Fluorescent Imaging Probe from Nanoparticle Made of AIE Molecule

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Fluorescent Imaging Probe from Nanoparticle Made of AIE Molecule Kuheli Mandal,† Debabrata Jana,‡ Binay K. Ghorai,*,‡ and Nikhil R. Jana*,† †

Centre for Advanced Materials, Indian Association for the Cultivation of Science, Kolkata 700 032, India Department of Chemistry, Indian Institute of Engineering Science and Technology, Shibpur, Howrah 711 103, India

‡

S Supporting Information *

ABSTRACT: Aggregation induced emission (AIE) active molecules are widely used for fluorescence “turn-on” detection applications with the unique advantages over conventional fluorescent probes. Transformation of AIE molecule into functional nanoparticle can greatly expand their biomedical application potential. Here we report an approach for preparation of 20−80 nm size functional nanoparticle made of AIE molecule. The approach involves aggregation of AIE active tetraphenylethene (TPE) molecule in the presence of functional TPE molecule. Following this approach we have synthesized TPE-based nanoparticle functionalized with polyethylene glycol, primary amine, aspartic acid, glucose, and arginine. Compared to reported methods of making AIE-based nanoparticles, presented nanoparticles have distinct advantages that they are composed of AIE molecules only (without any non-AIE molecule/polymer) and wide variety of surface functionalization can be achieved by this approach. Nanoparticles have good colloidal stability with fluorescence quantum yield of 12−15% and fluorescence remains intact in the presence of conventional quenchers. These nanoprobes are used as fluorescent cell labels and can be extended for preparation of AIE molecule-based different nanoprobes.

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INTRODUCTION Fluorescent nanoparticle-based bioimaging probes have attracted great attention for understanding biochemical activities down to cellular and molecular length scale.1−3 Variety of nanoprobes have been developed which are composed of quantum dot,1−3 doped semiconductor nanocrystal,4 fluorescent metal nanocluster,5−8 fluorescent carbon nanoparticle9 and silicon nanoparticle.10 These nanoprobes have been used for imaging-based detection of specific cell, subcellular organelle, tumor inside the body, and the activity of single molecule inside a live cell.11−15 These study shows that nanometer length scale plays unique role for in vitro/in vivo application and proves that the 50−200 nm size is ideal for in vivo targeting application,11,12 the 1−50 nm size is ideal for cellular and subcellular targeting application,13,14 and the 50% molar ratio produces polydispersed nanoparticle with low emission and 100 nm size with strong emission. However, they are completely insoluble in water and precipitate during preparation. The surface of the nanoparticle is decorated with different biomolecule/chemical, depending on functional TPE used in the synthesis processes. This is because functional TPE molecules are expected to link with nanoparticle through their TPE component and water-soluble biomolecule/chemical component would expose in water. Thus, nanoparticles are functionalized with PEG−NH2, aspartic acid, glucose, and arginine when they are synthesized using TPE−PEG−NH2, TPE−aspartic acid, TPE−glucose, and TPE−arginine, respectively. This functionalization is known to influence the cell−nanoparticle interaction. In particular, arginine functionalization is known to provide cationic surface charge and induce cell uptake,39 aspartic acid functionalization would provide anionic surface charge with lower cell uptake,13 and glucose functionalization would offer interaction with glycoprotein.40 Functionalization with

Figure 3. Demonstration of aggregation induced emission property of TPE and TPE−PEG−NH2, showing that emission increases in a methanol−water medium with the increasing water %. Same concentration of TPE/TPE−PEG−NH2 is taken in methanol−water media and then emission is measured using 390 nm excitation for TPE and 370 nm excitation for TPE−PEG−NH2.

water-soluble. We have varied the molar ratio of functional TPE between 5 to 80% to control the particle size and colloidal

Figure 4. (i) (a) Concentration-dependent fluorescence spectra of TPE−PEG−NH2 in water under 370 nm excitation. (b) Plot of emission intensity at 500 nm against the concentration showing critical aggregation concentration at ∼0.0045 mM. (ii) (a) Concentration-dependent fluorescence spectra of TPE−arginine in water under 370 nm excitation. (b) Plot of emission intensity at 550 nm against the concentration showing critical aggregation concentration at ∼0.005 mM. F

