Luminescence tunable fluorescent organic nanoparticles from polyethyleneimine and maltose: facile preparation and bioimaging applications

Abstract

Novel fluorescent organic nanoparticles (FONs) were facilely prepared via hydrothermal treatment of maltose and polyethyleneimine in water. These FONs exhibited suitable size and size distribution, high water dispersibility, excellent biocompatibility and tunable luminescence, making them highly potential for biological imaging applications.

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Semiconductor quantum dots have attracted great research interest for biomedical applications owing to their remarkable fluorescent properties, nanoscale size and multifunctional capability as compared with small organic dyes.^1,2 Since the first report of using semiconductor quantum dots for biomedical applications, a number of fluorescent nanoparticles especially fluorescent inorganic nanoparticles (FINs) have been fabricated and considered for various biomedical applications.^3–5 Based on their composition, these FINs could be mainly divided into semiconductor quantum dots, luminescent carbon nanoparticles, silicon quantum dots, metal clusters and lanthanide ions doped nanoparticles.^6–22 As a promising nanoprobe, it should be of suitable size and size distribution, excellent fluorescent properties, high water dispersibility, good biocompatibility and biodegredability.²³ Although FINs possess remarkable fluorescent properties and has been widely explored for bioimaging applications, most of FINs are non-biodegradable and some of them have verified toxicity to living organisms.²⁴ Therefore, the searching of alternative nanoprobes, which could overcome these problems of FINs is still highly desirable.

As compared with FINs, fluorescent organic nanoparticles (FONs) showed many advantages such as biodegradable potential, surface chemistry tailorability, ease of surface functionalization, designability of fluorescence and more important biocompatibility, which making them very promise candidates for bioimaging applications.^25–32 In recent years, a variety of FONs based on small organic dyes, conductor polymers and aggregation induced emission (AIE) dyes etc. have been developed and explored for various biomedical applications.^33–40 A general strategy for fabrication of FONs is preparation of dye contained amphiphilic copolymers, which can be self assembled into FONs in aqueous solution taken advantage of their hydrophobic interactions. After self-assemble, the hydrophobic dyes were encapsulated in the core of FONs, while hydrophilic segments were expanded into water and served as the shell, which rendered them water dispersibility. Although significant progress have been made, and some of them are promising for biomedical applications, the preparation of these FONs through conventional method is rather complex, tedious and time-consuming.^41–43 Therefore, a simple method for preparation of highly luminescent FONs with good water dispersibility and excellent biocompatibility should be of great research interest. Recently, a rather facile strategy for preparation of novel FONs based on polydopamine (PDA) was developed by our group.⁴⁴ We demonstrated that PDA based FONs with strong and tunable fluorescence can be obtained by self polymerization of dopamine and oxidation with concentrated H₂O₂. Thus obtained PDA FONs exhibited strong fluorescence and excellent biocompatibility, making them promising for bioimaging applications. This study open up a new orientation for preparation of FONs through a self polymerization of small organic molecules.

In this contribution, a rather facile strategy for preparation of FONs through hydrothermal treatment of maltose and polyethyleneimine (PEI) were developed for the first time (Scheme 1). To synthesize these FONs, we just needed to mix PEI and maltose in water and then heated to 100 °C for 2 h. The finally products (named PEI–Mal FONs) were obtained by purification with simple dialysis. To evaluate their biomedical application potential, the biocompatibility and cell uptake behavior of PEI–Mal FONs were also examined.

Scheme 1 Schematic showing the preparation of PEI–Mal FONs through hydrothermal treatment of polyethyleneimine (PEI) and maltose as the precusors and cell imaging applications of PEI–Mal FONs.

The morphology and fluorescent properties of PEI–Mal FONs were characterized by various techniques including transmission electron microscope (TEM), X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FT-IR) and fluorescence spectroscopy. As shown in Fig. 1, a number of organic nanoparticles with diameter less than 50 nm were observed through TEM images, evidencing the successful formation of PEI–Mal FONs. We have tried to obtain more detailed structural information about PEI–Mal FONs through improving magnification of TEM. However, the boundary of FONs became very blurred due to the low contrast of PEI–Mal FONs (Fig. S1†). On the other hand, we also found that the mixture of maltose and PEI was tuned into yellow during the preparation of PEI–Mal FONs, implying the successful reaction of PEI with Maltose. Due to the reaction temperature is 100 °C, which is much lower than carbonization temperature of maltose, therefore, these observed nanoparticles should be organic polymers from polymerization of maltose and PEI. Size is a very important parameter for biomedical applications of biomaterials. It has been demonstrated that the size of nanoparticles should be lower than 300 nm to efficiently enter target cells.⁴⁵ If the particle size larger than 300 nm, they can not enter the cells. In this work, the particle size of PEI–Mal FONs is much lower than 300 nm and large enough for avoiding renal clearance, which should be beneficial for in vivo biomedical applications of PEI–Mal FONs. Furthermore, the hydrodynamic diameter of PEI–Mal FONs was determined by a zeta Plus particle size analyzer. Results demonstrated that the size of PEI–Mal FONs determined by dynamic laser scatter is 468.9 ± 55.5 nm with a polydispersity index (PDI = 0.343) (Fig. S2†), which is much larger than that of TEM characterization. The possible reason is likely due to the shrinkage of PEI–Mal FONs when preparation of TEM samples.

