Cross-Linking Induced Self-Organization of Polymers into Degradable

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Crosslinking Induced Self-organization of Polymers into Degradable Assemblies Conghui Yuan, Bihong Hong, Ying Chang, Jie Mao, Yang Li, Yiting Xu, Birong Zeng, Weiang Luo, Jean-Francois Gerard, and Lizong Dai ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b02252 • Publication Date (Web): 07 Apr 2017 Downloaded from http://pubs.acs.org on April 8, 2017

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ACS Applied Materials & Interfaces

Crosslinking Induced Self-organization of Polymers into Degradable Assemblies Conghui Yuan †‡*, Bihong Hong †‡, Ying Chang †‡, Jie Mao†‡, Yang Li†‡, Yiting Xu†‡, Birong Zeng †‡, Weiang Luo†‡, Jean-François Gérard §, Lizong Dai †‡* † College of Materials, Xiamen University, Xiamen, 361005, China ‡ Fujian Provincial Key Laboratory of Fire Retardant Materials, College of Materials, Xiamen University, Xiamen, 361005, China § INSA de Lyon, IMP, Villeurbanne F-69621, France KEYWORDS: Self-assembly; Crosslinking polymers; Degradation; Stimuli-response; Cell uptake

ABSTRACT: Covalently stabilized polymer assemblies are normally fabricated from the selfassembly of polymer chains followed by a crosslinking reaction. In this report, we show that a crosslinking-induced self-assembly approach, in which boronate crosslinking sites are formed by the condensation reaction between boronic and catechol groups, can organize polymer networks into uniform assemblies. Self-assembly of these boronate crosslinked polymer networks adopts two different driving forces in water and methanol solutions. Hydrophobic aggregation of polymer networks in water solution affords spherical assemblies, while B-N dative bond formed

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between boronate and imine functionalities in methanol solution organizes the polymer networks into bundle-like assemblies. We not only demonstrate the intrinsic stimuli-responsive degradability of these crosslinked assemblies, but also show that their degradation can cause a controllable release of guest molecules. Moreover, bundle-like assemblies with rough surface and exposed boronate functionalities exhibit dramatically higher cell penetration capability than the spherical assemblies with smooth surface and embedded boronate functionalities.

INTRODUCTION Covalently crosslinked polymer assemblies possess an attractive capability of stable encapsulation of guests, as the three dimensional polymer networks can sequester molecules and prevent them from leakage

1-3

. To achieve a convenient encapsulation of guest, the crosslinking

reaction of the assemblies should be carried out in mild conditions such as room temperature, neutral pH, catalyst free and environmental friendly solvent

4, 5

. When targeting drug loading

applications, the functionalities for the crosslinking reaction need to be more elegantly designed to avoid side reactions with drugs as well as improve the biocompability of the assemblies. To meet these requirements, incorporation of specific functionalities that can induce the crosslinking of polymer chains during the polymer self-assembly has been demonstrated to be a relatively simple and effective route 6-10. Polymer assemblies crosslinked with irreversible covalent bonds are too stable to exhibit responding, adapting or evolving behaviors. Therefore, dynamic chemical interactions that are cleavable in response to external stimuli have emerged as promising candidates for the creation of decrosslinkable polymer assemblies

11-13

. During the past decades, numerous dynamic

chemical interactions including hydrazone 14, 15, boronate 16, 17, imine 18-21, disulfide bond 5, 9, 22-24 and metal-ligand coordination

