature overnight with stirring. The resulting brown solution was washed successively with 1 M HCl and brine, dried over anhydrous MgSO₄ for 12 h and then concentrated. The crude product was further purified by column chromatography using ethyl acetate/petroleum ether mixture and dried under vacuum at 40 °C for 10 h. The final pure product (brownish-yellow oil) was collected and characterized by ¹H NMR (Fig. S1†).

Synthesis of poly(AZO-co-OEGMA) amphiphilic copolymers

Monomer (4-(2-methylacryloyloxy)ethyloxy-4′-triuoromethoxy)azobenzene (AZO) was synthesized according to the method reported previously²⁴ and has been confirmed by ¹H NMR (Fig. S2†). In a typical procedure for the synthesis of poly(AZO-co-OEGMA), 475 mg OEGMA, 394 mg AZO, 44.1 mg alkyne-CTA, and 3.28 mg AIBN were dissolved in 4 mL THF under stirring within a 25 mL flask equipped with a magnetic stirring bar. After three freeze–pump–thaw degassing cycles, the solution was subjected to 70 °C for 24 h for RAFT polymerization, and then quenched in ice water and exposed to air for termination. The mixture was precipitated in hexane and redissolved three times in THF to obtain the poly(AZO-co-OEGMA) amphiphilic copolymers.

Self-assembly of poly(AZO-co-OEGMA) amphiphilic copolymers

10 mg poly(AZO-co-OEGMA) copolymer was dissolved in 1 mL THF at 30 °C under stirring, and 10 mL water was slowly added to the solution using a syringe pump within 1 h. The solution was then dialyzed (MWCO: 3.5 kDa) against water at 30 °C for 12 h to remove THF. To obtain the polymeric micelles containing Nile Red, poly(AZO-co-OEGMA) copolymer (4 mg) and Nile Red (0.1 mg) were dissolved into THF (0.5 mL), and water was then added dropwise to induce the formation of micelles encapsulating Nile Red with stirring for 12 h. The precipitation of unloaded Nile Red was removed by filtration through 0.22 μm membranes and the redundant THF was evaporated at 30 °C under stirring for 3 days to obtain polymeric micelles with Nile Red. A similar approach was carried out to obtain the polymeric micelles containing Rhodamine B or FITC. Poly(AZO₇-co-OEGMA₉) copolymer (10 mg) and Rhodamine B or FITC (0.1 mg) were dissolved into THF (1 mL), and 10 mL water was then added dropwise into the solution, the solution was dialyzed (MWCO: 3.5 kDa) against deionized water for 2 days to remove unloaded Rhodamine B or FITC.

Synthesis of core–shell NaYF₄:Gd/Yb/Tm@NaGdF₄ nanocrystals

NaYF₄:Gd/Yb/Tm nanocrystals were synthesized using a solvothermal method.²⁵ Briefly, 0.695 mmol YCl₃·6H₂O, 0.2 mmol YbCl₃·6H₂O, 0.1 mmol GdCl₃·6H₂O, and 0.005 mmol TmCl₃·6H₂O were added to a mixture of 6 mL OA and 15 mL ODE in a 100 mL three-neck round-bottom flask. This solution was heated to 120 °C under argon flow for 60 min to remove moisture and maintained at 150 °C for another 60 min to form a clear light yellow solution. After cooling to 50 °C, 10 mL methanol solution containing 4 mmol NH₄F and 2.5 mmol NaOH was added to this system. After vigorously stirring for 30 min, the white slurry was slowly heated and kept at 110 °C for 30 min to remove methanol and then was heated to 310 °C and kept for 1.5 h under an argon atmosphere. The product (NaYF₄:Gd/Yb/Tm nanocrystals) was dispersed into 30 mL ethanol, collected by centrifugation (12 000 rpm, 15 min), and washed with cyclohexane/ethanol three times. To synthesize core–shell NaYF₄:Gd/Yb/Tm@NaGdF₄ nanocrystals, 5 mL cyclohexane containing the as-prepared NaYF₄:Gd/Yb/Tm nanocrystals was mixed with 0.5 mmol GdCl₃·6H₂O, 6 mL OA and 15 mL ODE, and gently heated to 80 °C to remove cyclohexane. The final product was obtained by the precipitation–centrifugation process.

