Fabrication of AIE-active amphiphilic fluorescent polymeric nanoparticles through host–guest interaction

Abstract

Fluorescent polymeric nanoparticles (FPNs) have obtained more and more attention in recent years due to their excellent performance in the fields of bioimaging, biosensing, theranostics and many other biomedical applications. In this work, we reported a novel method to fabricate amphiphilic fluorescent copolymers through host–guest interactions based on an aggregation-induced emission (AIE) active dye (named as Ad-PhNH₂) and β cyclodextrin (β-CD) contained polymers, which were synthesized by free radical polymerization and subsequent ring-opening reaction. These AIE active copolymers can self assemble into FPNs (named as PEGMA–IA–β-CD/Ad-PhNH₂) due to their amphiphilic properties. The hydrophobic dye was aggregated in the core and therefore can emit strong fluorescent intensity due to its AIE feature. However, the hydrophilic polymers that covered the hydrophobic core can endow good dispersibility in pure aqueous solution. Biological evaluation results demonstrated that PEGMA–IA–β-CD/Ad-PhNH₂ FPNs can be effectively internalized into cells and they have shown low cytotoxicity. More importantly, the molar ratio of β-CD to Ad-PhNH₂ can be facilely adjusted and the surplus β-CD can be used for carrying chemical anticancer agents. Furthermore, a large number of carboxyl groups were generated during the ring opening reaction. These negative carboxyl groups can be potentially used for further conjugation reactions and for biological delivery. The above described features of PEGMA–IA–β-CD/Ad-PhNH₂ FPNs make them a prospect in biological imaging and delivery applications.

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1. Introduction

Biological imaging in modern medicine is an important tool to provide visual information on organs and biological processes.^1–6 A number of modern imaging techniques such as X-ray phase tomography, single-photon emission computed tomography, positron emission tomography, Raman imaging, magnetic resonance imaging, ultrasound and fluorescence imaging, etc. have been developed.^7–10 All of them have been applied and have had significant roles in a variety of biological applications like stem cell transplantation, cancer detection, drug delivery, therapeutic effect monitoring and so on.^11–16 Among them, the fluorescence imaging has attracted the most intensive attention because the fluorescence used in cells could be an output signal that provides obvious signal intensity and high resolution images at subcellular levels.^17–24 Moreover, the fluorescent probes had many properties such as low cost, well designability of dyes and final probes, desirable biocompatibility and so on. These characteristics make fluorescent imaging play an important role in biological imaging utilization.^25,26 Varieties of fluorescent nanoparticles with different components, fluorescent properties and surface coating have already been synthesized.^27–34 As compared with small organic dyes, fluorescent nanoparticles showed many advantages for biological applications including good biocompatibility, better chemical stability, enhanced photostability and multifunctional potential.^35–37 Among the fluorescent nanoparticles, fluorescent polymeric nanoparticles (FPNs) have already attracted increasing attention for their superiority in simple fabrication, peculiar luminescence properties and surface functionalization compared with fluorescence inorganic nanoparticles and organic dye-doped inorganic nanoparticles. Inorganic nanoparticles mainly contained the semiconductor quantum dots with great luminescent properties and rare earth-doped upconversion nanoparticles which can emit visible emission under excitation with a near-infrared laser.³⁸ Although many successes have been made in the research of fluorescent inorganic nanoparticles, some shortcomings cannot be ignored. For example, most of fluorescent inorganic nanoparticles are difficult to be biodegradable, and exhibited obvious toxicity to living organisms.³⁹ The basic principle for fabrication of FPNs is relied on the self assembly of amphiphilic dyes contained polymers in aqueous solution.^40–45 The hydrophobic components such as dyes were encapsulated in the core, which were covered by the hydrophilic segments. Therefore, these self assembly nanostructures displayed excellent water dispersibility in pure aqueous solution.^46,47 However, many FPNs based on the self assembly procedure still possess some problems. One of the most issues is the significant decrease of fluorescent intensity of FPNs based on conventional organic dyes. This phenomenon was known as aggregation caused quenching (ACQ) effect. The phenomenon is even more serious for dyes with emission in the far-red/near-infrared (FR/NIR) tissue transparency window.⁴⁸ A novel type of organic dyes that can emit stronger luminescence in the aggregation state was found by Tang et al. in 2001, which called aggregation-induced emission (AIE) dyes.⁴⁹ It provided a useful route for overcoming the ACQ effect and good chooses to fabricate FPNs with desirable luminescent performance. From then on, a huge number of dyes with AIE properties were synthesized and utilized for fabrication of luminescent polymeric nanoprobes.

