Artificially Smart Vesicles with Superior Structural ... - ACS Publications

Apr 18, 2017 - In acidic aqueous solution, the pores in the membrane of vesicles are opened, which is beneficial for transmembrane traffic. The pore ...
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Artificially Smart Vesicles with Superior Structural Stability: Fabrication, Characterizations, and Transmembrane Traffic Wen-Jian Zhang, Chun-Yan Hong,* and Cai-Yuan Pan* CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, Anhui 230026, China S Supporting Information *

ABSTRACT: Intelligent vesicles are fabricated at up to 30% solid content via an approach of polymerization-induced self-assembly and reorganization (PISR). Upon irradiation with UV light (365 nm), light-triggered dimerization of the coumarin moieties anchored in the membrane leads to the membrane cross-linking of the vesicles, which endows the vesicles with superior structural stability. Due to the tertiary amine groups in the membrane, the vesicles go through a swelling/deswelling change upon switching the pH values. In acidic aqueous solution, the pores in the membrane of vesicles are opened, which is beneficial for transmembrane traffic. The pore size in the membrane of vesicles is in accordance with the extent of membrane cross-linking, which can be conveniently regulated by the irradiation time of UV light (365 nm). The size range of the substance for transmembrane traffic is effectively enlarged; even 15 nm gold nanoparticles can be postloaded into the vesicles with lower extents of the membrane cross-linking through the diffusion method. Although the pores in the vesicle membrane are opened in acidic aqueous solution, transmembrane traffic is inhibited for the electropositive substance because of electrostatic repulsion but is allowed for the electronegative substance. These reported vesicles herein may be the smartest artificial vesicles to date due to their multiple selective permeability. KEYWORDS: polymerization-induced self-assembly and reorganization (PISR), RAFT dispersion polymerization, vesicles, selective membrane permeability, cross-linking

1. INTRODUCTION Selective substance channels are abundant in biological systems, which are essential in the evolution of life because they control substance exchange in organisms. Fabrication of the artificial nanostructures to mimic biological systems has gained increasing interests over the past decade.1,2 The most representative examples are lipid vesicles (liposomes) and block copolymer vesicles (polymersomes), which have become competitive candidates for nanoreactors, artificial organelles, and nanocarriers.3−7 Compared to liposomes, the polymersomes have better mechanical and chemical structure stability but always suffer from severe membrane impermeability issues because they are almost impermeable to small substances, ions, and even water molecules.8,9 A few approaches have been reported to improve the permeability of polymersomes such as postmodification of vesicle membranes,10,11 fabrication of stimuli-sensitive polymersomes,12−18 coassembly of oppositely charged block copolymers,19,20 etc. However, compared to the intelligent selective permeability of the cellular membrane, the artificial vesicles are still far from being satisfactory; for example, the transmembrane traffic of the artificial vesicles is usually affected by steric hindrance.13 Furthermore, adjusting pore size range in the artificial vesicles for enlarged size-selective transmembrane traffic is also difficult.21 Therefore, a type of polymersome which can separate a mixture of substances based not only on their size but also on their properties such as © XXXX American Chemical Society

electric charge is in high demand. For this purpose, we designed the types of vesicles shown in Scheme 1; their surface and core polymers are, respectively, poly(N-(2-hydroxypropyl) methacrylamide) (PHPMA) and poly(2-(diisopropylamino)ethyl methacrylate-co-7-(2-methacryloyloxy-ethoxy)-4-methylcoumarin) (PDIPEMA-co-PCMA). Under UV irradiation, dimerization of the coumarin groups in CMA units leads to crosslinking of the vesicular membrane (Scheme 1). The pore size of the membrane formed through cross-linking can be modulated in a broad range relative to reported polymersomes13 because both UV irradiation time and acidity of the solutions can change the pore size (Scheme 2). Enlarged size-selective transmembrane traffic was demonstrated by encapsulation of gold nanoparticles (AuNPs) with different sizes (5, 10, and 15 nm). The protonated membrane of the vesicles has high selectivity to the charged substances, and the membrane composed of the protonated PDIPEMA inhibits transmembrane traffic of the electropositive substance due to electrostatic repulsion (Scheme 2). For preparation of the polymersomes, a widely used selfassembly approach is selective solvation of the asymmetric block copolymer, which was achieved by displacement of the Received: February 28, 2017 Accepted: April 7, 2017