25-28

have been utilized to crosslink and stabilize the polymer


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assemblies. Based on these reversible crosslinkers, smart assemblies that are decrosslinkable in response to biologically relevant stimuli such as enzyme, carbohydrate and weak acidity have been developed to realize controlled and targeted drug release. Among these dynamic chemical interactions, boronates, especially those derived from the condensation reaction between boronic acid and catechol functionalities are even more attractive, as they are cleavable under the stimuli of both carbohydrate and weak acidity 29, 30. Normally, fabrication of crosslinked polymer assemblies includes two indispensable steps: (i) aggregation induced by amphiphilicity difference of polymer segments and (ii) crosslinking of the aggregates utilizing the reactive anchors appended on the polymer chains. From this perspective, assemblies should already be formed before the crosslinking reaction. Herein, we show that a synergetic approach in which the crosslinking reaction between a catechol functionalized polymer and a boronic acid containing polymer can easily bring the as-formed boronate polymer networks together to form uniform assemblies. The morphology of the assemblies has found to be highly dependent on the reaction solution. Spherical assemblies are formed in water solution, while bundle-like assemblies are created in methanol solution. These boronate crosslinked assemblies are degradable in response to D-glucose or weak acidity, and therefore cause an efficient release of guest molecules. Interesting, bundle-like assemblies have dramatically enhanced cell penetration capability in comparison with the spherical assemblies.

EXPERIMENTAL SECTION Materials: Methacryloyl chloride, 3, 4-dihydroxybenzaldehyde, 4-formylphenylboronic acid, triethylamine, p-phenylenediamine, polyethylene glycol monomethyl ether methacrylate (MAPEG, MW ~475), 2,2′-azobis(2-methylpropionitrile) (AIBN), 1,1'-dioctadecy l-3,3,3',3'-


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tetramethylindocarbocyanine perchlorate (DiI) and other conventional reagents were obtained from commercial sources and were used as received. Characterization: NMR spectra of synthetic monomer and resultant polymers in solutions were measured on a Bruker ARX 400MHz spectrometer. DLS measurements were performed on a Malvern Nanozetasizer. UV/vis absorption data of the samples were acquired in solutions by using UV spectrophotometry (Unico UV/vis 2802PCS). TEM measurements and electron diffraction experiment were performed with a JEM2100 at an acceleration voltage of 200 kV. To prepare the TEM samples, a small drop of sample solution was deposited onto a carbon-coated copper electron microscopy (EM) grid and then dried under room temperature at atmospheric pressure. SEM imaging and energy-dispersive X-ray spectroscopy (EDX) analyses were performed using a Hitachi SU-7 SEM instrument attached with an EDX (INCA, Oxford Instruments). The fluorescence emission spectra were measured by a FLS920 Fluorescence Lifetime and Steady State Spectrometer, fluorescent dye DiI was excited at wavelength 520 nm. Synthesis of N-(4-aminophenyl)methacrylamide (APMA): Methacryloyl chloride (2.19 g, 21.0 mM) and p-phenylenediamine (2.16 g, 20.0 mM) were dissolved in 10 mL and 50 mL of dichloromethane (DCM), respectively. Triethylamine (2.2 g, 22.0 mM) was added to the solution of p-phenylenediamine. Then, methacryloyl chloride DCM solution was added dropwise into the p-phenylenediamine solution at 5 oC. After stirring at room temperature for 12 h, the reaction mixture was washed with NaOH solution two times and with water three times to reach a neutral pH. The oil phase containing the key product was collected and dried with sufficient MgSO4. The crude product (yellow solid) was obtained after the removal of solvent by rotary evaporation. The pure product was purified by passing through a column chromatography using silica gel as stationary phase and mixture of ethyl acetate/hexane (2:1) with 2.0 vol%