Supermolecular chemistry has got much attention in the field of molecular recognition, supermolecular hydrogels and varieties of other functional materials by self assembly.^50–53 The process that gained the supermolecular polymers with typical dynamic polymeric structure was orthogonal assembly of monomers through supermolecular interactions at chain ends such as metal–ligand bonding, π-stacking, H-bonding and host–guest interaction. So far, the molecular recognition mechanism of cyclodextrin (CD) and its derivatives as an important part of supermolecular chemistry have gained widely concern.^37,54–59 Because CD owned many advantages such as good stability, excellent biodegradability, low toxicity, which was the ideal host molecules similar to enzymes and had the properties of enzymes' models. Among them, β-CD was commonly used for its better rigidity. The ester of organic molecules (host) with suitable size and shape would enter its cavity, formed as host–guest inclusion complexes by host–guest interaction. Although different strategies have been set up for fabrication of multifunctional AIE active polymeric nanoparticles, the fabrication of AIE active FPNs through host guest interaction has received rarely attention. The fabrication of AIE-active FPNs based on the β-CD contained copolymers and adamantane (Ad) terminated AIE dye has rarely been reported thus far.

In this contribution, we report for the first time that amphiphilic AIE-active supermolecules could be facilely formed taking advantage of host guest interaction between β-CD pendant copolymers (PEGMA–IA–β-CD) and Ad contained AIE dye (Ad-PhNH₂). As shown in Scheme 1, β-CD pendant copolymers can be synthesized by simple ring-opening reaction between amino groups of β-CD and anhydride group of poly(PEGMA–IA), which can be prepared by the free radical polymerization using itaconic anhydride (IA) and poly(ethylene glycol) methyl ether methacrylate (PEGMA) as the monomers. Then Ad-PhNH₂ is mixed with PEGMA–IA–β-CD to form the final products (PEGMA–IA–β-CD/Ad-PhNH₂ FPNs). The PEGMA–IA–β-CD/Ad-PhNH₂ are characterized by a number of techniques and examined for biological imaging applications.

Scheme 1 Schematic showing the preparation of PEGMA–IA–β-CD/Ad-PhNH₂ copolymers via host guest interaction.

2. Experiment

2.1 Measurements and materials

All of the chemical agents and solvents were obtained from commercial sources and used as received. All aqueous solutions were prepared with distilled water. β-CD (MW: 1135 Da, 99%), tosyl chloride (TsCl MW: 190.65 Da, 99%), ethylenediamine (EDA, MW: 60.10 Da, 99%), itaconic anhydride (IA, MW: 112.19 Da, 96%), poly(ethylene glycol) methyl ether methacrylate (PEGMA, MW: 950 Da, 98%), 2,2-azodiisobutyronitrile (AIBN, MW: 164.21 Da, 98%), phenothiazine (MW: 199.28 Da, 98%), 4-aminobenzyl cyanide (MW: 132.16 Da, 98%) and et al. were purchased from Aladdin (Shanghai, China). Anhydrous ethyl acetate and DMF were provided from Heowns (Tianjin, China). ¹H nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance-400 spectrometer with D₂O, TMS and DMSO as the solvents. The synthetic materials were characterized by Fourier transform infrared spectroscopy (FT-IR) using KBr pellets. FT-IR spectra were obtained using a Nicolet 5700 (Thermo Nicolet Corporation). Fluorescence spectra were measured on a PE LS-55 spectrometer with a slit width of 3 nm for both excitation and emission. UV-vis absorption spectra were recorded on UV/vis/NIR PerkinElmer Lambda 750 spectrometer (Waltham, MA, USA) using quartz cuvettes of 1 cm path length. Transmission electron microscopy (TEM) images were recorded on a JEM-1200 EX microscope operated at 100 kV. The TEM specimens were made by placing a drop of the nanoparticles suspension on a carbon coated copper grid. The hydrodynamic size distribution of the final products PEGMA–IA–β-CD/Ad-PhNH₂ FPNs in water was examined using dynamic laser scattering (DLS).

