MathJax.Hub.Config({tex2jax: {inlineMath: [['$','$'], ['\$','\$']]}}); Superparticles Formed by Amphiphilic Tadpole-like Single Chain Polymeric Nanoparticles and Their Application as an Ultrasonic Responsive Drug Carrier
 Chinese Journal of Chemical Physics  2017, Vol. 30 Issue (2): 211-218

#### The article information

Li Jiang, Hui-ya Li, Dao-yong Chen

Superparticles Formed by Amphiphilic Tadpole-like Single Chain Polymeric Nanoparticles and Their Application as an Ultrasonic Responsive Drug Carrier

Chinese Journal of Chemical Physics, 2017, 30(2): 211-218

http://dx.doi.org/10.1063/1674-0068/30/cjcp1611218

### Article history

Received on: November 15, 2016
Accepted on: November 30, 2016
Superparticles Formed by Amphiphilic Tadpole-like Single Chain Polymeric Nanoparticles and Their Application as an Ultrasonic Responsive Drug Carrier
Li Jiang, Hui-ya Li, Dao-yong Chen
Dated: Received on November 15, 2016; Accepted on November 30, 2016
The State Key Laboratory of Molecular Engineering of Polymers and Department of Macromolecular Science, Fudan University, Shanghai 200433, China
Author: Dao-yong Chen, E-mail:chendy@fudan.edu.cn
Abstract: Herein, we report self-assembly of tadpole-like single chain polymeric nanoparticles (TPPs) and the ultrasonic response of the resultant superparticles. The TPPs are with an intramolecularly crosslinked poly (2-(methacryloyloxy) ethyl pent-4-ynoate)-r-poly (hydroxyethyl methacrylate) (PMAEP-r-PHEMA) chain as the "head" and a poly (2-(dimethylamino) ethyl methacrylate (PDMAEMA) linear chain as the "tail", and are pre-pared simply and efficiently by Glaser-coupling of the pendant alkynes in the PMAEP-r-PHEMA block in the common solvent methanol. The formation of the TPPs was confirmed by gel permeation chromatograph, nuclear magnetic resonance spectroscopy, dynamic light scattering, static dynamic scattering, and transmission electron microscopy. In aqueous solution, the amphiphilic TPPs could self-assemble into regular superparticles, driven by ag-gregation of the hydrophobic "heads". Since in the structure there is no chain entanglement and the embedding of PDMAEMA chains disturb close-packing of the "heads", the superpar-ticles are responsive to a low-energy ultrasonic vibration, as evidenced by greatly enhanced release of the functional molecules from the superparticles by treatment of a low-energy ultrasound. Therefore, the superparticles should be very promising in the use as the drug carriers that can be manipulated from a long distance, considering that ultrasonic energy can be focused at a small area in a relatively long distance from the ultrasound-radiating source.
Key words: Single chain polymer nanoparticles     Superparticles     Ultrasonic response     Drug carrier
Ⅰ. NTRODUCTION