Preparation of colloidal nanoclusters

The poly(AZO₇-co-OEGMA₉) copolymer (20 mg) was dissolved in a mixture of THF (1 mL) and distilled water (10 mL), and then mixed with the oleic acid-stabilized UCNP nanocrystals (0.1 g) in cyclohexane. The two-phase suspension was sonicated at 70 °C for 30 min to evaporate cyclohexane and THF for the self-assembly of amphiphilic copolymers and nanocrystals. The obtained colloidal nanoclusters were centrifuged to remove the aggregation and further dialyzed against distilled water to produce hydrophilic UCNP nanoclusters. The co-assembly of QDs or SPIOs nanoclusters with poly(AZO₇-co-OEGMA₉) was carried out using the same procedure as above.

DOX loading and release

Doxorubicin hydrochloride (2 mg) and TEA (20 μL) were dissolved in 0.5 mL THF at 25 °C with stirring for 5 h. 5 mL colloidal UCNP nanoclusters with 10 mg copolymer were added to the abovementioned solution and the mixture was dialyzed against 7.4 PBS buffer at 25 °C for 3 h to remove THF, TEA, and unloaded DOX. The DOX-loading content was quantified by fluorescent spectrum. The encapsulation efficiency (EE%) was calculated as EE% = (W_total − W_unloaded)/W_total × 100%. The loading content (LC%) was calculated as LC% = W_loaded/(W_polymer + W_loaded) × 100%, where W_total, W_unloaded, W_loaded, and W_polymer refer to the weights of drug used, unloaded drug, drug encapsulated by micelles, and copolymer, respectively.

DOX-loaded colloidal nanoclusters were dispersed in 0.1 mol L⁻¹ phosphate buffer (pH 7.4) and transferred to a dialysis bag (MWCO: 3.5 kDa) immersed in the same buffered medium at 37 °C, 45 °C, and 45 °C with 0.1 M K₂SO₄. At certain time intervals, samples of the external medium were withdrawn and replaced with the same volume of fresh buffer solution. The concentration of DOX was calculated based on the fluoresce intensity of DOX at 590 nm. The cumulative amount of released drug was calculated, and the percentages of drug release from colloidal nanoclusters were plotted against time.

Characterizations

¹H NMR spectra were obtained on a Bruker spectrometer at 500 MHz NMR instrument using CDCl₃ or D₂O as the solvent. The UV-vis absorption spectra were acquired on a PerkinElmer Lambda 4C spectrophotometer. The molecular weight and molecular weight distribution were determined on a gel permeation chromatograph (GPC) with THF as the eluent, polystyrene as the standard at 35 °C and a flow rate of 1.0 mL min⁻¹. Fluorescent photographs were recorded by a Nikon C2 plus confocal laser scanning microscope (CLSM). The upconversion luminescence spectra were obtained on an Edinburgh LFS-920 spectrometer, where an external 0–3 W adjustable continuing wavelength (CW) laser at 980 nm (Connet Fiber Optics, China) replaced the xenon lamp as the excitation source. The X-ray diffraction (XRD) patterns were obtained on a Bruker D4 powder X-ray diffractometer using Cu Kα radiation (40 kV, 40 mA). The images of the polymer micelles, nanocrystals, and nanoclusters were scanned using a JEOL JEM-2010 transmission electron microscope at an accelerating voltage of 200 kV. Energy-dispersive X-ray analysis (EDX) of the samples was performed on a high resolution transmission electron microscope (HRTEM). Dynamic light scattering (DLS) measurements were carried out in a Malvern Instrument Nano ZS90.

Results and discussion

Synthesis and self-assembly of poly(AZO-co-OEGMA)

The amphiphilic poly (AZO-co-OEGMA) copolymers were synthesized via RAFT polymerization, as shown in Scheme 1, and confirmed by ¹H NMR spectroscopy and GPC. Fig. 1a demonstrates the ¹H NMR spectra of the copolymers with different amounts of OEGMA. The broad peak at 4.05 ppm (f) is assigned to –CH₂CH₂O–, the peak at 3.62 ppm (g) belongs to the repeating units of the OEGMA segment, and the singlet 3.37 ppm (h) is assigned to –OCH₃. The characteristic signals from 7 to 8 ppm are assigned to the resonances of the aromatic protons of the azobenzene group. The resonance signal from 3.94 to 4.72 ppm is ascribed to the methylene group of –CH₂–O– in the side chain. The other signals are attributed to the copolymer backbone and the RAFT agent. These results clearly indicate that the azobenzene-containing amphiphilic copolymer poly(AZO-co-OEGMA) has been successfully synthesized via RAFT polymerization.