2.2 Preparation of mono-6-deoxy-6-(p-tolysulfonyl)-β-CD

30 g β-CD (26.4 mM) and 20 g NaOH were dissolved in 666 mL water. The solution was immersed into an ice-water bath, and then 10 g TsCl was added slowly, causing the formation of white precipitates. After further stirring for 5 h at 0 °C, the unreacted TsCl was removed by suction filtration, then using the 10% hydrochloric acid solution adjusting the filtrate's pH at about 7. The suspension was refrigerated overnight at 0 °C in the refrigerator. The resulting precipitate was recovered by suction filtration and recrystallized from hot water 3 times getting the mono-6-deoxy-6-(p-tolysulfonyl)-β-CD (mono-6-OTs-β-CD). The product was dried under vacuum at 60 °C for 12 h.

2.3 Preparation of mono-6-deoxy-6-ethylenediamine-β-CD

The mono-6-deoxy-6-ethylenediamine-β-CD (EDA-β-CD) was synthesized according to the previous report.⁶⁰ In brief, 12 g of mono-6-OTS-β-CD was reacted with an excess amount of ethylenediamine (EDA) at 80 °C for 4 h. The solution was cooled down and most of the unreacted EDA was removed by rotary evaporation. The rest of the liquor was dissolved in water–methanol mixture and precipitated in acetone 3 times. The product was dried at 50 °C under vacuum for 24 h.

2.4 Synthesis of poly(PEGMA–IA)

The poly(PEGMA–IA) was synthesized according to the previous report.⁶¹ The poly(PEGMA–IA) was prepared via living free radical polymerization using PEGMA and IA monomers. In brief, PEGMA (3.2 mM, 3.04 g) and IA (8 mM, 960 mg) were mixed and dissolved in an anhydrous ethyl acetate solution (60 mL) at 80 °C for 10 min. AIBN (8 mM, 1.32 g) in 30 mL anhydrous ethyl acetate solution was quickly added into the reaction bottle and stirred at 80 °C for 24 h after the mixture was completely dissolved. The system was always maintained under a N₂ atmosphere before the reaction was accomplished. Then the crude product was purified by dialysis against an anhydrous ethyl acetate solution for 24 h. The product was dried under vacuum oven at 40 °C overnight.

2.5 Preparation of PEGMA–IA–β-CD

PEGMA–IA–β-CD was prepared by the ring-opening reaction between amine and anhydride in an anhydrous DMF solution. The preparation procedure could be summarized as follows. The previously obtained poly(PEGMA–IA) (1 g) was dissolved in anhydrous DMF (20 mL) and mixed with EDA-β-CD (8 g) in anhydrous DMF (40 mL). The system was stirred continuously at 40 °C for 10 h. The product was gotten through dialysis treatment with fresh water for 24 h and dried under vacuum oven at 40 °C for 24 h.

2.6 Preparation of Ph-NH₂ and Ad-PhNH₂

The synthetic of Ad-Ph-NH₂ was shown in Scheme S1 in detail.† The intermediate product 1 was synthesized according to the previous report using bromohexadecane and phenothiazine as templets.⁶² In 1000 mL bottles, POCl₃ (36 g, 0.24 mol) and DMF (11.7 g, 0.159 mol) was added to 30 mL ClCH₂CH₂Cl at 0 °C mixing for 30 min. The intermediate product 1 (7.59 g, 18 mM) was subsequently added and reacted at temperature of 90 °C overnight. The crude product was slowly added into cool water and extracted with ethyl acetate for three times. Then flash column chromatography using 1 : 10 mixture of ethyl acetate and petroleum ether as eluent was done and obtained the pure product 2. The intermediate product 2 (1.22 g, 3.2 mM) and 2-(4-amino phenyl) acetonitrile (1.68 g, 6.4 mM) were added into 40 mL ethanol solution and the mixture was stirring at room temperature. The tetrabutyl ammonium hydroxide solution (0.8 M, 10 drops) was added and mixture was refluxed for 2 h until a red solid was precipitated. The obtained red solids were washed with ethanol for several times until dark red solids were obtained. The Ph-NH₂ was obtained after dried for 24 h under vacuum oven. The adamantine terminated PhNH₂ (Ad-PhNH₂) was facilely obtained via amidation reaction between amino groups contained Ad-PhNH₂ and adamantine chloride. The purified PhNH₂ (1.13 g, 2.0 mM) and 1-adamantoyl chloride (0.796 g, 4.0 mM) were dissolved in dry THF (60 mL). Meanwhile, two drops of triethylamine were added in follow. The reaction mixture was reacted at room temperature overnight. Ad-PhNH₂ was obtained via filtration and further purified washing with water. The product was dried at 50 °C under vacuum for 24 h.