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triethylamine as eluent. Yield: 58 %. 1H-NMR (400 MHz, DMSO-D6) δ (ppm): 9.35 (s, 1H), 7.27 (d, 2H), 6.50 (d, 2H), 5.72 (s, 1H), 5.41 (s, 1H), 4.87 (s, 2H), 1.93 (s, 3H); 13C-NMR (300 MHz, DMSO-D6) δ (ppm): 166.41, 145.48, 141.12, 128.3, 122.53, 119.42, 114.07, 19.32. Synthesis of poly (N-(4-aminophenyl)methacrylamide-co-polyethylene glycol monomethyl ether methacrylate) (P(APMA-co-MAPEG)): To a 10.0 mL Schlenk tube, was charged 0.176 g APMA (1.0 mmol), 0.95 g MAPEG (2.0 mmol), 15.0 mg AIBN (0.09 mmol) and 5.0 mL tetrahydrofuran (THF). Three freeze-pump-thaw cycles were performed to eliminate the oxygen in the reaction mixture. After 24 h polymerization at 70 oC in argon atmosphere, the obtained product was purified by dissolving in THF and precipitating in n-hexane for three times. Pure product (yield: 66%) was obtained by drying at room temperature in vacuum for 24.0 h. The content of PAMA in this random copolymer was calculated to be 40 mol% from the 1H NMR spectrum. Incorporation of catechol or boronic functionality onto P(APMA-co-MAPEG): For the synthesis of catechol functionalized copolymer (referred as CP), 0.5 g of P(APMA-co-MAPEG) was dissolved in 10.0 mL of dichloromethane solution. To this mixture, excessive 3, 4dihydroxybenzaldehyde (0.138 g, 1.0 mmol) in 2 mL of methanol solution was added dropwise. After 6 h reaction at room temperature under vigorous stirring, the solution was concentrated by rotary evaporation to give crude product. Then, CP was obtained by dissolving the crude product in THF and precipitating in n-hexane for three times (yield: 71%). Boronic functionalized copolymer (referred as BP) was synthesized by using 4-formylphenylboronic acid (0.150 g1.0 mmol) as starting materials through the same synthetic procedure (yield: 73%). From the 1H NMR spectra of both CP and BP, it was calculated that all the amino groups were transformed into imine bonds, indicating that the disappearance of PAPMA segments.


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Preparation of BP-CP crosslinked assemblies: To fabricate spherical assemblies, BP and CP were dissolved in water to afford polymeric solutions with the same concentration. These two solutions were mixed together with equal volume under stirring at room temperature. Spherical assemblies were obtained after 2 h reaction. Bundle-like assemblies were fabricated through the same procedure using methanol as solvent. The as prepared bundle-like assemblies were collected from methanol solution by centrifugation and redispersed in water for further use. To investigate the effect of polymer concentration on the morphology of assemblies, the total concentration of BP+CP was changed from 0.1, 0.2 to 0.4 mg/mL. Also, this procedure was adopted for the crosslinking reaction between BP and CP in dichloromethane or in a solvent mixture containing 1:1 volume ratio of methanol and water. Encapsulation of guest molecule in BP-CP crosslinked assemblies: During the crosslinking of BP and CP, guest molecule DiI was added into the reaction solution. For bundle-like assemblies, the mixture was dialyzed against water (molecular weight cutoff 3500 g mol-1) for two days to remove methanol. Free DiI was removed by filtration. Cytotoxicity assay: The cell cytotoxicity of both bundle-like and spherical assemblies (derived from 0.2 mg/mL of BP+CP) against MC 3T3-E1 cells and Hela cells was determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The crosslinked assemblies were sterilized with UV irradiation for one day prior to use. The cells were seeded into a 96-well culture plate (Costar, IL, USA) at a density of 5×103 cells/well (0.1 mL). After culturing at 37 °C and 5% CO2 atmosphere for 12 h, the old medium was removed. Then the cells were respectively exposed to 20.0 µL of bundle-like or spherical assemblies with various concentrations for 24 h. After 24 h treatment, the cell viability of the cultured cells was assayed with MTT. The wells were washed with phosphate buffered saline (PBS, pH ≈7.4) twice, then


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10.0 µL of MTT supplemented with 90.0 µL culture medium was added to each well. After cells incubation for an additional 4 h, the medium was removed. To each well, 150.0 µL of DMSO was added to redisperse the cells. The assay plate was agitated on an incubator shaker at 37 °C for 20 min to dissolve formazan crystals. The absorbance of the wells was measured using a Microplate Reader (Model 680; Bio-Rad Laboratories Richmond, CA, USA) with wavelength at 570 nm and reference wavelength at 620 nm. Cell viability was calculated by the following equation:

