Supramolecular Assembly and Reversible Transition and of Chitosan Fluorescent Micelles by Noncovalent Modulation

Chitosan-based intelligent artificial systems have been of increasing interest for their biocompatibility, multifunctionality, biological activity, and low cost. Herein, we report the fabrication of supramolecular nanoparticles based on water-soluble chitosan (WCS) and 1,1′,1′′,1′′′-(ethene-1,1,2,2-tetrayl)tetrakis(benzene-4,1-diyl) tetrakis(azanediyl)tetraacetic acid (TPE-(NCOOH)4), which is capable of reversible transition between polyion complexes (PICs) and hydrogen bonding complexes (HBCs) with tunable aggregation-induced emission driven by pH value. The PIC micelles could be formed via electrostatic interaction between ammonium cations and carboxylate anions under mild alkaline conditions. The formation of the micelles dramatically blocks the nonradiative pathway and enhances the fluorescence of TPE moieties, and the maximum fluorescence intensity was achieved near the isoelectric point due to the restriction of intramolecular motion. In addition, the fluorescence intensity and size of the PIC micelles exhibited a temperature response in the range from 20 to 80°C. Upon adjusting the solution pH to 2, the PIC micelles were reconstructed into hydrogen-bonding complexes while the hydrogen bonding interaction between the protonated carboxyl groups of TPE-(N-COOH)4 and chitosan. Moreover, the size of the micelles underwent a remarkable decrease, whereas the fluorescence emission was further enhanced by ~6.25-fold. The pH actuated micellar transition from PIC to HBC with tunable fluorescence performance is fully reversible. This study provides novel multifunctional materials that are of great importance for their potential application in the fields of optoelectronic devices and chemical and biomedical sensors.


Introduction
Recently, stimulation responsive functionalized polymeric micelles have drawn considerable attention because they have distinctive photophysical properties and potential applications in life sciences [1][2][3]. Among them, the utilization of noncovalent interactions to self-assemble nature macromolecules with the formation of hierarchical assemblies is of enormous importance. In fact, the biomimicking of noncovalent interactions and their microenvironment triggered transitions in biological systems to fabricate biomedical materials has been a subject of considerable interest [4,5]. Accordingly, a great variety of supramolecular interactions have been developed and investigated to construct numerous functional architectures, including host-guest interaction, coordination interaction, electrostatic interaction, hydrophobic association, π-π stacking, and hydrogen bonding [6][7][8][9][10]. For example, the electrostatic interaction has performed an extensive effect on the controlled drug delivery and interprotein recognition [11,12], whereas hydrogen bonding interaction has dominated shape self-recovery and volume regulation of the vesicles constructed from polysaccharide and embodying cell like behavior [13]. The polyion complex (PIC) was constructed through electrostatic interaction originally proposed by Kataoka [14,15], while inter/intramolecular hydrogen bonding promoted the formation of hydrogen bonding complexes (HBCs). Thus, the advance of noncovalent interactions will not only enrich the family of biomimetic nanostructures, but also provide a new bridge between the colloidal and life sciences [7,[16][17][18][19].
Generally, the amphiphilic copolymers could selfassemble to form well-defined nanostructures with reversed and stimuli-responsive features. Taking advantage of dynamic nature of noncovalent interactions, a diversity of synthetic systems were developed for schizophrenic self-assembly behavior [20][21][22][23]. Stuart and co-workers used diblock copolymers, oligoligands, and metal ions to devise inverted nanoparticles with excellent salt stability and magnetic relaxation properties [24,25]. However, the syntheses of amphiphilic macromolecules by traditional covalent chemistry are typically time consuming and cost intensive. Along this line, the widespread attention has been paid to nature polymers with abundant functional groups. Especially, chitosan is considered as outstanding candidates, since it is rich groups, good biocompatibility, biodegradability, and wide source. To date, chitosan has been employed to assemble with functional molecules through the noncovalent interaction in solution resulting in supramolecular structures with various morphologies because of the convertibility between amino and ammonium salts [26,27]. Numerous chitosan-based supramolecular assemblies with special architectures and functions have been synthesized and investigated for applications in versatile fields, such as drug delivery, bioimaging, biosensing, antibacterial, wastewater treatment, and packing [28][29][30]. Given the crucial roles of noncovalent interactions in biological systems, we hypothesized that it was highly beneficial to construct new schizophrenic systems on the basis of the chitosan, which may be favorable for developing novel biomimetic materials.