Scheme 1 Synthesis of the poly(AZO-co-OEGMA) amphiphilic copolymer.

Fig. 1 (a) ¹H NMR spectra of the poly(AZO-co-OEGMA) copolymers, (b) GPC curves of the poly(AZO-co-OEGMA) copolymers.

The molecular weight and molecular weight distribution of these amphiphilic copolymers were determined by GPC. As shown in Fig. 1b, the relatively narrow and symmetric peaks indicate the relatively ideal macromolecular chain structures in these copolymers. The small shoulder of the traces is due to imperfect control of the molecule with the RAFT agent. With increasing amount of OEGMA, i.e., 475, 950 and 1425 mg, the number average molecular weights of the copolymers also increase from 7200, 14 000 to 15 100, respectively, which match well with their theoretical molecular weights (7900, 12 700 and 17 500, respectively, as shown in Table 1).

Table 1 Summary of the RAFT copolymerization of AZO and OEGMA with different ratio monomer and RAFT agents in THF

Polymer	RAFT agent (mg)	AIBN (mg)	AZO (mg)	OEGMA (mg)	AZO^a (% conv)	OEGMA^a (% conv)	Mn(GPC)^b	Mn(theor)^c	ÐM^b
a Conversions of monomers were calculated from intensities in ^1H NMR spectra of the crude samples.b The molecular weights (Mn,GPC) and molecular weight distributions (ÐM = Mw/Mn) were determined by GPC calibrated with PS standards.c Mn(theor) = [AZO]o/[RAFT agent]o × 394 × convAZO + [OEGMA]o/[RAFT agent]o × 475 × convOEGMA + M[RAFT agent], where Mn(theor) is theoretical molecular weight, convAZO and convOEGMA are the conversion of monomers, M[RAFT agent] is molecular weight of RAFT agent.
AZO7-co-OEGMA9	44.1	3.28	394	475	83	98	7200	7900	1.19
AZO7-co-OEGMA18	44.1	3.28	394	950	82	97	13 000	12 700	1.23
AZO7-co-OEGMA27	44.1	3.28	394	1425	81	96	15 100	17 500	1.30

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Polymeric micelles were prepared by the common solvent method using water and THF. When water was added slowly to the solution of amphiphilic copolymer with a little THF, self-assembly occurred, and the resulting dispersion was dialyzed in water to remove organic THF. It was found that the amphiphilic copolymer showed a tendency to form an aqueous solution of core–shell micelles, as shown in Fig. 2. During the self-assembly process, the copolymers have a high tendency to bury AZO into the interior of the micelles to form a hydrophobic core, which is due to the hydrophobic characteristic of the fluorinated azo chains, and the water-soluble OEGMA segments serve the outer hydrophilic shell as a stabilizer. Moreover, the mean size of these micelles increases as the mean molecular weight of copolymer increases.

Fig. 2 TEM images of the micelles with different molecular weights of poly(AZO-co-OEGMA). (a) Poly(AZO₇-co-OEGMA₉)copolymer, (b) poly(AZO₇-co-OEGMA₁₈), (c) poly(AZO₇-co-OEGMA₂₇).

Temperature responsive behavior of micelles

Fig. 3A presents the change in mean diameter of three copolymer versus temperature. As the temperature increases from 25 to 75 °C, the size of poly(AZO₇-co-OEGMA₉) gradually increases within less than 10 nm. The slight size increase is probably due to the weak hydrogen bonding interaction of the relatively decreasing OEGMA and increasing hydrophobic AZO. In contrast, the size of poly(AZO₇-co-OEGMA₁₈) and poly(AZO₇-co-OEGMA₂₇) copolymers shows a sharp increase at their LCST, 70 °C and 75 °C, respectively. At a hydrophobic PAZO molecular weight of 2700 and hydrophilic POEGMA molecular weight of 4200, this hydrophilic/hydrophobic balance does not exhibit obvious LCST. However, with the increase in the hydrophilic POEGMA molecular weight from 8500 to 12 800, the LCST transition can be enhanced by almost 5 °C. Moreover, the LCST can also be further verified by ¹H NMR spectra at different temperatures (Fig. S3†). With the increase in temperature, the hydrogen bond between OEGMA chains and water is destroyed, which decreases the hydrophilicity of the OEGMA shell and causes the aggregation of these polymer chain into large particles.²⁶ The LCST of the copolymer does not change after UV irradiation, indicating that the irradiation of UV light is unable to disrupt the polymeric size (Fig. S4†). The LCST can be further confirmed by the change in the appearance of the copolymer aqueous solution, which is yellow and transparent at room temperature, but becomes yellow-white turbid when the temperature exceeds LCST (the inserted image in Fig. 3A). This means that the balance between hydrophobic and hydrophilic moieties of amphiphilic copolymer has a strong influence on the LCST transition. The LCST has an obviously positive correlation with the OEGMA chains due to the stronger the hydrogen bonds.²⁷

