Biocompatible fluorescent polymeric nanoparticles based on AIE dye and phospholipid monomers

Abstract

Phospholipid monomer was utilized for the first time to construct aggregation induced emission (AIE) dye based cross-linked fluorescent polymeric nanoparticles. Such nanoparticles showed intense fluorescence and stable dispersibility in physiological solution below the critical micelle concentration, coupled with excellent biocompatibility, making them promising for cell imaging application.

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Fluorescence labeling is a well established methodology for bioimaging and monitoring biological species and processes in living systems.^1–3 Therefore, various fluorescent nanoparticles have been synthesized and extensively investigated for biomedical applications over the past few decades, which include semiconductor quantum dots, fluorescent proteins, carbon dots, and organic dyes.^4–10 However, semiconductor quantum dots often suffer from high cytotoxicity due to the accumulation of heavy metal ions (e.g., CdSe and CdTe) in the reticuloendothelial system;¹¹ while green fluorescent protein is usually subjected to poor photostability, small stokes shifts, easy enzyme degradation, and tedious transfection process.¹² Weak luminescence and non-functionalized feature often hamper carbon dots for cell imaging application.¹³ Furthermore, most of the conventional organic dyes often quench their fluorescence at aggregated phase due to the planar structures and strong intermolecular π–π interactions.¹⁴ In order to fabricate organic dyes based fluorescent nanoparticles with high water dispersibility and high fluorescence efficiency for cell imaging, aggregation induced emission (AIE) materials have been introduced instead of conventional organic dyes as an alternative material platform for potential bioimaging applications.^15,16 Due to the antiquenching effect in the condensed state of AIE dyes, various AIE fluorogens including siloles,^17–20 tetraphenylethene,^21–27 triphenylethene,^28–32 cyano-substituted diarylethylene,^33–36 and distyrylanthracene^37–43 have been developed for chemosensors and bioprobes. Furthermore, development of fluorescent polymeric nanoparticles (FPNs) based on AIE dyes has been considered as a burgeoning and promising strategy to construct cell imaging vehicles.⁴⁴

In recent years, some strategies for fabricating AIE based FPNs have been developed. Jen et al.⁴⁵ reported a simple non-covalent method utilizing synthetic amphiphilic block copolymers to encapsulate AIE dyes as bioprobes. Other non-covalent strategies have also been reported, silica nanoparticles, mesoporous silica nanoparticles, bovine serum albumin⁴⁶ and commercialized surfactant Pluronic F127^47,48 have been used to encapsulate AIE dyes to afford FPNs for cell imaging. With regard to the covalent methods, Tang et al.⁴⁹ have prepared tetraphenylethene-ended conjugated fluorescent polyelectrolytes as fluorescent visualizers for intracellular imaging. Reversible addition fragmentation transfer (RAFT) polymerization has been firstly utilized to construct AIE based FPNs in our group,^50,51 meanwhile, some tactics including emulsion polymerization⁵² and anhydride ring-opening polymerization⁵³ have also been developed to prepare various functional FPNs, and these FPNs based on covalent strategies have been demonstrated more stable than those of non-covalent ones to avoid the leakage of dyes. Although many impressive advances have been achieved in the construction of AIE FPNs, more robust architectures are still highly demanded, such as cross-linked copolymers, which can overcome the issue of critical micelle concentration (CMC) existing in linear polymers.^54–57

Phospholipids are a major component of all cell membranes that form lipid bilayers. As the structure of the phospholipid molecule generally consists of hydrophobic tails and a hydrophilic head, which can self-assemble into nano-films and other nano-structures such as micelles, reverse micelles and liposomes. Furthermore, phospholipids can be connected with other molecules via specific chemical linkages. These features, along with their excellent biocompatibility and transparency in visible light, have made phospholipids promising in nanotechnology and materials science.⁵⁸

In this contribution, a phospholipid monomer was utilized for the first time to construct AIE dye based cross-linked FPNs. As shown in Scheme 1, a hydrophilic phospholipid monomer of 2-methacryloyloxyethyl phosphorylcholine (MTP) and a new AIE dye based dimer (An) were copolymerized at the existence of chain transfer agent (CTA) via RAFT polymerization to form the amphiphilic cross-linked polymers (An–MTP). The obtained polymers are prone to self-assembly into stable FPNs and can be highly dispersed in physiological solution. Hereafter, a series of characterization methods, including gel permeation chromatography (GPC), Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), UV-visible absorption spectrum, fluorescence spectrum, transmission electron microscopy (TEM), and dynamic light scattering, were carried out to determine the performance of An–MTP FPNs. Furthermore, the biocompatibility and cell uptake behaviour of An–MTP FPNs were further conducted to evaluate their potential cell imaging application.