DOI: 10.1021/acs.jpcc.5b12682 J. Phys. Chem. C XXXX, XXX, XXX−XXX

Article

The Journal of Physical Chemistry C PEG−NH2 would decrease nonspecific interaction, and NH2 can be used for further functionalization.41 Functionalization of nanoparticle reported here is simple as compared to post functionalization approach. In post functionalization approach, nanoparticle is covalently conjugated with affinity molecule followed by dialysis to remove unbound molecules.2,8,13 This approach needs introduction of appropriate chemical functional group on nanoparticle surface, requirement of conjugation chemistry and dialysis step. In contrast presented approach of making functional nanoparticle is one step and does not require any dialysis or other purification step. However, a functional AIE molecule has to be synthesized first for each type of functionalization. Property of Functional Nanoparticle. Property of AIE-based functional nanoparticles are summarized in Table 1 and compared with the nanoparticles produced by two control methods. The size of the nanoparticles has been measured using scanning electron microscopy (SEM) and dynamic light scattering (DLS) study. SEM image shows the clear structure of the nanoparticle and DLS shows the size distribution of the nanoparticle. (Figures 1, 2 and Table 1) Average size of nanoparticle prepared by present approach varies between 50−80 nm. However, nanoparticles prepared by self-assembly approach (control 1) are relatively smaller in size (typically of 20−50 nm). In contrast size of insoluble particle prepared without using any functional TPE molecules (control 2) are greater than 100 nm. Surface charge of nanoparticles has been determined using ζ-potential measurement, and they are negative for all nanoparticles with the value between −36 and −46 mV, and no significant difference is observed for different functionalization. (Table 1) In contrast nanoparticles prepared by self-assembly approach (control 1) have relatively lower surface charge and varies between −4 and −12 mV. Fluorescence quantum yield has been measured using quinine sulfate as reference and the values are in the range of 12−15% for nanoparticles prepared by proposed approach as compared 8−10% for self-assembly-based nanoparticles. Overall the size, surface charge and fluorescence quantum yield of nanoparticles prepared by proposed approach are higher than nanoparticles prepared by self-assembly approach. Consistently higher fluorescence quantum yield and higher surface charge is possibly due to the relative larger size of nanoparticle. AIE property of TPE molecule and functional TPE molecules are shown in Figure 3. TPE and functional TPEs are nonfluorescent or weakly fluorescent in methanol due to their high solubility. However, green emission appears with the increasing water percent in the medium due to lowering of the solubility of TPE/functional TPE. This result indicates that TPE and functional TPEs exhibit AIE properties. Thus, it can be expected that TPE-based nanoparticles should exhibit green emission as aggregated forms of TPE molecules are present inside the nanoparticle. Self-assembly of functional TPE molecules has been studied by measuring the emission spectra in water with the increase of concentration. Functional TPE molecules start showing emission after attaining certain concentration, suggesting the assembly formation. (Figure 4) The critical aggregation concentration has been determined from this study which is in the range of 3−5 mM. Figures 5 and 6 show the optical property of functional nanoparticles prepared by the proposed approach and by the self-assembly (control I) approach. Result shows that all nanoparticles show intense green emission. However, the nature of the emission band, emission maxima, and emission

Figure 5. Absorption and fluorescence spectra of different functional nanoparticle prepared by proposed approach and by control I approach, showing that all nanoparticles show intense green emission due to aggregated TPE component. Excitations of 370 and 390 nm are used for nanoparticle made of proposed approach and control I, respectively.

intensity varies depending on the nature of the functional group, particle size, and method of preparation. Observed differences in the absorption and emission spectra of functional nanoparticles are possibly due to the difference in the nature of assembly/ aggregation of TPE.42 A nanoparticle prepared by the proposed method produces a stronger emission as compared to a nanoparticle prepared by the self-assembly approach. This can be observed from a digital image of nanoparticle solutions (Figure 6) and fluorescence quantum yields (Table 1), Colloidal stability and fluorescence stability of nanoparticle have been investigated under different buffer solution and cell culture media. (Supporting Information, Figure S8) Results show that nanoparticles do not precipitate on preservation for months and retain their fluorescence under these conditions. Fluorescence stability is further investigated under continuous UV exposure of a drop casted film of nanoparticle which show that fluorescence remain intact under continuous light exposure. (Supporting Information, Figure S9) These results suggest that fluorescence of nanoparticles would be stable under continuous excitation and useful for long-term imaging application. One of the critical issues of quantum dot based nanoprobe is that they often quench their fluorescence under adverse bioenvironment and in the presence of quenchers. For example, quantum dot based probes lose their fluorescence under acidic medium, in the presence of heavy metal ions or metal nanoparticles.43−45 G