A

DOI: 10.1021/acsami.7b02966 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Scheme 1. Fabrication of Vesicles with pH-Regulated Permeability by PISR and UV (365 nm) Irradiation Induced Membrane Crosslinking of the Vesicles

adjustable membrane permeability still remains a considerable challenge. On the basis of the above discussion, the PISR approach is used to fabricate the smart vesicles because this approach allows direct and highly efficient preparation of the vesicles at up to 30% solid content. RAFT dispersion copolymerization of the two functional monomers DIPEMA and CMA using PHPMA as macro-RAFT agent produces the vesicles, and the target vesicles are obtained after the coumarin moieties anchored in the membrane are dimerized to form cross-linked structure under UV irradiation (Scheme 1). The vesicles with pHregulated permeability are formed due to the tertiary amine in the membrane, and the PDIPEMA chains in the vesicle membrane undergo transition from an unprotonated and hydrophobic state to a protonated and hydrophilic state when the neutral aqueous solution is changed to acidic aqueous solution, which allows the transmembrane traffic of substances to occur. The pore size range for transmembrane traffic of the substances is effectively enlarged via decreasing cross-linking extent of the membrane.

Scheme 2. Multiple-Selective Transmembrane Traffic of Intelligent Vesicles in Acidic Solution (pH 4.6), Enlarged Size Selectivity, and Electric Charge Selectivitya

a

The pore sizes of the vesicle membrane are modulated in a broad range via adjusting the UV irradiation time. In the schematic, “yes” denotes successful transmembrane traffic, and “no” represents the transmembrane traffic is inhibited.

2. EXPERIMENTAL SECTION 2.1. Material. DIPEMA (Sigma-Aldrich, 97%) was purified by distillation under reduced pressure prior to use. N,N′-azobis(isobutyronitrile) (AIBN) was purchased from Sinopharm Chemical Reagent Co. Ltd. and purified by recrystallization from ethanol prior to use. Methacryloyl chloride (95%), sodium fluorescein, and doxorubicin hydrochloride (DOX·HCl, 98%) were purchased from Aladdin and used as received. Gold nanoparticles (5, 10, and 15 nm) were purchased from nanoComposix and used as received. 4-(4Cyanopentanoic acid) dithiobenzoate (CPDB),45 N-(2-hydroxypropyl) methacrylamide (HPMA),46 and 7-(2-methacryloyloxyethoxy)-4methylcoumarin (CMA),47 were synthesized according to the literature. All other reagents were of analytical grade and used as received. 2.2. Synthesis of PHPMA. HPMA (10.0 g, 70 mmol), CPDB (195 mg, 0.7 mmol), AIBN (23 mg, 0.14 mmol), and ethanol (20 mL) were added into a 50 mL polymerization tube with a magnetic bar. After three freeze−evacuate−thaw cycles, the tube was sealed under high vacuum, and then the sealed tube was placed in an oil bath at 70 °C while stirring for 20 h. After cooling to room temperature, the polymer solution was diluted with ethanol and then poured into excess diethyl ether while stirring. The precipitate was collected by filtration and then dissolved in ethanol, and the precipitation procedure was repeated

nonselective solvent with selective solvent in a relatively high dilution (typically less than 1% solids).22−25 Preparation of the fine structural nano-objects via such traditional methods normally requires multiple steps, including synthesis of block copolymer, boring purification, dissolving in a good solvent, and displacement of the good solvent with selective solvent cautiously.22−25 Recently developed polymerization-induced self-assembly and reorganization (PISR) allows efficient fabrication of polymeric vesicles at much higher concentration.26−39 To obtain the vesicles with superior structural stability, chemical cross-linking of the vesicle membrane is necessary and was achieved by copolymerization of cross-linker in the PISR process or employing postpolymerization crosslinking strategy.40,41 Although encapsulation of substance (such as silica nanoparticles, nile red, or proteins) into the vesicles during PISR was reported,42−44 fabrication of the vesicles with B