Scheme 1 Preparation of An–MTP copolymers from an AIE dye based dimmer (An) and a phospholipid monomer (MTP) via RAFT polymerization and self-assembly of An–MTP copolymers into FPNs for cell imaging applications.

The synthesis of An was shown in Scheme S1† with high yield (93%). The AIE characteristic of An was determined and shown in Fig. S1,† which demonstrated obvious AIE feature. To prepare An–MTP copolymers, a hydrophilic phospholipid monomer of MTP and a new AIE dye based dimer (An) were copolymerized at the existence of CTA via RAFT polymerization (Scheme 2). For the synthesis of An–MTP, the feed molar ratio of CTA, An, and MTP was designed as 1 : 1 : 10, respectively, while the designed degree of polymerization (DP) was 11. After the resulting cross-linked polymers were purified, the number average molecular weight (M_n) values of An–MTP were determined by GPC, which showed that the M_n values of An–MTP was 30 900 Da with narrow polydispersity index (PDI = 1.14). ¹H NMR spectrum of An–MTP dissolved in d₆-DMSO was conducted and shown in Fig. S2.† Although it is difficult to determine the chemical structure of the obtained cross-linked polymer from the ¹H NMR spectrum, the chemical shifts of the aromatic group and the phospholipid group can be found around 7.0–8.0 and 3.5–5.0, respectively. This determined molecular weight is not very high for the cross-linked polymers according to the GPC result, but it is worth mentioning that it is not a good thing to prepare polymers with ultra-high molecular weight for biomedical application, as it will encounter the problem of biodegradability.

Scheme 2 Synthetic routes of An–MTP: a new AIE dye based dimer (An) and a hydrophilic phospholipid monomer of MTP were copolymerized at the existence of CTA via RAFT polymerization to afford An–MTP.

FT-IR spectra were conducted to confirm the successful synthesis of An–MTP. As shown in Fig. 1A, in the sample of An, the C–H stretching vibrations of alkane group were observed located at 2915 and 2849 cm⁻¹, a series of peaks distributed between 1450-1650 cm⁻¹ can be ascribed to the stretching vibration of aromatic rings. For the sample of MTP, stretching vibrations of C=O located at 1722 cm⁻¹ and P=O located at 1254 cm⁻¹ were found, and the characteristic peaks P–O located at 1061 and 951 cm⁻¹ were also observed. After the RAFT polymerization to afford An–MTP, strong aromatic and alkyl C–H stretching vibration located at 3000, 2925 and 2850 cm⁻¹ were observed. Meanwhile, the stretching vibrations of aromatic rings were found located at 1603 and 1513 cm⁻¹. Furthermore, intense stretching vibrations of C=O located at 1722 cm⁻¹, P=O located at 1254 cm⁻¹, and P–O located at 1061 and 951 cm⁻¹ were observed, respectively. These results confirmed the successful formation of the cross-linked copolymers.

Fig. 1 (A) Normalized IR spectra of An, MTP and An–MTP, stretching vibrations of C=O located at 1722 cm⁻¹ and P–O located at 1061 and 951 cm⁻¹ were observed in the sample of An–MTP FPNs, suggesting successfully incorporation of An and MTP to afford An–MTP; (B) XPS spectra of MTP and An–MTP FPNs showing the presence of carbon, nitrogen, oxygen, phosphorus; (C) UV absorption spectrum of An–MTP FPNs dispersed in water, inset: visible images of An–MTP FPNs in water; (D) fluorescence excitation (Ex) and emission (Em) spectra of An–MTP FPNs, inset are the fluorescent images of An–MTP FPNs taken at 365 nm of UV light.