In recent years, solution-based self-assembly of nanoparticles has attracted great attention, because it can generate superparticles that are different from molecular assemblies (e.g., micelles and vesicles) in both structures and properties [1-6]. Generally, for the self-assembly in solutions, anisotropic interactions between the nanoparticles are necessary, which, in most cases, occur among the anisotropic nanoparticles that have chemical anisotropies on the surface [7-12]. As one kind of the anisotropic nanoparticles, tadpole-like single chain polymeric nanoparticles (TPPs) with a structure that a sphere is tethered with a single linear polymer chain are especially interesting since they are among a few anisotropic nanoparticles that can be conveniently and efficiently prepared; TPPs can be prepared by simply crosslinking one of the blocks of a diblock copolymer [13-17]. For solution-based self-assembly, TPPs have features of both flexible block copolymers and solid nanoparticles. The resultant superparticles formed in the selective solvent of the "tail" have not only the unique property and structure but also the good dispersibility required by any further solution-based application. However, although there are a lot of elegant studies related to TPPs [18-22], most of the studies focused on preparation of TPPs and the applications where TPPs were used in the form of small nanoparticles [23, 24]. The studies of self-assembly of TPPs, especially those of properties of the superparticles, are very limited [25-29]. In our previous study [30], we reported that the self-assembly of TPPs in the selective solvent for the "tails" formed the superparticles that dissociated into individual TPPs when being treated with ultrasonic vibration. To the best of our knowledge, this is the first example of ultrasonically induced dissociation of polymeric nanoparticles into the building blocks. However, in a previous study, the superparticles could disperse only in organic solvents and respond only to relatively high ultrasonic energy. The possible applications of the ultrasonic responsive superparticles remain unexplored. Considering that ultrasonic energy can be focused at a small area in a relatively long distance in front of the ultrasound-radiating source, ultrasonically responsive superparticles should be very promising in the use as a nanocarrier for drug delivery [31, 32]. Therefore, the superparticles that are water-dispersible and biocompatible, and can be used as the drug carrier responsive to low-energy ultrasound are very desirable. Herein, we report TPPs prepared by intrachain crosslinking of the pendant alkynes of PMAEP-r-PHEMA block of the diblock copolymer (PMAEP-r-PHEMA)-b-PDMAEMA, where PMAEP-r-PHEMA is poly (2-(methacryloyloxy) ethyl pent-4-ynoate)-r-poly (hydroxyethylmethacrylate), and PDMAEMA is poly (2-(dimethylamino) ethyl methacrylate), in the common solvent methanol. The amphiphilic TPPs are biocompatible [33] and able to self-assemble into regular superparticles that are well dispersible in aqueous solution. It is significant that, when subjected to a very gentle ultrasonic treatment, the release of the molecules encapsulated in the superparticles is remarkably enhanced. These make the superparticles very promising in the use as a nanocarrier for long distance controlled drug release.

Ⅱ. EXPERIMENTS A. Materials

4, 4'-azobis (4-cyanopentanoic acid) (ACVA) and 2, 2'-azobis (2-methylpropionitrile) (AIBN) were recrystallized from anhydrous ethanol twice before use. Sodium acetate, di (thiobenzoyl) disulfide, 4-pentynoic acid, dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP), tetramethyl-ethylene-1, 2-diamine (TMEDA), 8-anilino-1-naphthalenesulfonate (ANS) and triethylamine (TEA) were purchased from Sigma-Aldrich and used as received. 2-Hydroxyethyl methacrylate (HEMA, 98%, Aldrich) was purified twice by passing the monomer through a column filled with basic alumina to remove the inhibitor. 2-(Dimethylamino) ethyl methacrylate (DMAEMA, 99%, Aldrich) was purified by distillation under reduced pressure. Copper (Ⅰ) chloride (CuCl) was washed with acetic acid, followed by washing with methanol to remove impurities. DMF was dried with activated 4 Å molecular sieves overnight and distilled under reduced pressure. Hydrochloric acid and other solvents were purchased from Sinopharm Chemical Reagent Co. Ltd. and used as received.

B. Instruments and characterizations

Proton nuclear magnetic resonance (1H NMR) measurements were recorded with a Bruker Advance 400 spectrometer. Gel permeation chromatography-multiangle laser light scattering (GPC-MALLS) analysis was carried out with a Waters Breeze 1525 GPC analysis system with two PL mix-D columns, combined with a Wyatt Dawn Heleos Ⅱ LS detector, using DMF with 0.5 mol/L LiBr as eluent at the flow rate of 1 mL/min at 80 ℃, and PEO calibration kit (purchased from TOSOH) as the calibration standard. Astra software (Wyatt Technology Corp.) was used to determine the molecular weight characteristics from injected mass and assuming 100% mass recovery. Dynamic light scattering (DLS) measurements were performed using an ALV-5000 laser light scattering spectrometer. Before the measurements, all the samples were filtered through 0.45 μm Millipore filters (hydrophilic Millex-LCR, PTFE) to remove dust and then kept at 25 ℃ for 5 min. The cumulant mode of DLS analysis was applied to obtain the 〈Rh〉 values. TEM observations were conducted on a Philips CM120 electron microscope at an accelerating voltage of 80 kV. TEM samples were prepared by depositing a drop of the sample suspension onto a carbon-coated copper grid. The excess liquid on the copper grid was absorbed by filter paper immediately after the deposition. Then, the copper grid was allowed to dry under ambient conditions. The TPPs were stained by RuO4 for 15 min. Fluorescence spectra were measured by using a FLS920 spectrophotometer.