The traditional organic fluorescent materials show significantly light-emitting behaviors in dilute solutions but weak emission in concentrated solutions, which is generally considered a thorny obstacle to the practical applications. The concept of aggregation-induced emission (AIE) [31] or aggregation-induced emission enhancement (AIEE) [32] has emerged to be a powerful strategy for the design of novel types of fluorescent devices [33,34]. In order to have an indepth understanding of working mechanism, typical AIEactive molecules such as tetraphenylethene (TPE) have been employed to develop assemblies of various structures [35]. Obviously, aggregates formed by the noncovalent interactions between the multivalent polyelectrolytes and AIE molecules can provide an effective way toward the formation of reversible configuration and luminescent system. Therefore, it is expected that the applications of these "smart" assemblies have been expanded into many fields, such as bioprobes, fluorescence sensors, bioimaging, and organic lightemitting diodes [21,24,36].
In the present work, we report the fabrication of a novel supramolecular system comprising two functional components, namely chitosan and TPE derivative bearing four carboxyl acid moieties (TPE-(N-COOH) 4 ), that can form two distinct micelles with fully inverted structures in response to pH changes (Scheme 1). The chitosan provides biological activity to micelles, while TPE-(N-COOH) 4 endows them controllable fluorescence properties. Under mild alkaline conditions, the electrostatic association between the chitosan bearing amino cations and the carboxylate anions of TPE-(N-COOH) 4 can generate PIC micelles. Significantly, along with the PIC micelle formation, the fluorescence emission of TPE moieties was gradually "turn on" because of the restriction of intramolecular motion. Alternatively, after lowering the solution pH to 2, the protonated carboxyl groups of tetracarboxylated TPE serving as hydrogen bonding donor or acceptors can interact with the hydroxyl or amino groups in the chitosan to constitute the HBC micelles. Furthermore, the pH-mediated conformational transition of micelles enhanced the fluorescence emission of the system, presumably due to the deep fixation of the TPE chromophore within HBC micelles. Especially, the pH-dependent transition with reversed micellar structures and tunable fluorescence performance is fully reversible, as shown in Scheme 1.
Scheme 1: Schematic illustration of self-assembly between TPE-(N-COOH) 4 and chitosan via different interactions to form PIC and HBC micelles, respectively, under acid-based-controlled condition. The pH-mediated transformation of PIC to HBC micelles marked by AIE enhancement is reversible.  4 ). Synthesis schemes employed for the preparation of TPE-(N-COOH) 4 are shown in Scheme 2. 4,4 ′ ,4 ″ ,4 ″ ′-(ethene-1,1,2,2-tetrayl)tetraaniline (TPE-AM) was synthesized at first following the procedures reported previously with minor modifications [37]. Briefly, 0.8 g of 4,4 ′ -diaminobenzophenone was dissolved in 35 mL concentrated hydrochloric acid under heating and stirring. Then, 3.0 g of Tin powder was added, and the reaction mixture was stirred at 75°C for 6 h. After cooling, the mixture was filtered and washed with NaOH (1 M) and water. The product was obtained as a yellowish powder (685 mg, yield~85%) after dried under vacuum. TPE-AM was then reacted with chloroacetic acid to afford the target product, TPE-(N-COOH) 4 . In a typical procedure, 1.0 g of TPE-AM and 2.0 g chloroacetic acid were dissolved in 15 mL toluene under stirring, and triethylamine (3 mL) was then added. The mixture was heated to 85°C and kept stirring for 6 h. Thereafter, the resulting mixture was cooled to room temperature and washed with saturated NaHCO 3 aqueous solution. The product was collected by filtration, washed twice with ether, and dried to afford a yellowish power (830 mg) in 53% yield. The structure of TPE-AM and TPE-(N-COOH) 4 was confirmed by 1 H NMR and FT-IR spectra. 2.4. Characterization. All 1 H NMR spectra were recorded on a Bruker AVANCE III HD spectrometer with tetramethylsilane as internal standard. Fourier transform infrared (FT-IR) spectra were performed on a Nicolet 510p spectrometer. Morphology of the micelles was observed using transmission electron microscopy (TEM) (HT-7700, Hitachi) and scanning electron microscopy (SEM) (S-4800, Hitachi). A Brookhaven BI9000 AT system (Brookhaven Instruments Co., USA) was employed for dynamic light scattering (DLS) measurements, and the hydrodynamic diameter and distribution of micelles were computed. The zeta potential of the micelles was obtained with a Zetaplus (Brookhaven Instruments Corporation). All results were the average of triplicate measurements. The photoluminescence (PL) spectra were collected using an RF-5301PC spectrofluorometer (Shimadzu). The slit widths were set at 5 nm for both excitation and emission.