DOI: 10.1021/acsami.7b02966 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces three times. The resultant product was dried under vacuum at room temperature overnight to produce 3.57 g of red solids. 2.3. RAFT Dispersion Copolymerization of DIPEMA and CMA. A typical procedure of the RAFT dispersion polymerization for the total solid concentration of 20% is as follows: DIPEMA (170 mg, 0.8 mmol), CMA (58 mg, 0.2 mmol), PHPMA40-CPDB (60 mg, 10−2 mmol), AIBN (0.33 mg, 2 × 10−3 mmol), and solvent (1.15 g, the mass ratio of ethanol/water = 7/3) were added into a glass tube with a magnetic bar. After being degassed by three freeze−pump−thaw cycles, the glass tube was sealed under vacuum. The sealed tube was placed in an oil bath at 70 °C while stirring, and then the polymerization was quenched after reaction for 9 h. For the sake of description convenience, the sample obtained by the above description is simply denoted as H40D100-20%, wherein the H40 represent the PHPMA40-CPDB, the D100 denotes that the target degree of polymerization (DP) of P(DIPEMA-co-CMA) is 100, and the number after the hyphen represents that the total solid concentration is 20%. For the other RAFT dispersion polymerizations listed in Table S1, the same procedures were conducted beside the total solid concentration and the target DP of P(DIPEMA-co-CMA). For all the RAFT dispersion polymerizations, the molar ratio of AIBN/PHPMA40-CPDB was kept at 1/5. 2.4. Cross-Linking of the Block Copolymer Vesicles. The H40D100-20% vesicles were diluted 4-fold with ethanol/water (7/3 wt/ wt) to 50 g/L, and then 2 mL of the above solution was irradiated with UV at 365 nm (0.1 mW/cm2) under stirring for 2 h. The mixtures (50 μL) were removed and diluted 500-fold with ethanol/water (7/3) to 0.1 g/L at a predetermined time interval, and then characterized by UV−vis spectroscopy. The dispersions of vesicles were dialyzed against ethanol for 2 days to remove the unreacted monomers in the RAFT dispersion polymerization and then against deionized water for 24 h to remove the ethanol. The final product was obtained by lyophilization as a white powder. For fabrication of vesicles with a lower degree of cross-linking, the same procedures were conducted as above except for shorter UV irradiation time (herein, 15 and 30 min of UV irradiation were selected). 2.5. Encapsulation of Various Substances in Different Vesicles. 2.5.1. Encapsulation of Gold Nanoparticles (5, 10, and 15 nm) in Vesicles (UV 120 min). Vesicles (UV 120 min, 12 mg) were dispersed in acetate buffer solution (pH 4.6, 3 mL) and equally divided into three samples. Dispersions of gold nanoparticles (0.5 mL) of different sizes (5, 10, and 15 nm) were added into the above mixture. After the mixture was stirred at room temperature overnight, its pH was adjusted to about 8 via addition of NaOH aqueous solution (0.1 M). The gold nanoparticles outside of the vesicles were removed by centrifugation. 2.5.2. Encapsulation of Gold Nanoparticles (10 and 15 nm) in Vesicles (UV 30 min). Vesicles (UV 30 min, 8 mg) were dispersed in acetate buffer solution (pH 4.6, 2 mL) and equally divided into two samples. The 0.5 mL dispersions of gold nanoparticles of different sizes (10 and 15 nm) were added into the above mixture. After the mixture was stirred at room temperature overnight, its pH was adjusted to about 8 via addition of NaOH aqueous solution (0.1 M). The gold nanoparticles outside of the vesicles were removed by centrifugation. 2.5.3. Encapsulation of Gold Nanoparticles (15 nm) in Vesicles (UV 15 min). Vesicles (UV 15 min, 4 mg) were dispersed in acetate buffer solution (pH 4.6, 1 mL), and then 0.5 mL dispersions of gold nanoparticles (15 nm) were added into the above mixture. After the mixture was stirred at room temperature overnight, its pH was adjusted to about 8 via addition of NaOH aqueous solution (0.1 M). The gold nanoparticles outside of the vesicles were removed by centrifugation. 2.5.4. Encapsulation of Sodium Fluorescein in Vesicles (UV 120 min). Vesicles (UV 120 min, 4 mg) and sodium fluorescein (1 mg) were mixed in acetate buffer solution (pH 4.6, 1 mL). After the mixture was stirred at room temperature overnight, its pH was adjusted to about 8 via addition of NaOH aqueous solution (0.1 M). The sodium fluorescein outside of the vesicles was removed by dialysis against PBS buffer solution (pH 7.4, 20 mM) for 4 days (molecular