DOI: 10.1021/acs.jpcc.5b12682 J. Phys. Chem. C XXXX, XXX, XXX−XXX

Article

The Journal of Physical Chemistry C

dots significantly lose their fluorescence under most of these conditions. This result suggests that the AIE-based nanoparticle can be a better probe than the quantum dot under adverse conditions. Bio-Imaging Application Potential of AIE-Based Functional Nanoparticle. In order to explore the bioimaging application potential, AIE-based functional nanoparticles have been investigated as fluorescent cell labels. Typically, CHO cells are incubated with nanoparticles for 1 h and washed cells are imaged under fluorescence mode with blue excitation. Results are summarized in Figure 8 and the Supporting Information, Figure S10. Three general conclusions can be drawn from this imaging study. First, nanoparticles prepared by the proposed approach show stronger emission as compared to nanoparticles prepared by the self-assembly approach. This result suggests that nanoparticles prepared by the proposed approach are better candidates for labeling applications. Second, cells are labeled by all nanoparticles irrespective of surface functionality, although some differences are observed in the emission intensity of labeled cells. This result concludes that nanoparticles have a nonspecific binding problem with the cell, and further processing of surface chemistry is necessary for specific targeting application. Third, longer incubation of labeled cells under cell culture media shows that fluorescence of labeled cells decreases with time. (Supporting Information, Figure S10) This result suggests that nanoparticles either are exocytosized or gradually lose their assembled structures after attaching to the cell membrane, and further study is necessary for better understanding. A cytotoxicity study has been performed using CHO cell as reference and results show >85% cell viability for 24 h incubation of our nanoparticles in the labeling concentration range (Supporting Information, Figure S11). The most unique advantage of presented AIE nanoparticlebased fluorescence probes is that it does retain the fluorescence under acidic conditions and in the presence of quencher metal ion and nanoparticle. This aspect offers advantage over quantum dot based probes. For example quantum dot often lose their fluorescence under acidic endosomal compartment of cell, in the presence of metal nanoparticle and heavy metal ions.43−45 Retained fluorescence of AIE nanoparticle under these conditions can be exploited for development of an advanced optical probe. However, further advancement of probe development is necessary that should overcome following issues. First, nanoparticle should be more robust in nature so that assembled structure does not break during interaction with bioenvironment

Figure 6. Digital image of aqueous colloidal solution of nanoparticles prepared via proposed method and by control I and control II. Results show that proposed method and control I produces fluorescent colloidal nanoparticle but the emission is weaker for nanoparticles prepared by control I. Particles prepared by control II approach have strong emission but they precipitates in water. Colloidal nanoparticle can also be prepared in methanol−water media but they precipitate if methanol is removed by dialysis.

In contrast, fluorescence of presented nanoparticle remains intact under such an environment. Figure 7 shows comparative fluorescence stability of AIE-based nanoparticles and quantum dots under these conditions. It is observed that AIE-based probes retain their fluorescence in acidic medium and in the presence of quencher metal ion and metal nanoparticle. In contrast, quantum

Figure 7. Comparative fluorescence stability of AIE-based nanoparticles (a) and quantum dot (b) in the presence of different fluorescence quenchers. Glucose-functionalized AIE-nanoparticles (concentration 0.025 mg/mL) and polymer-coated CdSe-ZnS quantum dots are used for this study. Final concentration of metal ions is kept at 2 mM and concentrations of silver and gold in the respective nanoparticles are kept at 0.5 mM. H

DOI: 10.1021/acs.jpcc.5b12682 J. Phys. Chem. C XXXX, XXX, XXX−XXX

Article

The Journal of Physical Chemistry C

Figure 8. Fluorescence image of CHO cells labeled with different functional nanoparticle. Cells are incubated with nanoparticle for 1 h and washed cells are imaged under bright field (BF) and fluorescence mode (F) under blue excitation. Results show that cells labeled with nanoparticles prepared by proposed approach show stronger emission as compared to nanoparticles prepared by control I.

(cell membrane, for example) and they should have minimum nonspecific binding interaction. Second, size of the nanoparticle should be controllable in the 5−100 nm range and emission color should be tunable from visible to NIR range. With the development of a new generation of AIE molecules and advancement of nanoparticle synthesis, these issues are expected to be solved in near future.

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AUTHOR INFORMATION

Corresponding Authors

*(N.R.J.) E-mail: [email protected]. *(B.K.G.) E-mail: [email protected].

CONCLUSION We have developed an approach for preparation of AIE molecule-based functional nanoparticle. In this approach the AIE molecule is aggregated in the presence of a functional AIE molecule that restrict the size