Fig. 3 (A) Changes in the DLS size of poly(AZO-co-OEGMA) with different molecular weights in deionized water as a function of temperature. (B) Fluorescence spectra and images of different copolymers encapsulated with Nile Red (1.2 mg mL⁻¹).

Light- and temperature-responses of micelles

After encapsulating Nile Red, the solution of poly(AZO₇-co-OEGMA₉) copolymer exhibits the highest fluorescent intensity and is bright red, as shown in Fig. 3B, due to the most hydrophobic nature of the lowest MW polymer. Therefore, the poly(AZO₇-co-OEGMA₉) copolymer is used as the model and Nile Red as the fluorescent probe to investigate its light triggering response. As shown in Fig. 4a, upon UV irradiation for 200 s, the fluorescence intensity of Nile Red decreases presumably because of the migration of Nile Red from the micelle core to the external margin and the decrease in hydrophobicity of micelle cores associated with trans-to-cis isomerization.²⁸ The similar critical micelle concentration of polymeric micelles before and after UV irradiation can also confirm the Nile Red's movement within micelles, e.g., around 0.5 g L⁻¹ of the critical micelle concentration for poly(AZO₇-co-OEGMA₉) (Fig. S5†). However, this fluorescence intensity can recover to its initial value under the irradiation of visible light for 300 s, which should be attributed to the recovery of the polymeric micelles from the cis-to-trans isomerization (Fig. 4b). This variation of Nile Red via UV and vis irradiation can be carried out time and again, as shown in Fig. 4c and d. As for the trans–cis isomerization of AZO chromophores, UV irradiation caused a decrease absorbance at around 350 nm due to the π–π* transition and slightly increasing signals close to 440 nm corresponding to the n–π* transition with irradiation time (Fig. S6†), and the UV absorption spectrum was obtained several times via UV and vis irradiation, which confirm the stability of trans–cis isomerization.²⁹ The light response micelles loaded with other dyes FITC and RhB can be observed in the CLSM images in Fig. S7.† The morphologies of the micelles after UV irradiation with the trans-to-cis isomerization are comparable to those before irradiation, being less than 10 nm (Fig. S8†).

Fig. 4 Fluorescence spectra of poly(AZO₇-co-OEGMA₉) copolymer (2 mg mL⁻¹) solution encapsulated with Nile Red under the irradiation of UV light for 200 s (a), and visible light for 300 s (b), the fluorescence spectra of poly(AZO₇-co-OEGMA₉) copolymer solution by altering the irradiation of UV and visible light (c and d).

In addition, the fluorescence intensity of Nile Red encapsulated in the copolymer decreases when the solution is heated from 25 to 75 °C (Fig. 5a). This is attributed to the aggregation of polymeric micelles and the possible quenching effect,³⁰ as some Nile Red molecules approach each other inside the polymeric aggregations. However, when this solution is cooled from 75 to 25 °C, its fluorescent intensity increases again (Fig. 5b), due to the recovery of the size and morphology of these micelles. The better loading capacity at high temperature also accounts for the increase of fluorescent intensity. The control experiments of the fluorescence of Nile Red in response to UV and heat (Fig. S9†) display a decreasing intensity of fluorescence upon UV and heat. These results reveal that the micelles of the obtained copolymers can also be used for the loading and controlled release of guests through temperature response.

Fig. 5 Fluorescence spectra of poly(AZO₇-co-OEGMA₉) copolymer (1.8 mg mL⁻¹) solution encapsulated with Nile Red through a heating process from 25 to 75 °C (a) and the subsequent cooling process from 75 to 25 °C (b).