weight cutoff: 3500 Da) with exchanging of the solution by fresh buffer frequently. 2.5.5. Encapsulation of DOX·HCl in Vesicles (UV 120 min) in Acetate Buffer Solution (pH 4.6). Vesicles (UV 120 min, 4 mg) and DOX·HCl (20 mg) were mixed in acetate buffer solution (pH 4.6, 1 mL). After the mixture was stirred at room temperature overnight, its pH was adjusted to about 8 via addition of NaOH aqueous solution (0.1 M). The DOX·HCl outside of the vesicles was removed by dialysis against PBS buffer solution (pH 7.4, 20 mM) for 4 days (molecular weight cutoff: 3500 Da) with exchanging of the solution by fresh buffer frequently. 2.5.6. Encapsulation of DOX·HCl in Vesicles (UV 120 min) in DMSO. Vesicles (UV 120 min, 8 mg), DOX·HCl (40 mg), and triethylamine (1 mL) were mixed with 2 mL of DMSO. After the mixture was stirred at room temperature overnight, distilled water (20 mL) was added. The resultant mixture was divided into two parts and dialyzed against distilled water with exchange of fresh distilled water frequently until no DOX in the solution outside the dialysis bags can be detected. Then, one of them was further dialyzed against acidic water (pH value is about 3, which was prepared by HCl and distilled water) for 24 h. 2.6. pH-Regulated Release of Sodium Fluorescein. The above obtained vesicles (UV 120 min) loaded with sodium fluorescein were divided into two parts and transferred into two dialysis tubes (molecular weight cutoff: 3500 Da) and then dialyzed against 80 mL of PBS buffer (20 mM, pH 7.4) and 80 mL of acetate buffer (20 mM, pH 4.6), respectively. The buffer solution (2 mL) outside the dialysis tubes was sampled at selected time points and replaced with an equal volume of fresh buffer solution. The content of sodium fluorescein released was evaluated using the fluorescence spectrometer (excitation wavelength: 450 nm, emission wavelength: 520 nm). Note: before detecting fluorescence intensity, the samples taken from the acetate buffer (20 mM, pH 4.6) were adjusted to neutral via addition of NaOH aqueous solution (0.1 M). 2.7. Characterization. The 1H NMR (300 MHz) spectra were obtained on a Bruker DMX300 spectrometer; CDCl3, DMSO-d6, and CD3OD were used as the solvents, and tetra-methylsilane was used as an internal reference. A Waters 150C gel permeation chromatograph (GPC) equipped with two Ultrastyragel columns in series and an RI 2414 detector at 30 °C was used to measure the molecular weight (Mn) and Mw/Mn; DMF was used as eluent with a flow rate of 1.0 mL/ min, and monodispersed polystyrene standards were used in the calibration of molecular weight and Mw/Mn. The morphologies of the nano-objects were characterized with transmission electron microscopy (TEM), which was performed on a Hitachi H-800 electron microscope at an accelerating voltage of 100 kV. The samples for TEM observations were prepared by depositing 10 μL of the nano-object dispersion in ethanol on copper grids which were coated with thin films of Formvar and carbon successively. All the dynamic light scattering measurements were carried out on a commercial laser light scattering (LLS) spectrometer (Zetasizer Nano ZS90, Malven Instruments Ltd., Malvern, UK) equipped with a He−Ne Laser (4.0 mW, 633 nm) at 25 °C and a fixed angle of 90°, and all the data were averaged over 3 measurements. UV−vis measurements were carried out on a TU-1901 UV−vis spectrophotometer.

3. RESULTS AND DISCUSSION 3.1. Fabrication of Vesicles via PISR. PHPMA was synthesized by RAFT polymerization in ethanol at 70 °C using CPDB as the RAFT agent. Well-defined PHPMA with Mn = 6700 and Mw/Mn = 1.27 was obtained (Figure S1), and the degree of polymerization (DP = 40) of the PHPMA was calculated based on the 1H NMR data (Figure S2). Then, the resultant PHPMA40 was used as a macro-RAFT agent in the following RAFT dispersion polymerization. PDIPEMA has been widely used in fabrication of the stimuli-responsive nanoobjects due to its adjustable water solubility induced by pH.48,49 Therefore, DIPEMA was used in the RAFT dispersion C