Fig. 1 TEM images of PEI–Mal FONs with different magnificant, scale bar = 500 nm, the size of FONs is tens of nanometers.

The chemical information about PEI–Mal FONs was further characterized by XPS spectra. As shown in Fig. 2A, the survey XPS scan ranging from 0–1200 eV showed that elements including carbon (C), oxygen (O) and nitrogen (N) were present in the sample of PEI–Mal FONs, which given direct evidence that PEI was incorporated into PEI–Mal FONs. More detailed information of the XPS spectra of C, N and O is shown in Fig. 2B–D. Two major peaks of C1s with binding energy located at 285 and 287 eV can be observed, which can be mainly assigned to Sp2 and Sp3 carbon of PEI–Mal FONs, respectively. A binding energy peak located at 398.6 eV was found in N1s, indicating that C–N was existed in PEI–Mal FONs. On the other hand, O1s showed a binding energy peak located at 531.3 eV, suggesting the successful formation of doubly bond oxygen and C=O in PEI–Mal FONs. It has been reported that small molecular carbohydrates such as glucose and sucrose can be hydrolysis and formation of aldehyde bearing compounds.⁴⁶ These aldehyde bearing compounds could be further reacted with amino group of PEI via formation of Schiff base and formation of high molecular weigh polymers with luminescent properties. Furthermore, the element mass percentage of C, N and O was calculated based on the XPS spectra. The mass percentages of C, N and O are 67.6, 14.1 and 18.2, respectively. As compared the theoretical value of maltose, the mass percentage of C is significantly increased from 42.1% to 67.6%, while O is decreased from 51.5% to 18.3%, further confirming the successful formation of FONs. According to the mass percentage of N, we calculated that the mass ratio of maltose and PEI is about 1.67 : 1, which is very closed to their feed ratio (2 : 1).

Fig. 2 XPS spectra of PEI–Mal FONs. (A) XPS spectra, (B) C1s region, (C) N1s region, (D) O1s region.

The IR spectra of PEI and PEI–Mal FONs were shown in Fig. S3.† A series of absorbance bands located between 650–1600 cm⁻¹ in PEI were indentified from the IR spectra, which are possible ascribe to the stretching vibration of N–H band. On the other hand, the absorption peak located at 3278 cm⁻¹ assigned to stretching vibration of N–H was also found in the spectrum of PEI. Upon formation of PEI–Mal FONs, two feature peaks located at 2840 and 2920 cm⁻¹ with strong absorbance intensity in spectrum of PEI could still be observed, while their intensity is decreased, indicating successful incorporation of PEI into PEI–Mal FONs.^47,48 It should be worth to noting that a strong peak located at 1632 cm⁻¹ was emerged in PEI–Mal FONs, indicating that C=O was formation after hydrothermal treatment of maltose and PEI. These results are well consistent with the XPS results, which further confirmed the successful formation of reactive aldehyde groups and formation of PEI–Mal FONs.

Due to the existence of many hydrophilic functional groups such as –NH, –NH₂ and –OH on the surface of PEI–Mal FONs (Fig. S3†), they showed excellent dispersibility in aqueous solution. after they were deposited for one week, no perceivable aggregation was observed (left bottle of Fig. S4†). Strong blue fluorescence (right bottle of Fig. S4†) can be observed after PEI–Mal FONs were irradiated with UV lamp (λ = 365 nm). The photoluminescent (PL) properties of PEI–Mal FONs were detailedly characterized by fluorescent spectroscopy. As shown in Fig. 3, PEI–Mal FONs can be excited by various excitation wavelength ranged from 340–480 nm. The max intensity of excitation is located at 340 nm. When they were excited with different wavelength (340–480 nm), the emission peaks were redshift from 470–550 nm. However, their fluorescence intensity are decreased with increase of excitation wavelength correspondingly. On the other hand, we also found that the maximum fluorescence intensity can be obtained using 343 nm excitation wavelength based on excitation spectrum. The excitation-dependent fluorescent properties of PEI–Mal FONs is also reported by some other types of fluorescent nanoparticles, however the underline mechanism is still unclear.^44,49,50 UV-vis spectrum of PEI–Mal FONs was displayed in Fig. S5.† It can be seen that a small peak located at 323 nm was observed. These results suggested that conjugated carbonyl group were existed in PEI–Mal FONs, further indicating the formation of C=O after hydrothermal treatment of PEI and maltose. The quantum yield of PEI–Mal FONs was measured according to established procedure using quinine sulfate as ref. 51. Our results demonstrated that the quantum yield of PEI–Mal FONs is about 9.1%. On the other hand, the photostability of PEI–Mal FONs was measured recorded on a PE LS-55 spectrometer using time drive model. Results suggested that no significant fluorescent intensity decrease after PEI–Mal FONs were irradiated with 365 nm wavelength for 1800 S (Fig. S6†). As compared with conventional organic dyes, the excellent photostability made PEI–Mal FONs more suitable for practical biological imaging applications. Given the simple and scalable for preparation, high water dispersibility, strong and tunable fluorescence. Thus obtained PEI–Mal FONs should be of great potential for various biomedical applications.