2.7 Preparation of PEGMA–IA–β-CD/Ad-PhNH₂

The obtained PEGMA–IA–β-CD (500 mg) and Ad-PhNH₂ (150 mg) were blended in 50 mL anhydrous DMF solution and ultrasonically treated for 10 min. The reaction system was stirred continuously at room temperature overnight without any other treatment. Then anhydrous ether was added into the solutions. White precipitates were formed after refrigeration overnight at −20 °C. The PEGMA–IA–β-CD/Ad-PhNH₂ could be obtained via centrifugation and washing three times to remove the unreacted reagents. The product was dried under vacuum oven at 40 °C overnight.

2.8 Cytotoxicity evaluation of PEGMA–IA–β-CD/Ad-PhNH₂ FPNs

The cell viability of PEGMA–IA–β-CD/Ad-PhNH₂ FPNs towards HUVEC cells was determined by cell counting kit-8 (CCK-8) assay based on our previous reports.⁶³ The experimental procedure could be demonstrated by follows: HUVEC cells were put into 96-well microplates at a density of 5 × 10⁴ cells per mL in 160 μL of respective media containing 10% fetal bovine serum (FBS). After 24 h of cell attachment, the cells were incubated with different concentrations of FPNs (20–120 μg mL⁻¹) for 12 and 24 h. Then these uninternalized FPNs were removed by repeatedly washing with phosphate buffered saline (PBS) three times. 10 μL of CCK-8 dye and 100 μL of Dulbecco's Modified Eagle's medium (DMEM) cell culture medium were added to each well and incubated for 2 h at room temperature. Afterward, plates were analyzed using a microplate reader (Victor III, Perkin-Elmer). Measurements of formazan dye absorbance were carried out at 450 nm, with the reference wavelength at 620 nm. The values were proportional to the number of live cells. The percentage reduction of CCK-8 dye was compared to controls, which represented 100% CCK-8 reduction. Three replicate wells were used per microplate, and the experiment was operated for three times. Cell survival was expressed as absorbance relative to that of untreated controls. Results are presented as mean ± standard deviation (SD).

2.9 Confocal microscopic imaging

HUVEC cells were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM glucantamine, 100 U mL⁻¹ penicillin, and 100 μg mL⁻¹ of streptomycin. Cell culture was controlled at 37 °C in a similar humidified condition of 95% air and 5% CO₂ in culture medium. Culture medium should be updated every three days for maintaining the exponential growth of the cells. Before treatment, cells were seeded in a glass bottom dish with a density of 1 × 10⁵ cells per dish. On the day of treatment, the cells were incubated with PEGMA–IA–β-CD/Ad-PhNH₂ FPNs at a final concentration of 40 μg mL⁻¹ for 3 h at 37 °C. Afterward, the cells were washed three times with PBS to remove the PEGMA–IA–β-CD/Ad-PhNH₂ FPNs and then fixed with 4% paraformaldehyde for 10 min at room temperature. Cell images were obtained using a confocal laser scanning microscope (CLSM) Zeiss 710 3-channel (Zeiss, Germany) with the excitation wavelength of 405 nm.