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may serve as a “roadmap” for the reproducible fabrication of morphologically pure nano-objects. At solid contents from 10 to 30%, the spheres, nanowires, and vesicles could be obtained with target DP increasing of the P(DIPEMA-co-CMA) block (Figure 1E). Influence of the solid content on the morphology was also observed in the phase diagram of Figure 1E; at lower solid contents (10−20%), formation of the morphologically pure vesicles required higher target DP of the core-forming block in comparison with that of the polymerizations at solid contents of 25 and 30%. For example, when the target DPP(DIPEMA‑co‑CMA) equals 80, only a mixture of the vesicles and nanowires was formed at 10−20% solid contents, but pure vesicles were obtained for the solid contents of 25−30% because, at higher solid content, inelastic collision of the nanoparticles occurs easily, leading to internanoparticle fusion.50−52 Therefore, both copolymer composition and solid content have significant influence on the final morphologies, which is similar to that in the aqueous or alcoholic dispersion polymerization as previous reports.50−52 3.2. Cross-Linking of the Vesicles. Photo-cross-linking of the micelles, which is induced by phototriggered dimerization of the coumarin moieties incorporated in the polymer, has been reported.53−55 When the vesicles were exposed to UV irradiation at λ = 365 nm, the absorption band of coumarin moieties at around 320 nm continuously decreased with increasing irradiation time as shown in Figure 2A, and approximately 80% of the coumarin moieties in the vesicular membrane were taken part in dimerization within 2 h. 1H NMR spectra in Figure S6 show that the signal at 6.2 ppm assigned to coumarin moieties almost disappears after UV irradiation for 2 h, which indicates cross-linking of the vesicular membrane via dimerization of the coumarin groups. More evidence of the cross-linked polymersomes is provided in the following discussion. Owing to deprotonation and protonation of the tertiary amine groups in the PDIPEMA (pKa is around 6.3 as in previous reports48,49), the polymer chains undergo a transformation from a hydrophobic state at high pH value to a hydrophilic state at low pH. When the solution is above pH 6.70, the vesicles obtained before or after UV irradiation have a similar diameter (around 440 nm, Figure 2B) because of their unprotonated hydrophobic state. Due to protonation of the tertiary amine groups in the P(DIPEMA-co-CMA) chains at a low pH, such as below pH 5.74, the diameter of vesicles obtained after UV irradiation expanded to around 820 nm because the cross-linked P(DIPEMA-co-CMA) chains of the vesicle walls became hydrophilic; thus, the vesicles are highly swollen, while the vesicles without UV irradiation were disaggregated to molecular dissolution at these conditions (Figure 2B), demonstrating that photo-cross-linked membrane endows the vesicles with more robust structure. Briefly, the P(DIPEMA-co-CMA) blocks are hydrophobic in alkaline solution, so the vesicle morphology kept well at pH 8 (Figure S5). In acidic solution such as pH 4.6, the block chains became hydrophilic, and the well-defined morphology of the crosslinked vesicles still remained as shown in the TEM image of Figure 2C, which further illustrated the superior structural stability of the cross-linked vesicles. Even suffering from the challenge of dimethyl sulfoxide (DMSO), which is a good solvent for both blocks, the morphology of vesicles kept well, as evidenced by the TEM image in Figure 2D. High stability of the vesicular cross-linked structure during the swelling (in acidic solution)−shrinking (in alkaline solution) cycles is very important for repeating use of the

polymerization to fabricate vesicles with pH-regulated permeability. The solvent is very important for the RAFT dispersion polymerization because the fundamental criterion for this type of polymerization is that the monomer has good solubility, but the in situ formed polymer has poor solubility in the solvent. Both DIPEMA and PDIPEMA are well-soluble in ethanol, but they are water-immiscible, so a mixture of ethanol and water may be an appropriate polymerization media. The mixed solvent with ethanol/water = 7/3 (weight ratio) was used in the present study. The PHPMA was selected as macro-RAFT agent because this polymer is well-soluble in aqueous or alcoholic solution. CMA was used as the comonomer to anchor the coumarin group in the membrane of vesicles for the postpolymerization cross-linking via ultraviolet-triggered dimerization. The CMA is insoluble in the mixture of ethanol/water = 7/3 (weight ratio) at room temperature, but its solubility is well-improved at 70 °C. With consideration of the significant influence of cross-linking degree on the pore size of membrane, the feed molar ratio of DIPEMA/CMA = 8/2 was used in all RAFT dispersion copolymerizations of the DIPEMA and CMA. All of these polymerizations were proved to be well-controlled and efficient; low polydispersities of the obtained polymers and >90% of monomer conversions were routinely achieved, as judged by GPC and 1H NMR studies (Table S1 and Figure S3). The morphologies of the obtained nano-objects were dependent on the diblock copolymer composition and the solid content (Table S1); the spherical micelles, nanowires, nanowires/vesicles, and vesicles were successively obtained with target DP increasing of the core-forming P(DIPEMA-co-CMA) block at 20% of the solid content (Figures 1A−D, Figure S4). A phase diagram in Figure 1E was constructed by systematical variation of the solid content and the DP of P(DIPEMA-coCMA) block for preparation of various morphologies, which