Elemental composition of MTP and the prepared An–MTP have been determined by XPS studies, which are shown in Fig. 1B and S3.† The XPS result showed the presence of carbon as major component along with other minor components like nitrogen, oxygen and phosphorus. The overall wt% of elements present in MTP was C–N–O–P ∼ 60 : 4 : 31 : 5, while that in An–MTP was C–N–O–P ∼ 69 : 3 : 25 : 3. It was obvious to find the increase of carbon component and the decrease of other three components after RAFT polymerization. The C 1s XPS spectrum of An–MTP was observed a slight shift compared with that of MTP, while its spectrum could be deconvoluted into 2 components (Fig. S3A†). The main peak at ∼285 eV and shoulder peak ∼287 eV could be attributed to carbon with sp² and sp³ carbon atoms, respectively. The other three elements also showed slight shifts between MTP and An–MTP. The N 1s spectrum of An–MTP showed peak centered at ∼403 eV (Fig. S3B†), while the P 2p spectrum showed peak at ∼133 eV (Fig. S3D†). The O 1s also could be deconvoluted into 2 components located at ∼531 and ∼532 eV (Fig. S3C†). The above results confirm that An and MTP were successfully combined to afford An–MTP copolymers.

Thanks to the amphiphilic feature of MTP, when the resulting An–MTP copolymers were dispersed in aqueous solution, self-assembly occurred into polymeric nanoparticles with hydrophilic phosphorylcholine groups covered at the surfaces and the hydrophobic AIE components aggregated into the cores, so these obtained FPNs were expected to show high dispersibility in aqueous environment. Thus the UV absorption spectrum of An–MTP FPNs dispersed in water was shown in Fig. 1C, which could be found that the absorption peak was located at 440 nm. It is noteworthy that the entire spectrum started to increase from 700 nm, which is ascribed to the Mie effect, indicating the dispersion of nanoparticles in the solution. The insets of Fig. 1C also showed the high water dispersibility of such FPNs directly as they were readily dispersed in water with high transparency. Due to the aggregation of An inside the nanoparticles, these FPNs showed strong yellow-green fluorescence in water (inset of Fig. 1D). The PL spectra of An–MTP FPNs in water were carried out to quantitatively determine the excitation and emission wavelengths (see Fig. 1D). The maximum emission wavelength was located at 501 nm, while the fluorescence excitation wavelength was located at 368 and 436 nm. The fluorescence quantum yield values of the FPNs were determined using quinine sulfate as the reference dye, which showed 61% for An–MTP FPNs. This intense fluorescence was greatly benefited for the potential cell imaging applications.

Self-assembly of polymeric materials into nanoparticles is a promising strategy for various biomedical applications including imaging and therapy.⁵⁹ Thus, the TEM study was conducted to further confirm the formation of the resulting An–MTP FPNs (Fig. 2A). Some spherical nanoparticles with diameters ranging from 30 to 50 nm can be clearly identified, which demonstrated the formation of FPNs from the synthetic copolymers. Meanwhile, the size distribution of An–MTP FPNs in phosphate buffer solution (PBS) was determined using a zeta Plus particle size analyzer, showing that the result of size distribution was 141.7 ± 11.6 nm, with a polydispersity index of 0.332. As compared with the size distribution in PBS, the particle size characterized by TEM was somewhat smaller, which might be due to the drying-causing shrinkage of the self-assembly process.

Fig. 2 (A) TEM image of An–MTP FPNs dispersed in water; (B) intensity of the aggregate emission vs. the logarithm of the concentration of An–MTP (λ_ex = 405 nm, λ_em = 500 nm).

In this work, cross-linked architecture of the polymers has been constructed in order to conquer the trouble of CMC. The response of the AIE-active fluorescence behavior toward the concentration of the aqueous FPNs solutions was used to locate the CMC.⁶⁰ The maximum fluorescent emission (500 nm) due to the aggregated cross-linked polymers was used to track the CMC values. Thus, the intensity of the aggregate emission vs. the logarithm of the concentration of An–MTP was carried out to evaluate the CMC. At low concentrations below CMC, the detected intensity of aggregate emission is very low, but at concentrations above CMC, the aggregate emission increases abruptly. The result showed 0.138 mg mL⁻¹ of the CMC for An–MTP FPNs (Fig. 2B). Surprisingly, when we studied the size distribution of An–MTP FPNs by dynamic light scattering (DLS), the FPNs showed very stable dispersion in extremely dilute physiological solution. Even the concentration of the FPNs was ultimately diluted to 0.1 μg mL⁻¹, the FPNs could also be detected by DLS. This result demonstrates that cross-linking strategy is greatly benefited for the stable dispersibility of FPNs in physiological solution below the critical micelle concentration.