3. Results and discussion

Due to the sixth hydroxyl of β-CD strongest alkaline and nucleophilicity, we can easily get the sixth mono replacement of the derivatization for the β-CD. And the Ad-PhNH₂ was facilely obtained via an amidation reaction. The structure information of these obtained products were first characterized and confirmed by ¹H NMR spectroscopy. The structure of mono-6-OTS-β-CD could be confirmed from ¹H NMR (DMSO) spectra. The result of ¹H NMR spectra from Fig. 1 is described as follows. δ = 2.41 ppm (3H, –CH₃), δ = 3.18–3.42 ppm (14H, H-2,4), δ = 3.44–3.78 ppm (28H, H-3,5,6), δ = 4.14–4.48 ppm (6H, OH-6), δ = 4.72 ppm (3H, H-1), δ = 4.81 ppm (4H, H-1), δ = 5.44–5.90 ppm (14H, OH-2,3), δ = 7.40 ppm (2H, aromatic protons), δ = 7.71 (2H, aromatic protons) demonstrate the successful preparation of mono-6-OTS-β-CD. The resulting data of EDA-β-CD from ¹H NMR (D₂O) spectra: δ = 2.69 ppm (2H, NH₂CH₂–CH₂), δ = 2.90 ppm (2H,–CH₂NH-β-CD), δ = 3.30–3.51 ppm (14H, H-2,4), δ = 3.64–3.84 ppm (28H, H-3,5,6), δ = 4.92 ppm (7H, H-1) demonstrate the successful preparation of EDA-β-CD. The chemical structure of Ad-PhNH₂ was also confirmed by ¹H NMR spectroscopy. ¹H NMR spectrum of Ad-PhNH₂ in CDCl₃ is shown in Fig. S1.† The shifts ranged from 1.70 to 2.1 ppm were consistent with Ad protons. The peaks at about 6.80–7.80 ppm were found due to the protons of benzyl. The peak at 1.25 ppm represents the methylene group of the side chain (–CH₂–CH₂–). Furthermore, the shift at 3.93 ppm was observed due to the methylene protons connected with heterocyclic ring. All the above results confirmed the successful synthesis of Ad-PhNH₂.

Fig. 1 ¹H NMR spectra of mono-6-OTS-β-CD and EDA-β-CD.

For preparation of PEGMA–IA–β-CD/Ad-PhNH₂, the hydrophilic copolymers (PEGMA–IA) were synthesized using PEGMA and IA as the monomers and AIBN as the initiator via free radical living polymerization. EDA-β-CD was facilely linked with the IA of PEGMA–IA via ring-opening reaction with EDA-β-CD at 40 °C. Then PEGMA–IA–β-CD and Ad-PhNH₂ were reacted via host–guest interactions. The successful preparation of PEGMA–IA, PEGMA–IA–β-CD and PEGMA–IA–β-CD/Ad-PhNH₂ could be confirmed by the ¹H NMR spectra (Fig. 2). The structure of PEGMA–IA could be confirmed. The data from ¹H NMR spectra are described as follows (DMSO). The peak at δ = 1.1–1.3 ppm (–CH₃), δ = 1.7–1.8 ppm (–CH₂–CH₂), δ = 3.20 ppm (–CO–CH₂), δ = 3.60 ppm (–O–CH₂), δ = 4.21 ppm (–COO–CH₂) demonstrate the successful preparation of PEGMA–IA by free radical living polymerization. For the structure of PEGMA–IA–β-CD, different peaks are summarized as follows (DMSO). The peak at δ = 1.15–1.30 ppm (–CH₃), δ = 1.75 ppm (–CH₂–CH₂), δ = 2.9 ppm (–CO–CH₂), δ = 3.2 ppm (–O–CH₂–), δ = 3.34 ppm (NH–CH₂), δ = 4.05 ppm (COO–CH₂), δ = 5.60 ppm (O–CH–O), δ = 7.92 ppm (–CO–NH). Furthermore, we can also know that the PEGMA–IA–β-CD/Ad-PhNH₂ was successfully synthesized. The results of ¹H NMR for the PEGMA–IA–β-CD/Ad-Ph-NH₂ are described as follows (DMSO). The peak at δ = 1.15–1.30 ppm (–CH₃), δ = 1.75 ppm (–CH₂–CH₂), δ = 2.86 ppm (–CO–CH₂–), δ = 3.16 ppm (–O–CH₂–), δ = 3.32 ppm (NH–CH₂), δ = 4.09 ppm (COO–CH₂), δ = 5.65 ppm (O–CH–O), δ = 7.10 ppm (S–C–CH=), 7.60 ppm (Ph–), 7.70–7.86 ppm (N–C–CH=), 7.95 ppm (–CO–NH–).