C. Synthesis of RAFT agent 4-cyanopentanoic acid dithiobenzoate (CPADB)

Freshly distilled ethyl acetate (20.0 mL), dry 4, 4'-azobis (4-cyanopentanoic acid) (3.4 g, 12.2 mmol) and di (thiobenzoyl) disulfide (2.5 g, 8.1 mmol) were added to a 100 mL round-bottomed flask. The mixture was heated at reflux temperature for 18 h. After the reflux, the reaction solution was evaporated in vacuo to remove the ethyl acetate. The crude product was purified by flash chromatograph on silica, eluting with ethyl acetate/petroleum ether (V/V=1:1). The red fractions were collected and dried over anhydrous sodium sulfate overnight. The solvent was removed under vacuum, and the red oily residue was placed in a freezer at-20 ℃, whereupon it crystallized. The target compound CPADB was recrystallized from benzene.

1H NMR (CDCl3): δ/ppm 1.86 (s, 3H), 2.45 (m, 2H), 2.75 (m, 2H), 7.42 (m, 2H), 7.57 (m, 1H), 7.91 (dd, 2H).

D. Synthesis of macro RAFT agent PHEMA and block copolymer PHEMA-b-PDMAEMA

CPADB (26.1 mg, 0.094 mmol), HEMA (9.09 mL, 75 mmol), 15 mL DMF, and 51 mL sodium acetate buffer (pH=5.2) were mixed in a 250 mL round-bottomed flask. Then, to the mixture, 5.25 mg ACVA was added by pipetting 3.75 mL of the freshly prepared ACVA solution in methanol (the concentration was 1.4 mg/mL) into the flask. After having been purged with Ar for 30 min, the sealed flask was immersed into an oil bath set at 70 ℃ for 2.5 h. Then, the reaction mixture was quenched in liquid nitrogen and exposed to air. The product was precipitated by excess ether, washed several times, and dried in a vacuum oven for 24 h.

To synthesize the block copolymer, 364 mg macro RAFT agent PHEMA dissolved in 2 mL DMF, 1.6 mL sodium acetate buffer (pH=5.2), 0.56 mg ACVA (0.4 mL ACVA solution in NaAc buffer at 1.4 mg/mL), and the solution containing 2.5 mmol DMAEMA (for preparing the DMAEMA solution, DMAEMA was mixed with NaAc buffer at pH of 5.2 in a flask immersed in an ice bath. Then, HCl was added to adjust the pH value to 5.0) were mixed in a 50 mL round-bottomed flask. The concentration of DMAEMA in the mixture was 0.42 mol/L. Subsequently, the flask was purged with Ar for 30 min and immersed into an oil bath set at 70 ℃. Then, the reaction mixture was quenched in liquid nitrogen and exposed to air after 1.5 h. The block copolymer was purified by dialysis against water, and was dried by lyophilization.

E. Synthesis of polymer precursor (PMAEP-r-PHEMA)-b-PDMAEMA (P0)

Firstly, the thiocarbonyl-thio group at the chain end of the PHEMA-b-PDMAEMA was completely removed by mixing the block copolymer (284 mg) with excess AIBN (5 mg) in 10 mL distilled DMF, followed by purging the mixture with Ar for 30 min and then heating the system at 80 ℃ for 12 h. Secondly, PHEMA block was changed to the PMAEP-r-PHEMA block by random esterification of hydroxyl groups of the HEMA units with 4-pentynoic acid; the esterified HEMA units became the MAEP units and each of the MEAP unit had an alkyne pendant group; the target modification ratio was 30%. For the esterification, the just mentioned mixture was cooled, mixed with 4-pentynoic acid (61.7 mg, 0.63 mmol), DCC (130 mg, 0.63 mmol) and DMAP (7.7 mg, 0.063 mmol), and then heated at 30 ℃ for 48 h. The impurities were removed by dialysis in methanol after the precipitates were filtered out. The solution was concentrated and dried in vacuo in a PTFE vessel.