Preparation and Characterization of PIC Micelles.
As one of the typical AIE-active molecules, TPE derivatives have been widely used in sensing field with analytes ranging from thiols, gas, heavy-metal ions to biomacromolecules [22,38]. Here, to build fluorescent micelles based on the electrostatic interaction, tetracarboxylated TPE derivative (TPE-(N-COOH) 4 ) was synthesized at first via two steps according to Scheme 2. The 1 H NMR spectra of the synthesized TPE derivatives are shown in Figure 1. As marked in Figure 1, the characteristic signals from TPE-AM (δH a 6.82 ppm, 8H ArH; δH b 6.43 ppm, 8H ArH; and δH c 3.54 ppm, 8H -NH 2 ) and TPE-(N-COOH) 4 (δH d 6.57 ppm, 8H ArH; δH e 6.26 ppm, 8H ArH; δH f 4.84 ppm, 8H -CH 2 COO-; and δH g 3.32 ppm, 4H -NHCH 2 -) can be clearly observed, indicating the successful synthesis of the compounds. The three peaks of TPE-AM at 6.82 ppm, 6.43 ppm, and 3.54 ppm were assigned to the protons of the phenyl ring (a and b) and amino group (c), respectively. By comparison, the 1 H NMR spectrum of TPE-(N-COOH) 4 shows a new peak at 4.84 ppm, corresponding to the protons on the methylene (f) of the acetyl group.
Being a cationic biopolymer bearing amino group, chitosan chains undergo counterion complexation in aqueous solution with many multivalent anionic substances, such as EDTA and poly(acrylic acid) [26,27], to fabricate colloidal aggregates. Here, the inter-and intramolecular linages occurred between carboxyl groups from TPE-(N-COOH) 4 and positively charged amino groups of WCS, resulting in the formation of micelles with hydrophilic WCS coronas. Advances in Polymer Technology of a broader size distribution. Also, the zeta potential of the micelles shows a negative value in the range of feed ratio observed. The micelle structure was also confirmed by FT-IR analysis, as shown in Figure 2(b). For PIC micelles, the intensities of amide band II at 1521 cm -1 and amide I at 1637 cm -1 , which can be observed clearly in pure chitosan, decrease dramatically, and two new characteristic peaks appeared at 1776 cm -1 and 1656 cm -1 , which were assigned to the absorption of the carboxyl groups of TPE-(N-COOH) 4 (the absorption peak of carboxyl groups in pure tetracarboxylated TPE appears at 1622 cm -1 ), and the NH 3 + absorption of WCS, respectively, are observed. These changes in the FT-IR spectrum indicate that the carboxylic groups of TPE-(N-COOH) 4 are dissociated into COOgroups which complex with protonated amino groups of WCS through electrostatic interaction to form the PIC micelles.