cell viability =

Abss × 100% Abscontrol

where Abss was the absorbance of the wells containing the cells incubated with the micelles suspension and Abscontrol was the absorbance of the wells containing the cells incubated with PBS. Cellular uptake: Hela cells were seeded in a 6-well plate at a density of 1×105 cells per well at 37 °C for 24 h. Then, the medium was replaced with 1.0 mL of bundle-like or spherical assembly suspensions at a concentration of 0.01 mg mL−1 in serum-free media. The cells were incubated again for different predesigned time at 37 °C, washed with PBS three times and stained with 50.0 µL of Hoechst 33258 for 10 min. The cells were then washed twice with PBS, fixed with 4% formaldehyde for 15 min and further washed twice with PBS. Finally, the cells were observed using a Leica TCS SP5 confocal laser scanning microscopy (CLSM, Leica Microsystems, Mannheim, Germany).

RESULTS AND DISCUSSION Morphology control The crosslinking anchors, viz. boronic and catechol functionalities, were incorporated onto poly

(N-(4-aminophenyl)methacrylamide-co-polyethylene

glycol

monomethyl

ether


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methacrylate) (P(APMA-co-MAPEG), Mn≈12400 and Đ=1.62) through a Schiff base formation reaction to afford BP and CP, respectively (Scheme S1). The amount of crosslinking anchors appended on the polymer chains were ~40 mol%, as calculated from the 1H NMR results (Figure S1-3). BP and CP have high solubility in methanol or water. As the condensation reaction between catechol and boronic groups has a high efficiency to afford boronate

29, 31

, we envision

that the crosslinking reaction between BP and CP may produce crosslinked nano-assemblies. Indeed, Spherical and bundle-like assemblies can be easily fabricated through this route in water and methanol solutions, respectively (Scheme 1). We first performed the crosslinking reaction of BP and CP in water solution at room temperature. TEM images shown in Figure 1a-c indicated that all the assemblies are spherical. An increase in particle size was observed from TEM images and dynamic light scattering (DLS) results (Figure 1d), when increasing the polymer concentration from 0.1, 0.2 to 0.4 mg/mL. Inter-particle crosslinking became evident at polymer concentration 0.4 mg/mL, as revealed by TEM image (Figure 1c) and the dramatic increase of polydispersity index (PDI) in the DLS result (Figure 1d). Moreover, these assemblies seemed to have a darker core and a lighter shell in the TEM images, indicating that the existence of component heterogeneity. EDX line scan analysis of a typical nano-sphere clearly indicated that boron element was evidently concentrated in the assembly core (Figure 1e). These results implied that the boronate esters derived from the condensation reaction of catechol and boronic acid moieties along with the unreacted boronic groups migrated from out-layer into the interior of the assemblies during crosslinking reaction. This is possible as boronate ester and boronic acid moieties are hydrophobic. Therefore, the spherical assembly was probably constructed by a boronate ester core and a PEG chain enriched shell.


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The formation of boronate ester between BP and CP was verified by the concurrent emergence of absorption bands at 1315 (B-O), 1248 (C-O) and 1030 cm-1 (B-C) in the FT-IR spectrum of frozen-dried BP-CP assemblies (Figure S4a). Evident evolution in UV-vis absorption was also observed along with the formation of BP-CP assemblies. While CP showed characteristic absorptions at 365 and 256 nm, and BP had characteristic absorptions at 326 and 275 nm, the BP-CP assemblies exhibited new absorption bands at 350 and 252 nm (Figure S4b). We then performed the crosslinking reaction in methanol solution. It was observed that 0.1 mg/mL of polymers led to the formation of irregular assemblies (Figure 2a). Improving the polymer concentration to 0.2 mg/mL resulted in the formation of bundle-like assemblies (Figure 2b and its inset). These assemblies grew further and inter-particle crosslinking occurred when the polymer concentration reached 0.4 mg/mL (Figure 2c). DLS results further indicated that the diameter and PDI of these assemblies increased with the increase of polymer concentration (Figure 2d). The inset of Figure 2e gives the SEM image of a typical assembly, from which nano-rods were found to bound together to form a bundle-like structure. EDX line scan analysis of a typical bundle-like assembly revealed that both carbon and boron elements were homogenously distributed throughout the entire assembly (Figure 2e), which was completely different from the element distribution in spherical assemblies shown in Figure 1e. This result implied that there was no migration of boronate or boronic containing polymer chains during the crosslinking reaction between BP and CP in methanol solution. Since we targeted to the biological applications, a methanol solution of these assemblies was not feasible. Therefore, we collected the bundle-like assemblies from methanol solution and redispersed them in water solution. We found that bundle-like assemblies in a water solution were also stable. No evident