Fig. 2 ¹H NMR spectra of PEGMA–IA, PEGMA–IA–β-CD and PEGMA–IA–β-CD/Ad-PhNH₂.

The functional groups on PEGMA–IA–β-CD/Ad-Ph-NH₂ were evaluated by FT-IR spectroscopy. As displayed in Fig. 3, a strong peak at about 2925 cm⁻¹ was observed in all the samples, which can be attributed to the stretching vibration of CH₂ and CH₃. However, the intensity of the peak was obviously decreased in the sample of PEGMA–IA–β-CD and PEGMA–IA–β-CD/Ad-PhNH₂ as compared with PEGMA–IA. On the other hand, there are some tangled peaks appearing in the range of 650 to 780 cm⁻¹, which means the fingerprint region of β-CD. Furthermore, the peak at 3397 cm⁻¹ can be assigned to stretching vibration of hydroxyl group. As compared with PEGMA–IA, the peak intensity of at 3397 cm⁻¹ was significantly enhanced in the samples of PEGMA–IA–β-CD and PEGMA–IA–β-CD/Ad-PhNH₂. The obvious stretching vibration of C–N at 1027 cm⁻¹ also proved that Ad-PhNH₂ and β-CD had being linked on the polymer. Furthermore, compared with the sample of PEGMA–IA–β-CD, a new peak that appeared at 1158 cm⁻¹ can be ascribed to the fingerprint region of C–S. All of these results confirmed the successful formation of PEGMA–IA–β-CD/Ad-Ph-NH₂ through host–guest interactions.

Fig. 3 FT-IR spectra of PEGMA–IA, PEGMA–IA–β-CD and PEGMA–IA–β-CD/Ad-PhNH₂.

The optical properties of PEGMA–IA–β-CD/Ad-PhNH₂ were examined by UV-vis and FL spectroscopy. The UV-vis spectrum of PEGMA–IA–β-CD/Ad-PhNH₂ was displayed in Fig. 4A, the maximum absorption peaks of PEGMA–IA–β-CD/Ad-PhNH₂ dispersed in pure water was located at about 206 nm, which can be attributed to the π → π* transition. On the other hand, a shoulder peak at about 230 nm was also found in the UV-vis spectrum. This peak can be assigned to the band Π of benzene amino derivatives. Furthermore, because PEGMA–IA–β-CD is absent of adsorption between 200 and 400 nm, therefore we could further conclude that Ad-PhNH₂ has incorporated into PEGMA–IA–β-CD/Ad-PhNH₂ successfully through host–guest interaction. The final copolymers (PEGMA–IA–β-CD/Ad-PhNH₂) are tended to self assembly due to their amphiphilic properties. The hydrophobic Ad-PhNH₂ will be encapsulated in the core while the hydrophilic segments such as PEGMA and IA will expanded into water. It is therefore the self assembly nanostructure will emit strong luminescence and good water dispersibility for the AIE active properties of Ad-PhNH₂ and hydrophilic PEGMA and IA. The AIE properties of Ad-PhNH₂ were displayed in Fig. S2.† It can be seen that the fluorescent intensity of Ad-PhNH₂ was changed corresponding with the fraction of water to THF. When the water fraction is 60%, the fluorescent intensity was reached to the lowest value. The fluorescent intensity was rapidly increased when further increase the fraction of water. The enhanced fold is greater than 1.5. The above results clearly showed that Ad-PhNH₂ is an AIE active dye. Furthermore, the fluorescent intensity of the final FPNs in the solution with different ratios of water and DMF was also examined by PL spectroscopy. As shown in Fig. S3,† the fluorescent intensity change of PEGMA–IA–β-CD/Ad-PhNH₂ showed similar trend like the Ad-PhNH₂. The maximum fluorescent intensity was achieved when the water fraction was 80%. The intensity value is about 3 folds of minimal fluorescent intensity. All of the above results suggested that both Ad-PhNH₂ and PEGMA–IA–β-CD/Ad-PhNH₂ possess desirable AIE properties. This is very important for fabrication of FPNs with desirable fluorescent properties. As evidenced by insets of Fig. 4A, good light transmittance was found after PEGMA–IA–β-CD/Ad-PhNH₂ FPNs were dispersed in water (left cuvette in Fig. 4A-a). After the PEGMA–IA–β-CD/Ad-PhNH₂ water suspension was irradiated by UV lamp at 365 nm, uniform red fluorescence was observed (right cuvette in Fig. 4A-b). The above results clearly demonstrated that the successful formation amphiphilic AIE active copolymers through supermolecular interactions. Fig. 4B showed the detailed information about the fluorescence of PEGMA–IA–β-CD/Ad-PhNH₂. The maximum emission wavelength was located at about 573 nm, while the fluorescence excitation wavelength of FPNs was appeared at 432 nm when the emission wavelength was set at 573 nm. The photostability is another very important factor for the FPNs for bioimaging applications. A number of previous reports have demonstrated that AIE active FPNs could display better photostability both in solution and internalization into cells.^11,64