F. Typical procedure for preparing tadpole-like single chain polymeric particles (TPPs)

The TPPs were prepared by crosslinking the PMAEP-r-PHEMA block of the block copolymer (PMAEP-r-PHEMA)-b-PDMAEMA (denoted as P0) in its common solvent methanol by the reaction of Glaser coupling among pendant alkyne groups of MAEP units. In a typical process for the crosslinking, P0 (50.0 mg, 0.0004 mmol) was dissolved in 20 mL of methanol and then the solution was transferred to a 20 mL syringe. Meanwhile, the catalyst for the coupling reaction was prepared by mixing CuCl (7.5 mg, 0.075 mmol), TMEDA (11.3 μL, 0.075 mmol), TEA (20.8 μL, 0.15 mmol), and methanol (80 mL) together in a 250 mL flask. The crosslinking reaction was conducted by drop-wise addition of the methanol solution of P0 into the 250 mL flask containing the catalyst solution over 6 h at room temperature by using a syringe pump, and further reaction for 24 h at room temperature.

G. Self-assembly of P0 and TPPs in water

Under mild stirring, 4.0 mL of water was added drop-wise into 1.0 mL of methanol solution of P0 or TPPs (1.0 mg/mL) using a micro-syringe at a speed of 5.0 mL/h. Then, the suspensions were dialyzed against water to remove the methanol.

H. Ultrasonic response of the superparticles formed by self-assembly of the TPPs in water, using the micelles of P0 as the control

8-anilino-1-naphthalenesulfonate (ANS) was loaded into the superparticles of the TPPs or the micelles of P0 via co-assembly process. 200 μL of ANS solution (2.0 mmol/L in methanol) was mixed with 1.8 mL of methanol solution of TPPs or P0 at 1.0 mg/mL. Then the same process for self-assembly of P0 or TPPs was conducted; the concentration of ANS in the final suspension was 33 μmol/L. The suspension was dialyzed against ANS saturated water to avoid leaking of the dye during dialysis. All the samples were protected from light to avoid photobleaching.

The obtained suspensions were then treated ultrasonically in an ultrasonic bath (DL-360D) operating at 40 kHz with a power output of 36 W or 72 W for different time.

Ⅲ. RESULTS AND DISCUSSION A. Synthesis and characterization of the block copolymer PHEMA-b-PDMAEMA

As the precursor of diblock copolymer (PMAEP-r-PHEMA)-b-PDMAEMA, the diblock copolymer PHEMA-b-PDMAEMA was synthesized firstly by two-step RAFT polymerizations in mixed solvent of DMF and sodium acetate buffer. Based on GPC measurement (Fig. 1(a)), the macro RAFT agent PHEMA obtained at the first step RAFT polymerization had a number-average molecular weight (Mn.GPC) of 38 kg/mol and a polydispersity index (PDI) of 1.11; the block copolymer PHEMA-b-PDMAEMA produced at the second step had an Mn.GPC of 52 kg/mol and a PDI of 1.18 (Fig. 1(a)). The polymerization degrees for the two blocks were also determined by 1H NMR analysis. As shown in Fig. 1(b), according to the relative signal intensities of peaks of the RAFT agent CPADB (the peaks at 7.90, 7.61, and 7.44 ppm are assigned to the respective Ar-H atoms B, C and A of CPADB, as indicated in inset of Fig. 1(b)) and peak D of ethylene group of the HEMA units (-O-CH2-groups at 4.07 ppm), the polymerization degree of the PHEMA block was calculated to be 524. Similarly, the polymerization degree of the PDMAEMA block was calculated to be 246 according to the relative signal intensities of peaks D and E, which are assigned to ethylene signals in the HEMA and DMAEMA units, respectively. The molecular weights for the PHEMA and PDMAEMA blocks calculated based on the 1H NMR results are consistent with the respective values obtained by the GPC measurements. The final block copolymer was denoted as PHEMA524-b-PDMAEMA246.