Figure 1. TEM images of the PHPMA40-P(DIPEMA-co-CMA)x nanoobjects prepared by RAFT dispersion copolymerization of DIPEMA and CMA (DIPEMA/CMA = 8/2, molar ratio) at 70 °C: (A) H40D4020% (sphere micelles), (B) H40D60-20% (nanowires), (C) H40D80-20% (the mixture of nanowires and vesicles), and (D) H40D100-20% (vesicles), where H40 denotes PHPMA40, D denotes P(DIPEMA-coCMA), the subscript number of D represents the targeting DP of P(DIPEMA-co-CMA), and 20% represents the solid concentration. (E) Phase diagram constructed by systematically varying the copolymer solid content and the DP of the P(DIPEMA-co-CMA) block, wherein s = sphere micelles, nw = nanowires, and v = vesicles. D

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Figure 2. Characterization of the sample H40D100-20% vesicles before and after phototriggered membrane cross-linking: (A) UV−vis absorbance spectra recorded under UV irradiation of the vesicle dispersion in ethanol/water (7/3, wt/wt) for different times (0−120 min). (B) Variation of hydrodynamic diameter of the vesicles obtained before and after cross-linking with different pH values of the solution (solid concentration is 0.1 mg/ mL). (C) TEM image of the cross-linked vesicles in acidic aqueous solution (pH 4.6). (D) TEM image of the cross-linked vesicles in dimethyl sulfoxide (DMSO). The samples for B−D were obtained by lyophilization the vesicle dispersions without or with UV irradiation at 365 nm for 120 min and then dispersion in the corresponding solvent.

states of the pores in the vesicle membranes can be adjusted via CO2 stimuli.13 However, the pores are switched only at two states of opening and closure, and adjusting the size range of the opened pores is still difficult. Huang etc. reported adjustment of the size range of the opened pores by using different lengths of cross-linkers, but the extent of cross-linking (EC) was difficult to quantify, and the transmembrane traffic of the 11 nm-substance was inhibited even when using the longest cross-linker.21 In this study, the vesicle membrane was crosslinked via photoinduced dimerization of coumarin, and the reaction extent is easily controlled by the irradiation time so that better quantification of the EC can be achieved. To demonstrate herein that the size range of the opened pores in the vesicle membrane is adjustable, encapsulation tests of the AuNPs with different diameters into the vesicles in an acetate buffer solution (pH 4.6) were conducted. The AuNPs with the diameter (D) of 5 nm were successfully encapsulated into the vesicles obtained after UV irradiation for 120 min (UV 120 min), as shown in Figures 4A and B, but the transmembrane traffic of AuNPs with D of 10 and 15 nm is inhibited (Figures 4A, S7A, and S7B). The above results indicate that swelling of the vesicles in the acetate buffer solution (pH 4.6) induces the formation of pores in the vesicular membrane, and their size is larger than 5 nm but smaller than 10 nm. Another problem is whether the size range of the opened pores in the vesicle membrane can be controlled. Figure 2A reveals that the EC increases with the UV irradiation time increasing, so the EC is easy to control through adjusting the UV irradiation time. Upon UV irradiation for 30 min (UV 30 min), approximately 50% of the coumarin moieties in the membrane of vesicles took part in dimerization (Figure 5A),

vesicles in transmembrane traffic of the substances. Thus, we studied the diameter change with switching pHs of the solution between pH 8 and 4, and the results in Figure 3 display no

Figure 3. Reversible change in diameter of the vesicles (UV 120 min) between pH 4 and 8.