Fig. 4 (A) UV-vis spectrum of PEGMA–IA–β-CD/Ad-PhNH₂ FPNs, insets were the visible image of such FPNs in water; “a” and “b” are represented the optical images of PEGMA–IA–β-CD/Ad-PhNH₂ FPNs without and with UV lamp irradiation. It can be seen that the symbol of cuvette can be observed from the inset “a”, indicating good water dispersibility of PEGMA–IA–β-CD/Ad-PhNH₂ FPNs. The inset of “b” is the optical image of PEGMA–IA–β-CD/Ad-PhNH₂ FPNs after irradiation with UV lamp at 365 nm. (B) Fluorescence excitation (Ex) and emission (Em) spectra of PEGMA–IA–β-CD/Ad-PhNH₂ FPN (λ_Ex = 432 nm, λ_Em = 573 nm).

The morphology of micelles appeared as spheres with suitable size ranged from 300 to 500 nm. Based on the TEM images, we calculated the size distribution of PEGMA–IA–β-CD/Ad-PhNH₂ and suggested that the size distribution is about 385.8 ± 90.2 nm, which suggested that the resulting amphiphilic copolymers were self-assembled into nanoparticles in aqueous solution (Fig. 5). The probable mechanism forming spherical PEGMA–IA–β-CD/Ad-PhNH₂ could explain that the Ad-PhNH₂ was hydrophobic and curled into a sphere in water, while the hydrophilic PEGMA–IA–β-CD were covered on the surface of the hydrophobic core and contact with water, causing the good water dispersibility. All the results showed the FPNs had equal size and excellent dispersibility in aqueous solution, good prospect for biomedical applications. Furthermore, the hydrodynamic size distribution of PEGMA–IA–β-CD/Ad-PhNH₂ FPNs in water was determined to be 453.59 ± 144.4 nm with relative broad polydispersity index of 0.235 (Fig. S4†). As compared with the TEM characterization, the size distribution is relative large, which may be ascribed to the expand polymers in water.

Fig. 5 TEM image of PEGMA–IA–β-CD/Ad-PhNH₂ FPNs. The size of PEGMA–IA–β-CD/Ad-PhNH₂ FPNs is ranged from 300 to 500 nm.

Considered the high water dispersibility and desirable AIE active fluorescent properties, the potential applications of these PEGMA–IA–β-CD/Ad-PhNH₂ FPNs in biomedical fields were evaluated. First, the cytotoxicity of PEGMA–IA–β-CD/Ad-PhNH₂ FPNs was determined for exploring their potential biomedical applications. A preliminary biocompatibility evaluation was conducted using the CCK-8 assay to evaluate the effect of the biomaterials on the cell viability. As shown in Fig. 6, it showed no significantly decreased after HUVEC cells were incubated with distinct concentrations of PEGMA–IA–β-CD/Ad-PhNH₂ FPNs (0–120 μg mL⁻¹) for 12 and 24 h. Moreover, no significant cell viability decrease was detected after 24 h incubation even the concentration of PEGMA–IA–β-CD/Ad-PhNH₂ FPNs was as high as 120 μg mL⁻¹. The cell viability values at all experimental concentrations are obviously greater than 90%. The IC₅₀ values based on cell viability examination is greater than 500 μg mL⁻¹. Based on the cell viability evaluation, we could conclude that PEGMA–IA–β-CD/Ad-PhNH₂ FPNs show negative toxic effect on HUVEC cells. The excellent biocompatibility of PEGMA–IA–β-CD/Ad-PhNH₂ FPNs makes them promising candidates for biomedical applications.