 FIG. 1 GPC curves (a) and 1H NMR spectra (b) of the macro RAFT agent PHEMA and the block copolymer PHEMA-b-PDMAEMA. The 1H NMR spectra were recorded in methanol-D4. The inset is the magnified spectrum of the PHEMA in the range from 7.38 ppm to 7.98 ppm.
B. Preparation of the diblock copolymer P0 and the TPPs

As the precursor of the TPPs, the diblock copolymer (PMAEP-r-PHEMA)-b-PDMAEMA (denoted as P0) was prepared by random esterification of a part of hydroxyl groups of HEMA units of the PHEMA block of PHEMA524-b-PDMAEMA246 with 4-pentynoic acid to change the hydroxyl groups into alkyne groups; after the esterification, PHEMA block becomes (PMAEP-r-PHEMA) block, where the alkyne groups are randomly distributed. Due to strong hydrophobicity of alkyne groups, (PMAEP-r-PHEMA) block is hydrophobic and P0 is thus amphiphilic; PDMAEMA is a well-known hydrophilic polymer. The fraction of the HEMA units that had been converted to MAEP units was calculated based on the 1H NMR spectrum of P0. Comparing the spectrum of P0 in Fig. 2(a) with the spectrum of PHEMA524-b-PDMAEMA246 (Fig. 1(b)), we can clearly see that, after the esterification intensity of the ethylene signals of the HEMA units decreased and accompanied by the appearance of new ethylene signals (peaks F and G at 4.39 and 4.24 ppm, respectively) assigned to MAEP. According to the relative signal intensity of the ethylene groups of the MAEP units and that of the HEMA units in the spectrum of P0 (Fig. 2(a)), the fraction of the esterified HEMA units (relative to total amount of the HEMA units before the esterification) was calculated to be 35.7%, which was sufficient to obtain an efficient chain-compaction by crosslinking the MAEP units. P0 was finally determined as (PMAEP188-r-PHEMA336)-b-PDMAEMA246. The GPC curve of P0 in Fig. 2(b) was symmetrical and unimodal, indicating that no considerable coupling reaction of alkynes occurred during the esterification procedure. Mn.GPC (54 kg/mol) and PDI (1.24) of P0 increased slightly due to the introduction of 4-pentynoic acid.

 FIG. 2 1H NMR spectra (a), and GPC (b) and DLS curves (c) of the P0 and the TPPs. The 1H NMR spectra were recorded in methanol-D4.

The PMAEP-r-PHEMA block of P0 was crosslinked by Galser coupling reaction among the alkyne groups of the MAEP units in the common solvent methanol at the concentration of P0 of 0.5 mg/mL; at the concentration, P0 was molecularly solubilized in methanol according to dynamic light scattering (DLS) characterization (Fig. 2(c)). Due to the low concentration of P0 and the protection from the PDMAEMA chains [30], the interchain crosslinking was largely avoided. This was confirmed by further characterizations, as detailed below.

In the case of crosslinking individual polymer chains of a polymer in its dilute solution, it is well-known that when the crosslinking is intramolecular, the hydrodynamic volume of the polymer chains will decrease, and thus the elution time of GPC peak of the polymer (using the same solvent as the eluent) will increase, due to the collapse of the polymer chains after being intramolecularly crosslinked. As exhibited in Fig. 2(b), the GPC peak of the as-crosslinked P0 is unimodal and shifts to longer elution time compared with the GPC peak of P0. This suggests that the crosslinking reaction of P0 mainly occurred intramolecularly. Besides, as indicated in Fig. 2(c), after crosslinking, the 〈Rh〉 of the polymer chains decreases apparently. Since the Z-averaged 〈Rh〉 is very sensitive to the aggregates of larger mass, the apparent decrease in 〈Rh〉 after the crosslinking strongly support the conclusion that the crosslinking reaction of P0 mainly occurred intramolecularly. Additionally, this conclusion was also supported by the measurements of the absolute molecular weights of P0 and the as-crosslinked P0 by multi-angle laser light scattering, which are 97 and 99 kg/mol, respectively.

TEM observations confirmed that single chain particles formed after the crosslinking reaction. As indicated in Fig. 3, after the crosslinking, P0 chains became small particles with a diameter of 5.8±1.5 nm and a relatively narrow size distribution; the small particles were stained by RuO4 before the TEM observations. The size of the particles is close to the size calculated based on the molecular weight of the (PMAEP-r-PHEMA) block (by supposing that the particles are spheres and density of the particles is 1.0 g/cm3, the diameter of the particles is calculated to be 6.2 nm). This also supports the conclusion that the crosslinking reaction mainly occurred intramolecularly.