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changes in morphology, size and element distribution were observed to these assemblies (Figure S5). The above results indicated that the crosslinking reaction between BP and CP in water and methanol solutions led to the formation of nano-assemblies with different morphologies. In our previous work, we have found that boronate can interact with imine to form B-N dative bond, therefore bringing small molecules together into uniform assemblies with tunable morphologies in methanol solution 30. We envisaged that B-N dative bond might also be formed along with the crosslinking reaction between BP and CP in methanol solution (Scheme 2a), thus orderly organizing the crosslinked polymer networks into bundle-like nano-architectures. To verify this, we performed the crosslinking reaction of BP and CP in methanol-D4 and monitored the

11

B

NMR spectrum of the reaction mixture (Figure 2f). Note that BP itself showed a typical tricoordinated boron signal at δ=28.0 ppm in its

11

B NMR spectrum, bundle-like assemblies

formed by 0.2 mg/mL polymers exhibited a broaden peak (δ=11.2 ppm) derived from the methoxylated B-N dative bond 30, 32. Similarly, the 11B NMR spectrum of the reaction system of BP and CP in D2O was measured. As shown in Figure 2f, we could only find a tricoordinated boron signal at δ=27.2 ppm, implying that B-N dative bond was not formed between boronate and imine moieties in a water solution. These results in combination with the element analyses, indicated that the formation spherical assemblies in water solution was promoted by the hydrophobic aggregation of polymer chains, while the formation of bundle-like assemblies in methanol solution was related to the B-N dative bond. Moreover, control experiments were conducted by using other solvents to further confirm the relationship between B-N dative bond and the morphology of the BP-CP assemblies. For example, we performed the crosslinking reaction between BP and CP in dichloromethane, as it is also a convenient solvent to facilitate


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the formation of B-N dative bond

33, 34

. The as formed BP-CP assemblies showed bundle-like

morphology (Figure S6a). These assemblies had large diameter ( ＞ 1µm in length) and precipitated from the reaction solution easily, thus making them unsuitable for biomedical application. Interesting, when adopting a solvent mixture with 1:1 volume ratio of methanol and water for the crosslinking reaction, only spherical BP-CP assemblies were formed (Figure S6b). Probably, this was due to the fact that water could prevent the formation of B-N dative bond among the polymer chains. Degradation capability Since the building bridges of BP-CP crosslinked networks comprise both imine and boronate moieties, the cleavage of these dynamic bonds may induce the disassembly of the BP-CP assemblies. Previous investigations have already showed that imine bond is cleavable in weakly acidic solutions (pH≤5.5) 18-20, while boronate moiety is degradable in response to D-glucose 29

16,

. Here, we not only demonstrate the possibility of the degradation of BP-CP crosslinked

assemblies under the stimuli of acidic pH or D-glucose, but also show that the assembly morphology has impact on the degradation kinetics. The degradation of BP-CP assemblies at acidic pH or with the presence of D-glucose was first confirmed by monitoring the evolution of UV-vis spectra. Taking bundle-like assembly (formed by 0.2 mg/mL of BP and CP) as an example, the constant UV-vis absorption at pH=7.4 without D-glucose during 12 h implied their relatively high stability (Figure S7). At pH=6.0, 16.5 % decrease in intensity of the characteristic absorption at 350 nm could be observed after 12 h (Figure 3a). The decrease rate of the characteristic absorption was accelerated at pH=5.0 (24.8 % decrease within 12 h, Figure S8a), indicating that the evolution of UV-vis spectra of bundlelike assemblies was indeed caused by acidic pH. With the addition of D-glucose, this evolution