Fig. 6 The biocompatibility evaluation of HUVEC cells after incubating with distinct concentrations (0–120 μg mL⁻¹) of PEGMA–IA–β-CD/Ad-PhNH₂ FPNs.

The interaction between PEGMA–IA–β-CD/Ad-PhNH₂ FPNs and HUVEC cells was determined using CLSM. As shown in Fig. 7, strong fluorescence could be observed from the HUVEC cells after they were incubated with 40 μg mL⁻¹ of PEGMA–IA–β-CD/Ad-PhNH₂ FPNs for 3 h (Fig. 7B). Because the free PEGMA–IA–β-CD/Ad-PhNH₂ FPNs in cell culture medium were almost completely removed by repeatedly washing, the fluorescent signal in CLSM should be the AIE active FPNs that were internalized by cells. Moreover, we could also found that the location of fluorescent signal is covered on the location of cells (Fig. 7C). Therefore, we can confirm that PEGMA–IA–β-CD/Ad-PhNH₂ FPNs can be used internalized by cells and the fluorescence intensity is strong enough for biological imaging. Furthermore, many dark areas were surrounded by the areas with strong fluorescent signal. The dark areas should be the location of cell nucleus. Due to the absent of targeting agents on the surface of PEGMA–IA–β-CD/Ad-PhNH₂ FPNs, the internalization of PEGMA–IA–β-CD/Ad-PhNH₂ FPNs is mainly ascribed to the nonspecific endocytosis, which has been demonstrated by Liu et al.⁶⁵ It is well known that the cell nucleus pore is less than 30 nm, which is much smaller than the size of PEGMA–IA–β-CD/Ad-PhNH₂ FPNs. Therefore, we think that PEGMA–IA–β-CD/Ad-PhNH₂ FPNs are difficult to enter cell nucleus directly and mainly distributed in the cytoplasm. Considered the properties included their high water dispersibility, low toxicity and desirable AIE active optical properties, PEGMA–IA–β-CD/Ad-PhNH₂ FPNs are expected to be promising candidates for biomedical applications.

Fig. 7 CLSM images of cells incubated with 40 μg mL⁻¹ of FPN for 3 h. (A) Bright field, (B) fluorescent image, which was excited with 405 nm laser, (C) merged image. Scale bar = 20 μm.

4. Conclusions

In summary, FPNs based on AIE dye and supermolecular chemistry were prepared via a novel strategy relied on free radical polymerization, ring-opening reaction and host–guest reactions. In this reaction system, the AIE dye (named PhNH₂) with amino group served as the fluorogen. The gained FPNs had not only strong fluorescence, uniform size and high water dispersibility, but also possessed excellent biocompatibility, making them promising candidates for various biomedical applications. Furthermore, because of the existence of carboxyl groups at the PEGMA–IA, multifunctional platform for the introduction of many other components like drugs and targeting agents could be provided to the AIE FPNs. On the other hand, β-CD could also be utilized for encapsulation of some anticancer agents (e.g. paclitaxel, curcumin) or fabrication of supermolecular hydrogels through host guest interaction. Furthermore, the obtained FPNs with red emission show important for fabricating fluorescent probes because of their long excitation wavelength, which largely reduce the injury for cells. Therefore, PEGMA–IA–β-CD/Ad-PhNH₂ FPNs are expected to have a profound impact on the biomedicine fields for above described advantages. Finally, many other AIE active supermolecular nanostructures can also be fabricated relied on the host guest interactions between β-CD and Ad using different β-CD derivatives and Ad-capped molecules. Taken together, combined all the advantages of the FPNs as being mentioned, the AIE active fluorescent supermolecular assemblies were believed to be promising candidates for fabrication of many multifunctional biomaterials.

Acknowledgements

This research was supported by the National Science Foundation of China (No. 51363016, 21474057, 21564006, 21561022), and the National 973 Project (No. 2011CB935700).

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Footnotes

† Electronic supplementary information (ESI) available: The synthetic routes of amino-terminated β-CD and Ad-PhNH₂ and ¹H NMR spectrum of Ad-PhNH₂. See DOI: 10.1039/c6ra08677b

‡ These authors contributed equally to this work.

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