Particle morphology was then studied by electron microscopy. The SEM and TEM observations demonstrated that the obtained micelles were presented as spherical nanoparticles at the isoelectric point (Figures 2(c) and 2(d)). It is apparent that the nanoparticle size observed under SEM and TEM is smaller than the DLS result due to the shrinking in dry state. Hence, we can conclude that the most stable and compact PIC micelles can only be fabricated at the isoelectric point, which is consistent with the results of predecessors [20]. Additionally, due to the restriction of the intramolecular rotation of TPE-(N-COOH) 4 moieties within the PIC core, the self-assembling of micelles could be understood by the turn-on emission of TPE chromophores. Specific fluorescence titration spectroscopy with varying charge ratios revealed that the maximum emission intensity was achieved at the [NH 3 + ]/[COO -] molar ratio of 1.0, as shown in Figure 3. The result is in accordance with the trend of the hydrodynamic diameter variation of micelles.
Therefore, we infer that the fluorescence intensity of the PIC micelles is strongly affected by the degree of electrostatic interaction between chitosan and tetracarboxylated TPE.

Response Behavior of Micelles.
In view of the fabrication of PIC micelles, the research on the temperature response of micelles will be conducive to the development of their applications. Subsequently, the properties of self-assembled micelles against temperature variations were further conducted. Figure 4 shows the changes of the micelle size and light scattering intensity with temperature were monitored with the samples inside the DLS cuvette. On increasing the temperature of PIC micelle solution formed at the isoelectric point from 20 to 80°C, the scattered intensity underwent a pronounced drop though the hydrodynamic diameters kept almost constant over the same temperature range. This result indicates that the PIC micelles underwent gradual disintegration subjected to temperature rise. In other words, high temperature weakens the electrostatic interaction between opposite charges, leading to disintegration of micelles. As mentioned earlier, the electrostatic interaction between ammonium groups and carboxylate anions within micelles restrains the rotation of the phenyl groups of TPE-(N-COOH) 4 because of the spatial constraint, giving the enhanced fluorescence emission. Thus, it can be found that the fluorescence intensity of TPE moieties within the cores of PIC micelles gradually decreased subjected to temperature rise, as shown in Figure 4(c). On the other hand, high temperature weakens the restriction of rotation of the phenyl groups of TPE, resulting in a decrease of the fluorescence emission. Therefore, the PIC micelles incorporating TPE-(N-COOH) 4 fluorophores with unique temperature responsive characteristics could be employed as thermal sensors with tunable fluorescence emissions.

Advances in Polymer Technology
As mentioned above, the increase in temperature can significantly weaken the stability of PIC micelles, leading to the disassembly of the WCS micelles and loss of TPE fluorescence. Based on similar considerations, it was postulated that the PIC micelles would probably disassemble with the decrease of solution pH as well, especially when the pH was lower than the pKa of carboxyl groups, due to protonation of carboxylate anions and thus eradication of electrostatic interaction between cationic chitosan and TPE-(N-COOH) 4 . To further confirm the assumption, the performance of the micelles under acidic milieu was then evaluated. Under acidic condition, the TEM showed that the morphology of micelles became irregular but the particle size was significantly decreased (Figure 5(a)). Specifically, the hydrodynamic diameter of the micelles decreased from 65 nm to~26 nm upon pH drop from 8 to 2. Key determinants in the changes of particle size and intramicellar interaction were the ionization degrees of WCS and TPE-(N-COOH) 4 in different pH solution. The pKa values of carboxyl groups in TPE-(N-COOH) 4 and WCS are 2.6 and 6.5, respectively [26,39]. At low pH, the carboxyl groups of tetracarboxylated TPE was almost protonated. It is noteworthy that the WCS with proton sponge effect can maintain complete or partial ionization in a wide pH range [40]. This result was confirmed by zeta potential measurements, as shown as in Figure 5(b). It can be seen that zeta potential of the micelles gradually increased from -23.9 mV to 42.2 mV with the decrease of pH from 8 to 2. The negative zeta potentials at a pH above 7.0 were mostly caused by the adsorption of anions, such as the OH - [39]. Meanwhile, the fluorescence intensity of the micellar solutions exhibited a further 2.13-fold increase as compared to that of PIC micelles at pH = 8, as schematically shown in Figure 5 6 Advances in Polymer Technology leads to a fluorescence enhancement feature but also a slight blue shift of 14 nm. The AIE enhancement feature accompanied by blue shifts occurred generally in systems containing small molecules with heteroatom-based groups [41].