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in UV-vis spectra was even faster (Figure 3b and Figure S8b), as the characteristic absorption of bundle-like assemblies almost disappeared after 12 h. 73.8 % And 86.8 % decays in the absorption intensity could be observed with the addition of 0.1 mM of D-glucose at pH=6.0 and 5.0, respectively. The attenuation of the characteristic absorption was much faster (92.3% decay after 12 h) when increasing the concentration of D-glucose to 0.6 mM (Figure S8c). Spherical assemblies had the same evolution of UV-vis spectra under the stimuli of acidic pH or D-glucose (Figure S9). Figure 3c illustrated the UV-vis absorption reduction kinetics of both bundle-like and spherical assemblies in response to these stimuli. Bundle-like assemblies exhibited much faster decay in characteristic absorption than spherical assemblies in response to the same stimuli. The size evolution of these assemblies was measured by DLS to further verify their degradability. As shown in Figure 3d, the particle size of both bundle-like and spherical assemblies increased gradually in acidic solutions, the rate and extent of this size variation became larger at lower pH value. An addition of 0.1 mM of D-glucose into these weakly acidic solutions could cause a different size evolution of the assemblies. The diameter of all the assemblies increased rapidly within the first 2 hours and decreased dramatically with the further prolonging of time. This size evolution was evidently accelerated when the concentration of D-glucose was increased to 0.6 mM. Importantly, the rate and extent of these size evolutions accorded well with that of the UVvis evolutions. In this regard, bundle-like assemblies degrade more easily than spherical assemblies. This can be explained well by the structural difference between these two assemblies. For the spherical assemblies, boronate and imine containing polymer chains are embedded in interior of the assemblies, and therefore their degradation under the stimuli in solution is slowed down. In contrast, the boronate moieties are homogenously distributed throughout the bundle-


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like assemblies, which can facilitate the interaction of boronate or imine groups with solution stimuli, as a result leading to an enhanced degradability. The morphology evolution of these assemblies during degradation was confirmed by TEM. As shown in Figure S10, bundle-like assemblies in a solution with 0.1 mM of D-glucose at pH=5.0 swelled evidently within 2.0 h and already disassembled after 8.0 h. The difference between the size evolutions triggered by acidic pH and caused by the combinative trigger of acidic pH and D-glucose (Figure 3d), can be explained by the different cleavage sites of the polymer networks (Scheme 2b). Weakly acidic condition can promote the cleavage of imine bond but unlikely has evident impact on the boronate moiety. The dynamic feature of imine bonds only allows a part of them to be cleaved. Moreover, the hydrophilicity difference between imine bond and its decomposition product (primary amine and aldehyde) is not evident. Therefore, although the crosslinking density is reduced, BP-CP assemblies can still hold their structure and only exhibits a certain degree of swelling at acidic pH. In an acidic solution with the presence of D-glucose, both imine and boronate moieties are cleavable. Specifically, the combination between D-glucose and boronic group may evidently increase the hydrophilicity of the polymer chains. As a result, the BP-CP assemblies first swell extensively and then disassemble. To demonstrate the potential application of these assemblies in drug delivery, their guest encapsulation and controlled release behavior were monitored by using DiI as a probe. The encapsulation of DiI in bundle-like assemblies seemed to be stable at pH=7.4 without D-glucose, as the emission intensity did not change with time (Figure S11a). Since the UV-vis spectrum evolution and DLS results confirmed that the assemblies could swell in weakly acidic solution, it was important to verify that whether this type of swelling could cause the release of DiI. Indeed,