Salt-responsive behavior of micelles

Fig. 6 illustrates the light transmittance at 550 nm of a solution in deionized water and in a salt solution (K₂SO₄ as the model salt solution). With the increase in temperature, the transmittance of the aqueous solution poly(AZO₇-co-OEGMA₉) copolymer decreases slightly, while those of poly(AZO₇-co-OEGMA₁₈) and poly(AZO₇-co-OEGMA₂₇) copolymers show a sudden decrease in transmittance at their LCST (Fig. 6a). This is consistent with the changes in chemical shift and micelle size as discussed above. However, in the K₂SO₄ solution, the first copolymer also displays an obvious LCST at around 45 °C, whereas the other two copolymers present a decreasing LCST, 55 °C and 65 °C (Fig. 6b). This is because more chains are strongly affected by Hofmeister ions owing to the high interfacial water structuring normally associated with OEGMA.³¹ It is well known that different salts affect the phase transition of the polymers by changing the water structure and forming a hydration shell and this effect is more pronounced for anions than for cations. In the salt aqueous solution, SO₄²⁻ become strongly hydrated with adjacent water molecules and will thus reduce the hydrogen bonding interaction between copolymer chains and water molecules. Consequently, the hydrogen bonding between hydrophilic chain segments becomes dominant, which leads to a strong tendency for the polymers to associate and therefore decreases the temperature of the LCST. Moreover, the presence of SO₄²⁻ will increase the hydrophobic–hydrophobic interaction, which also causes a strong tendency for polymer chain aggregation and decrease in the LCST.³² In particular, SO₄²⁻ ions, with higher electrovalence and larger electronegativity, can reduce the LCST of the polymer. These results can be further confirmed by the change in DLS size, as shown in Fig. S10.† When low state salts, e.g., NaCl and KCl, were used, although the poly(AZO₇-co-OEGMA₉) copolymer showed a LCST at 55 °C, the other two copolymers have no obvious shift in LCST (Fig. S11†).

Fig. 6 Transmittance at 550 nm of solutions of poly(AZO-co-OEGMA) in deionized water (a) and 0.1 M K₂SO₄ solution (b).

Self-assembled colloidal nanoclusters

Owing to the amphiphilic property, poly(AZO–OEGMA) copolymers can self-assemble to form a supramolecular micelles structure to provide interior space in aqueous solutions, where the fluorinated segments of polymers could tightly integrate with the hydrophobic oleic acid molecules on the nanoparticles through hydrophobic–hydrophobic interactions. Specifically, fluorinated segments of polymers show good compatibility with oleic acid of the nanocrystals,³³ whereas the hydrophilic OEGMA segments are the toward outside to improve water solubility. Therefore, colloidal clusters derived from multifunctional micelles possess the superiority for drug delivery. The self-assembly of the amphiphilic copolymer and nanocrystals is illustrated in Scheme 2. In addition, this amphiphilic copolymer can also be used to assemble with other hydrophobic nanocrystals, e.g., SPIO (Fig. S12†) and QD (Fig. S13†), easily converting them from hydrophobicity to hydrophilicity for biological applications.

Scheme 2 Schematic for the preparation of colloidal nanoclusters by self-assembly.

As shown in Fig. 7a and b, the oleic acid-stabilized UCNP nanocrystals have very good dispersion in cyclohexane. The apparent enlargement of average nanoparticles size from 29 nm to 40 nm indicates the successful deposition of NaGdF₄ crystals onto the outer surface of NaYF₄:Gd/Yb/Tm. The high crystalline and hexagonal phases of the core–shell nanocrystals were confirmed by powder XRD and EDX (Fig. S14–15†). After being assembled with a poly(AZO₇-co-OEGMA₉) copolymer, all the nanocrystals are organized into apparently spherical nanoclusters, which are finely dispersed in an aqueous phase with an average size of about 350 nm (Fig. 7c) and further confirmed by the elemental mapping images (Fig. 7e–j). Under excitation at 980 nm, the upconversion emissions at 345, 360, 450, and 475 corresponding to the ¹I₆–³F₄, ¹D₂–³H₆, ¹D₂–³F₄, and ¹G₄–³H₆ transitions can be observed from both UCNP nanocrystals and nanoclusters (Fig. 7d). However, the upconversion emission of the core–shell nanocrystals was significantly enhanced by 6 times as much as that of the NaYF₄:Gd/Yb/Tm core alone. Moreover, compared to the core–shell UCNP nanocrystals in cyclohexane, the emission intensity of UV from their nanoclusters in a copolymer solution is remarkably depressed, indicating that the emission intensity of UV from the UCNP nanocrystals have been greatly absorbed by azobenzene groups in the copolymer.³⁴