obvious diameter change after at least five cycles, which indicates that the vesicular cross-linked structure is very stable. As we discussed above, this cycling is entirely due to the deprotonation/protonation changes of the PDIPEMA chains in going from a hydrophobic entangle state at high pH value to a hydrophilic state at low pH value, and the cross-linking unit of coumarin dimer prevents the vesicle-to-unimer disassembly in hydrophilic state of the PDIPEMA chains. 3.3. Modulation of the Pore Size in the Vesicle Membrane and Selective Transmembrane Traffic. Modulation of the pore size in the vesicle membrane is essential for selective transmembrane traffic. Yuan and coworkers fabricated CO2 responsive vesicles, and open/closure E

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Figure 4. (A) Schematic encapsulation of gold nanoparticles with different sizes by the vesicles with various EC in an aqueous solution of pH 4.6. TEM images of (B) the vesicles (UV 120 min) loaded with 5 nm gold nanoparticles, (C) the vesicles (UV 30 min) loaded with 10 nm gold nanoparticles, and (D) the vesicles (UV 15 min) loaded with 15 nm gold nanoparticles. The scale bars in the inserted images in B−D are 20, 40, and 40 nm, respectively.

which illustrates that lower EC of the vesicles (UV 30 min) were obtained compared to the cross-linked vesicles (UV 120 min). The lower EC of vesicles did not reduce their stability; the morphology of vesicle (UV 30 min) in DMSO, which is a good solvent for both of the blocks, still kept well (Figure 5B), although Figure 5B reveals the shape difference from the images in Figure 4 and Figure S7. The image in Figure 5B shows the ball with deep dents because the vesicle walls were swollen in DMSO and became thick; evaporation of the solvent in the inner hole of vesicles causes formation of the dents. This is further evidenced by dynamic light scattering results. The hydrodynamic diameter of the vesicles (UV 30 min) in an acetate buffer solution (pH 4.6) is about 1150 nm, which is much larger than that (820 nm) of the vesicles (UV 120 min) in the same medium (Figure 6). The above result indicates that lower cross-linking density in the membrane of vesicles (UV 30 min) was obtained, which may induce formation of bigger pores in the vesicle membrane. This reasonable deduction was demonstrated upon encapsulation of the larger AuNPs, and the TEM image in Figure 4C shows that the AuNPs with D = 10 nm are successfully encapsulated into the vesicles (UV 30 min). However, the transmembrane traffic of AuNPs with D = 15 nm is still inhibited (Figures 4A and S7C), which illustrates that the pores in the cross-linked vesicle membrane (UV 30 min) in acetate buffer solution (pH 4.6) are bigger than 10 nm but

smaller than 15 nm. Upon further reducing the UV irradiation time from 30 to 15 min, the pores with D > 15 nm are formed in the vesicle membrane in an acetate buffer solution (pH 4.6); therefore, the 15 nm AuNPs were successfully encapsulated into the vesicles (UV 15 min), as shown in Figure 4D. Although only about 30% of the coumarin moieties in the membrane of vesicles (UV 15 min) were dimerized, as shown in Figure 5C, the morphology of vesicles (UV 15 min) in good solvent (DMSO) was also good (Figure 5D). As with the vesicles (UV 30 min), the TEM image in Figure 5D reveals the balls with deep dents. The hydrodynamic diameter of the vesicles (UV 15 min) in an acetate buffer solution (pH 4.6) is about 1560 nm (Figure 6), which is much larger than that of the vesicles (UV 30 min), and the larger pores are ascribed to lower cross-linking density of the vesicle membrane. The hydrodynamic diameters of the three cross-linked vesicles (UV 120, 30, and 15 min) in neutral aqueous solution (pH 7.4) have no obvious differences (Figure 6) because the PDIPEMA polymer chains are hydrophobic at this condition. For encapsulation of AuNPs with D = 5, 10, and 15 nm in neutral aqueous solution (pH 7.4), the transmembrane traffic was inhibited for all three vesicles due to closure of the pores in the vesicle membrane. The above results reveal that robust vesicles with pH- and size-selective permeability were successfully fabricated, and the pore sizes for transmembrane traffic of F

DOI: 10.1021/acsami.7b02966 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Table 1. LC% and LE% of Several Substances in the Vesicles (UV 120 min) under Different Conditionsa sodium fluoresceinb LC% LE%

4.62 18.5

DOX·HClb DOX·HClc 0.058 0.012

311.5 62.3

DOX·HCld

DOX·HClc

300 60

323 64.6

a

LC% was the ratio of the weight of the substances encapsulated in vesicles to the weight of the vesicles. LE% was the ratio of the weight of the substances encapsulated in vesicles to the total weight of the substances in feed. bEncapsulation of the substances by the vesicles (UV 120 min) in aqueous solution at pH 4.6. cCalculated according to the fluorescence spectra. dObtained by directly measuring the weight of the vesicles loading with DOX·HCl after lyophilizing.