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the fluorescence intensity of DiI decreased slowly with time at pH=6.0 (Figure 4a) and this intensity attenuation was accelerated slightly at pH=5.0 (Figure S11b), implying that the swelling of bundle-like assemblies could only cause the release of a small amount of DiI. In comparison, when 0.1 mM of D-glucose was introduced into the acidic solution, the degree of DiI release was significantly increased at pH=6.0 and 5.0 (Figure 4b and Figure S11c). The release of DiI could be further enhanced when the concentration of D-glucose was increased to 0.6 mM in acidic solution at pH=6.0 (Figure S10d). The same stimuli-responsive release of DiI was also observed for spherical assemblies (Figure S12). The release kinetics was then calculated to quantitatively compare the release of DiI under different stimuli (Figure 4c). Obviously, the kinetics of DiI release was consistent with that of UV-vis spectra and particle size. In acidic solution with pH=5.0, the release of DiI from bundle-like and spherical assemblies could reach 32.8 and 17.7 % within 12 h, respectively. However, with the present of 0.1 mM of D-glucose, 62.7 and 40.8 % of DiI could be released from bundle-like and spherical assemblies after 12 h, respectively. When the concentration of D-glucose was increased to 0.6 mM at pH=6.0, the release of DiI from bundle-like assemblies could reach 65.2 % after 12 h. Cytotoxicity assay and cellular uptake The BP-CP assemblies are constructed by random copolymers containing PEG chains, we envisioned that they might have good biocompatibility. Therefore, their cytotoxicity was evaluated by the cell-killing performance. MC 3T3-E1 cells (normal cell lines) and Hela cells (cancer cell lines) were adopted to test the cytotoxic effects of the BP-CP assemblies using a standard MTT assay. As shown in Figure 5, after treating with bundle-like or spherical assemblies with concentration ≤ 0.01 mg/mL for 24 h, the cell viabilities of MC 3T3-E1 cells and Hela cells were above 90%, indicating that the BP-CP assemblies have no significant


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adverse effect among this concentration range. A significantly higher cytotoxicity on both normal cell lines and cancer cell lines could be observed for both bundle-likeandspherical assemblies at concentration ≥0.1 mg/mL. These results implied that the BP-CP assembly might be a promising nano-carrier for drug delivery when its concentration ≤ 0.01 mg/mL. Comparing Figure 5a and b, no apparent cell viability difference could be observed between bundle-like and spherical assemblies, indicating that the morphology of the BP-CP assemblies had no impact on the cytotoxicity. Note that the cytotoxicity tests were performed with diluted BP-CP assembly solutions, it was important to evaluate the stability of these assemblies against dilution. As shown in Figure S13, both bundle-like and spherical assemblies exhibited no evident size evolution in water or in PBS solutions at pH=7.4 even after 20 times dilution, revealing that dilution of the stock solution could not cause the dissociation of BP-CP assemblies. Therefore, it is reasonable to consider that the cytotoxicity tests reflect the cytotoxic effect of BP-CP assemblies themselves instead of the polymers. Nano-carriers formed by the self-assembly of multi-component copolymers comprising PEG chains generally have high biocompability, low cell and tissue cytotoxicity and long term circulation35-37. Nevertheless, the PEG chain can specifically weaken the cell penetration capability of the nano-carriers 38-40. Our findings further confirmed this argument, as no cellular uptake was observed for spherical assemblies which were coated with PEG chains (Figure S14). Interesting, although the bundle-like assemblies have the same composition with that of spherical assemblies, they exhibit an unexpected high cell penetration capability. As shown in Figure 6a, bundle-like assemblies derived from 0.1 mg/mL of BP and CP polymers were internalized successfully in HeLa cells after 1.5 h. The amount of the internalized assemblies increased evidently with the prolonging of time from 1.5 h to 4.5 h, implying that the cell uptake


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of bundle-like assemblies was time dependent. With the same treatment, bundle-like assemblies derived from 0.2 mg/mL of BP and CP polymers likely had higher cell penetration ability, as much more assemblies were internalized in HeLa cells after 1.5 h (Figure 6b). When using the bundle-like assemblies derived from 0.4 mg/mL of BP and CP polymers, their cell internalization could be further enhanced (Figure 6c), obvious cell internalization could be observed within 0.5 h. In comparison with spherical assemblies, bundle-like assemblies have two advanced features. First, their asymmetrical structure endows the assemblies with rough surface, therefore facilitating their internalization. Second and more importantly, the boronate containing polymer chains are uniformly dispersed in the bundle-like assemblies. As the boronate moiety is dynamic, some of them would dissociate into boronic and catechol groups. The exposure of catechol moiety can enhance the binding affinity of the assemblies to the cell membrane and improve their cell penetration capability 41, 42.