 FIG. 3 (a) A typical TEM image of the TPPs prepared by the intrachain crosslinking of the (PMAEP-r-PHEMA) block of P0 (stained by RuO4 for 15 min). (b) Size distribution of the TPPs based on TEM observations, obtained by averaging over more than 100 particles.

The as crosslinked-P0 was further characterized by 1H NMR. After the crosslinking reactions, the peaks F and G at 4.39 and 4.24 ppm, which were assigned to the MAEP units and detected in the spectrum of P0, almost disappeared after the crosslinking (as indicated by the circle in Fig. 2(a)). Obviously, after the crosslinking, mobility of protons on the MAEP units was seriously restricted so that the signals of these protons were undetectable by liquid 1H NMR. Meanwhile, the crosslinking remarkably decreases the mobility of protons of the HEMA units in the (PMAEP-r-PHEMA) block, as indicated by apparent decrease in signal intensities of peaks D and H assigned to the PHEMA units (Fig. 2(a)). These results reveal collapsed structure of the (PMAEP-r-PHEMA) block after the crosslinking. Furthermore, it is notable that the intensities of proton signals due to the PDMAEMA block remain unaffected after the crosslinking (peak E in Fig. 2(a)), demonstrating that the PDMAEMA block is not wrapped by the intrachain crosslinking and thus remains solvated after the crosslinking. Therefore, we conclude that the as-crosslinked P0 are tadpole-like single chain polymeric particles with the intramolecularly crosslinked (PMAEP-r-PHEMA) block as the "head" and the PDMAEMA block as the "tail", denoted as TPPs hereafter.

C. Self-assembly of TPPs and ultrasonic response of the resultant superparticles

We confirmed that both P0 and the TPPs were amphiphilic and capable of self-assembly in water. Through addition of 4-fold volume water into methanol solution of P0 or the TPPs (at 1.0 mg/mL), followed by dialysis against water to remove methanol in the system, P0 micelles or superparticles of the TPPs were obtained. P0 micelles are spherical particles with an average diameter of 33 nm according to TEM observations (Fig. 4(b)). The average hydrodynamic diameter 〈Rh〉 of P0 micelles was measured by DLS to be 19 nm (PDI=0.22) (Fig. 4(d)), close to the diameter observed by TEM. Of course, in the P0 micelles, the PMAEP-r-PHEMA block chains form the core and the PDMAEMA block chains form the shell, since the former is hydrophobic and the latter is hydrophilic.

 FIG. 4 (a) Schematic illustration of the respective processes for preparing the TPPs, self-assembly of P0, and self-assembly of the TPPs. (b, c) TEM images and (d, e) DLS curves of P0 micelles (b and d) and the superparticles of the TPPs (c, e) formed in water.

According to TEM observations, the TPP superparticles are spheres with an average diameter of 130 nm (Fig. 4(c)). The 〈Rh〉 of the superparticles measured by DLS is 80 nm (PDI=0.28) (Fig. 4(e)). Judging from the small size of the TPPs, the relatively large size of the superparticles suggests a structure similar to large compound micelles, i.e., a substantial proportion of the TPPs were embedded inside the superparticles. Therefore, the structure of the superparticles is quite different from that of the P0 micelles.

To investigate the structure and property of the superparticles, in comparison with those of the P0 micelles, 8-anilino-1-naphthalenesulfonate (ANS) was encapsulated in the superparticles and the P0 micelles by the co-assembly method and used as the fluorescent probe. ANS has a very weak fluorescence emission at a λmax of 525 nm in water. However, when it is encapsulated in a non-polar environment, the emission will be greatly enhanced and the λmax shifts to a relatively low wavelength. As indicated in Fig. 5(a), the fluorescence emission of ANS in water at pH=6.5 is very weak and the λmax is 525 nm (the black curve). Differently, the fluorescence emissions are relatively strong in the systems of ANS/superparticles (the red curve in Fig. 5(a), λmax=487 nm) and ANS/P0 micelles (the blue curve in Fig. 5(a), λmax=485 nm), indicating encapsulation of ANS in the particles. The superparticles encapsulated much more ANS than the P0 micelles, since the red curve is much larger than the blue curve. It is known that the superparticles resulted from the aggregation of the TPPs with a relatively rigid "head"; the rigidity was revealed by the disappearance of the MAEP signals in 1H NMR spectrum of the TPPs (Fig. 2(a)). Therefore, there should be small stacking voids in the structure of the superparticles, which make the superparticles more capable in encapsulating ANS molecules that have a relative large size, compared with the P0 micelles of which the core should be composed of fully fused polymer chains.