The biocompatibility was proceeded to evaluate the potential biomedical applications of An–MTP FPNs. First of all, the influence of An–MTP FPNs to A549 cells was examined by optical microscopy after the cells were incubated with different concentrations of these FPNs for 24 h (Fig. 3A–C). The result showed that the cells grew well when incubated with 20 or even 80 μg mL⁻¹ of An–MTP FPNs, evidencing that the FPNs were biocompatible with cells. To further confirm the cytocompatibility of An–MTP FPNs, cell viability of such FPNs to A549 cells was determined by cell counting kit-8 (CCK-8) assay.⁸ As shown in Fig. 3D, no decrease of cell viability was observed when the cells were incubated with An–MTP FPNs at the range of 10–120 μg mL⁻¹ for 8 h and 24 h. Even when the concentration reached up to 120 μg mL⁻¹, the value of cell viability still maintained above 90%, which confirmed good biocompatibility of these FPNs. All the above results proved that the prepared FPNs were highly potential for cell imaging.

Fig. 3 Biocompatibility evaluations of An–MTP FPNs. (A–C) Optical microscopy images of A549 cells incubated with different concentrations of An–MTP FPNs for 24 h: (A) control cells, (B) 20 μg mL⁻¹, (C) 80 μg mL⁻¹; (D) cell viability of An–MTP FPNs for 8 h and 24 h.

Then cell imaging applications of An–MTP FPNs were further explored. The cell uptake behaviour of An–MTP FPNs was evaluated by Confocal Laser Scanning Microscope (CLSM). As shown in Fig. 4, intense yellow-green fluorescence could be observed inside the cells after incubating with 10 μg mL⁻¹ of An–MTP FPNs. Inside the cells, many areas with relative weak fluorescent intensity were found due to the location of cell nucleus (Fig. 4B), which indicated An–MTP FPNs were facilely uptaken by the cells along with the cytoplasm stained by the FPNs. Additionally, these FPNs were considered as uptakes through the endocytosis process of the cells as comparing with the size of FPNs and nucleus pore of the cells. Therefore, we could expect the An–MTP FPNs be promising candidates for cell imaging with potential advantages including stable dispersion, intense fluorescence, and excellent biocompatibility.

Fig. 4 CLSM images of A549 cells incubated with 10 μg mL⁻¹ of An–MTP FPNs for 3 h. (A) Bright field, (B) excited with 405 nm laser, (C) merged image of (A) and (B). Scale bar = 20 μm.

In summary, a hydrophilic phospholipid monomer of MTP and a new AIE dye based dimer (An) were copolymerized via RAFT polymerization to form the amphiphilic cross-linked polymers (An–MTP). An–MTP were prone to self-assemble into uniform FPNs with diameters ranging from 30 to 50 nm, and showed high water solubility and intense fluorescence in aqueous solution due to the hydrophilic phosphorylcholine groups covered at the surfaces and the AIE components aggregated into the cores. These FPNs also showed stable dispersibility in physiological solution below the CMC. Biocompatibility evaluation and cell imaging results demonstrated that these FPNs were biocompatible for bioimaging applications. More importantly, the introduction of phospholipids into the construction of cross-linking FPNs reported in this work provides a new way to prepare robust and biocompatible bioimaging materials and expand the scope of their real biomedical applications.

Acknowledgements

This research was supported by the National Science Foundation of China (no. 21134004, 21201108, 51363016), and the National 973 Project (no. 2011CB935700), China Postdoctoral Science Foundation (2012M520243, 2013T60100), High-level Science Foundation of Qingdao Agriculture University (6631334).

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Footnote

† Electronic supplementary information (ESI) available: Detailed information about materials and measurements; synthesis of An; preparation of An–MTP FPNs; AIE characteristic of An; ¹H NMR spectrum of An–MTP; XPS spectra of MTP and An–MTP FPNs. See DOI: 10.1039/c4ra03092c

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