CONCLUSIONS In summary, we have developed a simple crosslinking induced self-assembly pathway to fabricate stimuli-responsive nano-assemblies through a condensation reaction between a boronic containing polymer and a catechol bearing polymer. The morphology of the assemblies depends largely on the reaction solution. Hydrophobic aggregation of the crosslinked polymer networks in water solution creates spherical nano-assemblies; while the B-N dative bond organizes the crosslinked polymer networks into bundle-like assemblies in methanol solution. The crosslinked assemblies are degradable due to the cleavage of imine and boronate moieties, and have the ability to release the encapsulated guest molecule on demand. Acidic pH can only induce the swelling of the assemblies, while the combination of acidic pH and D-glucose can trigger a total disassembly. Importantly, our findings indicate that polymeric assemblies designed with


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asymmetrical structure or surface functionalized with catechol moiety may have significantly enhanced cell penetration ability.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org. 1H NMR spectra of the polymers; TEM, SEM images of the assemblies; UVvis and fluorescence spectra; CLSM images. Corresponding Author *Conghui Yuan, E-mail: [email protected]; *Lizong Dai, E-mail: [email protected]; Tel: +86 592 21836178, Fax: +86 592 2183937. Acknowledgements This work was supported by the National Natural Science Foundation of China (51403176, 51373142, 51511130130, PICS 07002 pour le CNRS); Technological Innovation Platform of Fujian Province (2014H2006); Natural Science Foundation of Fujian Province (2015J01220).

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Scheme 1. Chemical structures of BP and CP random copolymers; self-assembly of BP-CP crosslinked networks.


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Figure 1. (a-c) TEM images of spherical assemblies prepared in water solution using 0.1, 0.2 and 0.4 mg/mL of BP and CP. (d) Diameters of the assemblies. (e) EDX line scan analysis of boron and carbon of a spherical assembly, the inset is the SEM image of a typical spherical assembly.


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Figure 2. (a-c) TEM images of bundle-like assemblies prepared in methanol solution using 0.1, 0.2 and 0.4 mg/mL of BP and CP. (d) Diameters of the corresponding assemblies. (e) EDX line scan analysis of boron and carbon of a typical bundle-like assembly and its corresponding SEM image. (f) 11B NMR spectra of BP, bundle-like and spherical assemblies.


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Scheme 2. (a) Chemical structure of B-N dative bond formed between boronate and imine moieties in the bundle-like assemblies; (b) possible chemical process of the hydrolysis of imine bond in response to weak acidity and the cleavage of boronate moiety caused by D-glucose.


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Figure 3. UV-vis spectrum evolution of bundle-like assemblies under different stimuli: (a) pH=6.0, (b) pH=5.0 with 0.1 mM of D-glucose. (c) UV-vis absorption reduction kinetics of bundle-like and spherical assemblies with different stimuli. (d) Diameter evolution of bundle-like and spherical assemblies caused by different stimuli. These assemblies were prepared from 0.2 mg/mL of BP and CP.


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Figure 4. Fluorescence spectra trace the release of DiI from bundle-like assemblies in response to different stimuli: (a) pH=6.0, (b) pH=5.0 with 0.1 mM of D-glucose. (c) Release kinetics of DiI from bundle-like and spherical assemblies under different stimuli. These assemblies were prepared from 0.2 mg/mL of BP and CP.


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Figure 5. Cell viability of (a) MC 3T3-E1 cells and (b) Hela cells after treating with bundle-like and spherical assemblies for 24 h.


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Figure 6. CLSM images Hela cells incubated with bundle-like assemblies prepared using different concentrations of BP and CP. For each panel, the images from left to right show cell nuclei stained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI, blue), DiI loaded BP-CP assemblies (red) in cells, and overlays of the two images. The concentration of the assemblies used for the cell uptake experiment was 0.01 mg mL−1.


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Table of Contents


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Cross-Linking Induced Self-Organization of Polymers into Degradable

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