Fig. 7 HRTEM images of NaYF₄:Gd/Yb/Tm and NaYF₄:Gd/Yb/Tm@NaGdF₄ (a and b), core–shell UCNP nanocrystals in cyclohexane (b, inset: image), UCNP nanoclusters in aqueous phase (c, inset: image). Fluorescence spectra of NaYF₄:Gd/Yb/Tm and NaYF₄:Gd/Yb/Tm@NaGdF₄ nanocrystals in cyclohexane, and UCNP nanoclusters in a copolymer aqueous solution (d). Elemental (Yb, Gd, Y, F, Na, and Tm) mappings of the UCNP nanoclusters (e–j).

NIR and temperature DOX release

With DOX as a model therapeutic drug, we further explored the photoinduced controllable release property of the DOX-loaded colloidal clusters. The loaded DOX content is designed to be ∼13 wt%. As shown in Fig. 8A, the release amount reaches ∼65% within 24 h under 980 nm laser light irradiation with power of 3 W cm⁻² at pH 7.4, because of the trans-to-cis isomerization of azobenzene groups. On the contrary, less than 15% of DOX was leached into the aqueous solution in the control experiment without NIR irradiation. This suggests that the colloidal nanoclusters of the amphiphilic copolymer can be used to convert NIR into UV lights for controllable drug release.

Fig. 8 Drug release in PBS under NIR light irradiation (b) and dark condition (a) (A), drug release in PBS at 37 °C, 45 °C, 45 °C with 0.1 M K₂SO₄ (B).

In addition, the in vitro drug release of DOX from the loaded hydrophilic clusters was also carried out at 37 °C, 45 °C, and 45 °C with 0.1 M K₂SO₄. As shown in Fig. 8B, the DOX-loaded nanoparticles display well temperature-responsive drug release. At 37 °C, the drug release is suppressed and the release rate decreases after 10 h. The accumulated release amount is less than 15% after 24 h, whereas at 45 °C, the drug release is accelerated drastically. The accumulated release amount is about 23% within 15 h, which could be attributed to the enhanced mobility of the AZO segment and weakened hydrophobic interactions between polymer chains and DOX with the increase in temperature; therefore, DOX could migrate easily to the aqueous solution from the clusters upon heating. Moreover, the accumulated release amount further increase to 38% in the presence of K₂SO₄ after 24 h, which is probably because the salt further disrupts the hydrogen-bonding and hydrophobic interactions of the polymer chains sharply upon the same heating. Thus, temperature can also be used as a switch to trigger the drug release in this system.

Conclusions

We demonstrated one-step synthesis of novel multiresponsive amphiphilic copolymers by RAFT polymerization of hydrophilic OEGMA and hydrophobic azobenzene-containing methacrylate. The amphiphilic copolymers can not only self-assemble easily into micelles in the aqueous phase, but also exhibit excellent responses to temperature, UV/vis light, and ions, and occupy a LCST. The LCST can be tuned by the amounts of hydrophilic OEGMA segments. This multi-responsive property can make this facile copolymer a very promising candidate for loading and controlled release of dyes. This multi-responsive copolymer can readily assemble with hydrophobic nanocrystals, such as SPIO, QD, and UCNP, to form hydrophilic nanoclusters, by intermolecular force of the oleic acid chains of nanocrystals and the hydrophobic fluorinated azobenzene segments of copolymers. The UCNP colloidal nanoclusters can be utilized in near-infrared (NIR) light and temperature triggers the release of the anticancer drug, which has a great potential in drug delivery.

Acknowledgements

Financial supports of this research from the National Natural Science Foundation of China (Grants 51133001 and 21374018) and the Science and Technology Foundation of Shanghai (13JC1407800) are appreciated.

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Footnote

† Electronic supplementary information (ESI) available: Other characterization, including ¹H NMR of CTA, alkyne-CTA, and AZO, EDX and XRD of UCNP, UV absorption of the poly(AZO-co-OEGMA) copolymer, DLS of the poly(AZO-co-OEGMA) copolymer at variable temperatures under UV irradiation, and CLSM of the poly(AZO-co-OEGMA) copolymer with FITC and RhB, DLS of the poly(AZO-co-OEGMA) copolymer at variable temperatures in NaCl and KCl solutions, assembly with SPIO and QD, are supplied. See DOI: 10.1039/c6ra17622d

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