Fig. 3 PL spectra of PEI–Mal FONs (in water), emission peak of PEI–Mal FONs is range from 470–550 nm, and the excitation peak is located at 343 nm when the emission peak was 480 nm. When PEI–Mal FONs were excited by various wavelength, intensity of PEI–Mal FONs was changed correspondingly.

To explore their potential biomedical applications, biocompatibility of PEI–Mal FONs with human lung adenocarcinoma epithelial (A549) cells was examined by optical microscopy observation and cell viability measurement.^52–56 As shown in Fig. S7,† cells still strong adhered to cell plate after they were incubated with different concentrations of PEI–Mal FONs for 24 h. As compared with control cells, no obvious cell morphology change was observed after cells were incubated with PEI–Mal FONs at the concentration of 120 μg mL⁻¹. The optical microscopy observation suggested that PEI–Mal FONs are good biocompatible with A549 cells. To further verified their biocompatibility, cell viability of PEI–Mal FONs was examined by cell counting kit-8 (CCK-8) assay.^57–60 As shown in Fig. 4, almost no cell viability decrease was observed when cells were incubated with 10–120 μg mL⁻¹ of PEI–Mal FONs for 8 and 24 h. Even the concentration of PEI–Mal FONs is as high as 120 μg mL⁻¹, the cell viability values of PEI–Mal FONs are still greater than 90%. Cell viability measurement further confirmed the excellent biocompatibility of PEI–Mal FONs. The excellent biocompatibility of PEI–Mal FONs endow them great potential for biomedical applications.

Fig. 4 Biocompatibility evaluation of PEI–MAL FONs. Cell viability of PEI–Mal FONs with A549 cells were determined by CCK-8 assay (concentrations of PEI–Mal FONs are ranged from 10–120 μg mL⁻¹).

The potential bioimaging applications of PEI–Mal FONs were examined by their cell uptake behavior using Confocal Laser Scanning Microscope (CLSM).^61,62 As showed in Fig. 5, cell internalization of PEI–Mal FONs could be clearly observed after cells were incubated with 10 μg mL⁻¹ of PEI–Mal FONs for 3 h (samples were irradiated with 405 and 458 nm laser). According to PL spectra, the emission peaks of PEI–Mal FONs should belocated at about 475 and 540 nm, respectively. Therefore, they should emit blue (Fig. 5B) and green (Fig. 5C) fluorescence after irradiation with 405 and 458 nm laser. The PEI–Mal FONs are therefore have some advantages over conventional dyes for bioimaging applications due to their tunable luminescence. On the other hand, many dark areas with relative weak fluorescence intensity were also observed by CLSM images (Fig. 5B and C). These dark areas are possible the location of cell nucleus. Due to the size of PEI–Mal FONs is obviously larger than that of cell nucleus pore, PEI–Mal FONs should be internalized into cells through phagocytosis. More importantly, the cell uptake of PEI–Mal FONs could be clearly observed even the concentration of FONs was as low as 10 μg mL⁻¹. If the surface of PEI–Mal FONs was further conjugated with targeting agents. The dosage for cell imaging could be further decreased. Therefore, we believed that PEI–Mal FONs were biocompatible enough for practical biological imaging applications.

Fig. 5 CLSM images of A549 cells when they were incubated with 10 μg mL⁻¹ of PEI–Mal FONs for 3 h. (A) Bright field, (B) excited with 405 nm laser, (C) excited with 458 nm laser, (D) merge image of A, B and C, scale bar = 20 μm.

In summary, PEI–Mal FONs were facilely prepared through hydrothermal maltose in the present of PEI for the first time. This reaction can be occurred under room temperature and air atmosphere without needing catalysts, which is rather simple and scalable. The possible reason for the formation of PEI–Mal FONs is likely due to hydrolysis of maltose and formation of aldehyde bearing compounds, which can be further reacted with the amino group of PEI through Schiff base. Thus obtained FONs exhibited high water dispersibility, strong and tunable luminescence and excellent biocompatibility. On the other hand, many other functional components such as targeting agents, drugs and functional polymers could also be further conjugated with PEI–Mal FONs because there are various surface functional groups on their surface. Thus multifunctional imaging and theranostic systems based on PEI–Mal FONs can be fabricated. It is therefore PEI–Mal FONs are expected highly potential for various 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 (no. 2012M520388, 2012M520243, 2013T60100, 2013T60178).

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Footnote

† Electronic supplementary information (ESI) available: FT-IR spectra of PEI and PEI–Mal FONs optical images of PEI–Mal FONs water dispersion and optical microscopy images of A549 cells were provided in ESI. See DOI: 10.1039/c4ra03103b

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