 FIG. 5 (a) Fluorescence spectra of ANS/superparticles in water, ANS/P0 micelles in water and pure ANS in water. pH value is 6.5. Fluorescence spectra of ANS in the superparticles (b) and the P0 micelles (c) in water at pH of 6.5 measured after being treated by the ultrasound of 40 kHz at different output powers for different time.

As mentioned above, the superparticles were stabilized by aggregation of the relatively rigid "heads"; there should have no entanglement among the TPPs within the structure of the superparticles. Besides, there were soluble "tails" embedded within the superparticles, which disturb close-packing of the "heads". This should make the superparticles ultrasonically responsive. To test ultrasonic response of the superparticles using the P0 micelles as the control, the two suspensions of the ANS/superparticles and ANS/P0 micelles were subjected to ultrasonic treatments of 40 kHz at different output powers for different time (Fig. 5(b) and (c)). It is significant that after being treated by the ultrasound of 36 W for 1 min, about half of ANS originally encapsulated within the superparticles was released. Increase in either the output power or the time of ultrasonic treatment has no remarkable effect on the release behavior of ANS from the superparticles (Fig. 5(b)). In contrast, in the system of ANS/P0 micelles, the ultrasonic treatments only slightly increase the release of ANS from the P0 micelles. In our previous study, we confirmed that the superparticles formed by tadpole-like single chain particles in organic solvents could be dissociated after being treated by the ultrasound of higher output power (216 W) for 10 min. In the present study, no dissociation of the superparticles was observed by TEM; the TEM sample was prepared by transferring a drop of the suspension under ultrasonic treatment to the copper grid very quickly, freezing the copper grid with the sample on it promptly by liquid nitrogen, and freeze-drying the TEM sample in a freeze-dryer at-50 ℃. Therefore, the release of ANS in response to the gentle ultrasonic treatments should be caused by the change in the structure of the superparticles. Although no dissociation of the superparticles occurred, the gentle ultrasonic treatments induced the structural change of the superparticles that caused the release of the ANS encapsulated, which should be accounted for by the abovementioned structural features of the superparticles. Differently, in the core of the P0 micelles, there should be entanglements among the core-forming PMAEP-r-PHEMA linear chains. Besides, the PMAEP-r-PHEMA linear chains are flexible enough to fuse together with each other. Therefore, the gentle ultrasonic treatments have no remarkable effect on structure of the P0 micelles as well as release behavior of the ANS in the micelles (Fig. 5(c)). To the best of our knowledge, the present study represents the first example of drug-release from polymeric nanoaggregates in response to ultrasonic treatment.

Ⅳ. CONCLUSION

In summary, we prepared (PMAEP188-r-PHEMA336)-b-PDMAEMA246 and well-defined TPPs via simply intramolecularly crosslinking (PMAEP188-r-PHEMA336) block of the block copolymer. Both the block copolymer and the TPPs are amphiphilic and form regular spherical micelles and superparticles, respectively, in water. The superparticles can encapsulate much more ANS molecules relative to the micelles; due to existence of stacking voids among the "heads" of the TPPs, the superparticles are more capable of encapsulating ANS molecules that have a relative large size. It is very significant that the superparticles are sensitive to a gentle ultrasonic treatment, as indicated by the greatly enhanced release of ANS molecules from the superparticles when a gentle ultrasonic treatment was applied. These should make the superparticles very promising in the use as long-distance controllable nanocarrier for controlled drug release, considering that ultrasonic energy can be focused at a small area in a relatively long distance from the radiating source.

Ⅴ. ACKNOWLEDGMENTS

This work was supported by the National Natural Science Foundation of China (No.21334001 and No.91127030).

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