To elucidate the underlying mechanism on aggregationinduced emission enhancement and the hydrodynamic diameter decrease subject to pH drop from 8 to 2, the fluorescence intensity changes of the pure TPE-(N-COOH) 4 solution under varying pH were detected as the control ( Figure 5(d)). Upon decreasing pH from 8 to 2, the fluorescence intensity of TPE-(N-COOH) 4 fluorophores demonstrated a cumulative 6.25-fold increase, higher than that of the mixture of WCS/TPE-(N-COOH) 4 at the charge molar ratio (+/-) of 1.0 with the same TPE-(N-COOH) 4 concentration. Moreover, there was no shift of the emission band while the fluorescence was enhanced. It is concluded that there was a specific interaction between chitosan and pro-tonated TPE-(N-COOH) 4 in acidic milieu, which prevented the hydrophobic TPE-(N-COOH) 4 molecules from precipitation. Considering the formation of much smaller assemblies at pH = 2, a possible common mechanism was introduced, the protonated TPE-(N-COOH) 4 served as the hydrogen bond donors or acceptors interacted with chitosan to form HBC micelles, on the surface of which positively charged WCS stabilized the hydrogen-bonded inner cores. Therefore, the hydrophobicity of AIE molecules and hydrogen bonding interaction within the HBC cores impeded the intramolecular rotation of TPE moieties and further boosted the fluorescence, because of the elimination of electrostatic repulsion of negatively charge TPE-(N-COOH) 4 under basic condition and the formation of more compact micelles with smaller size. Interestingly, the pH-mediated transitions in fluorescence intensity and hydrodynamic diameter were reversible with pH cycling between 8 and 2. The results  Figure 6 illustrate that the TPE-(N-COOH) 4 moieties were fully protonated and hydrophobic at pH = 2, and the electrostatic interaction disappeared between the neutral TPE-(N-COOH) 4 and cationic chitosan. In fact, the micelles were treated five times with the acid-based cycle, and both the fluorescence intensity and diameter were highly reversible under the acid-based-controlled condition.

Conclusions
In summary, nature cationic chitosan polyelectrolyte and TPE-(N-COOH) 4 with an AIE characteristic were used to construct fascinating micelles, exhibiting pH reversible transition between PIC and HBC aggregates. The highlight of this structure is the use of chitosan homopolymer not only as stabilized hydrophilic coronas but also as one indispensable building block of the aggregate cores. Additionally, under mildly basic condition (such as pH = 8), the positively charge chitosan can electrostatic interact with deprotonated TPE-(N-COOH) 4 to form PIC micelles, whereas the micelles were transformed to HBC with fully inverted micellar structures containing hydrogen upon pH decrease from 8 to 2. The diameter and fluorescence emission of the PIC micelles can be manipulated by changing the [NH 3 + ]/[COO -] molar ratio. Moreover, the fluorescence emission of PIC micelles weakened as temperature rises, reflecting their temperature responsiveness. It is noteworthy that pH-driven formation of HBC with flipped micellar structures compressed the particle sizes and further intensified the fluorescence emission of TPE-(N-COOH) 4 moieties thanks to the elimination of electrostatic repulsion of carboxyl groups within the micelles. Furthermore, the pH-actuated fascinating transformation between PIC and HBC micelles with AIE enhancement feature was completely reversible under the control of acid-based cycle. The surface of the micelles was chemically active and provided the functional sites with chemical groups for subsequent chemical reactions (e.g., binding of biomolecules). Accordingly, this robust architecture of micelle may be expected to hold great potential for many different applications in a variety of fields including, for example, colloids, pH sensors, biomarkers, and antimicrobial materials.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declare that they have no conflicts of interest. Advances in Polymer Technology