Figure 5. Characterization of the sample H40D100-20% vesicles after UV irradiation for different time: (A) UV−vis absorbance spectra of the vesicles after UV irradiation for 0 and 30 min. (B) TEM image of the vesicles (UV irradiation for 30 min) in dimethyl sulfoxide (DMSO). (C) UV−vis absorbance spectra of the vesicles after UV irradiation for 0 and 15 min. (D) TEM images of the vesicles (UV irradiation for 15 min) in dimethyl sulfoxide (DMSO).

Figure 7. (A) ζ-potentials in aqueous solution at pH 4.6 of (a) vesicles (UV 120 min), (b) DOX·HCl, and (c) sodium fluorescein; in DMSO of (d) vesicles (UV 120 min) and (e) DOX·HCl; of (f) the mixture of vesicles (UV 120 min), triethylamine, and DOX·HCl in DMSO stirring overnight with redundant triethylamine removed and a large amount of distilled water added; and of (g) the mixture as in (f) after being dialyzed against acidic aqueous solution at pH 3. (B) Cumulative release profiles of fluorescein from the fluorescein-loaded vesicles (UV 120 min) in PBS buffer solution (20 mM, pH 7.4) and acetate buffer solution (20 mM, pH 4.6).

When encapsulation of the DOX·HCl by the vesicles (UV 120 min) was conducted in DMSO, extraordinary high LC% (311.5%) and LE% (62.3%) were obtained (Table 1). The ζpotentials for both of the vesicles and DOX·HCl are close to zero in DMSO (Figures 7Ad and Ae), which can be regarded as electroneutral. Therefore, the transmembrane traffic of the DOX·HCl is allowed in DMSO because the size of DOX·HCl is much less than 5 nm. After addition of a large amount of the distilled water into the mixture of vesicles (UV 120 min), DOX· HCl, DMSO, and triethylamine, the ζ-potential of the resultant mixture is about −28 mV (Figure 7f). This is due to deprotonation of the DOX·HCl via reaction with triethylamine (Figure S8). A vast amount of the electronegative DOX was encapsulated into the vesicles via electrostatic attraction; as a result, much higher LC% (311.5%) and LE% (62.3%) were achieved. The resultant solution of DOX-loaded vesicles (UV 120 min) was freeze-dried, and the obtained solid was weighed for calculation of LC% and LE%, which are, respectively, 300 and 60% (Table 1). It can be seen that LC% and LE% measured by fluorescence and weight methods are very close. To further check the results obtained with the encapsulation method, release of the DOX or fluorescein-loaded vesicles was used to study selectivity of the vesicular membrane. When the solution of vesicles (UV 120 min) loaded with electronegative DOX was dialyzed against acidic aqueous solution at pH 3 for 24 h, LC% and LE% of the DOX-loaded vesicles were measured again, and they were, respectively, 323 and 64.6% (Table 1), which are in accordance with the corresponding results before dialysis. This result indicates that the encapsulated DOX cannot escape from the vesicles. During

Figure 6. Hydrodynamic diameters of the vesicles after UV irradiation for different times in the aqueous solutions of pH 7.4 (red columns) and pH 4.6 (green columns).

substances can be controlled through adjusting the EC (the lower EC resulted in larger pore size of the vesicle membrane in acidic solution). Because the AuNPs (5 nm) can be efficiently encapsulated into the vesicles (UV 120 min), it is reasonable to speculate that a small substance such as sodium fluorescein or doxorubicin hydrochloride (DOX·HCl) (both of them are much smaller than 5 nm) also could be encapsulated. Table 1 shows that the loading content (LC%) and loading efficiency (LE%) of sodium fluorescein in the vesicles (UV 120 min) are 4.62 and 18.5%, respectively, which indicates the successful encapsulation of sodium fluorescein. However, LC% and LE% of the DOX·HCl are negligible (0.058 and 0.012%, Table 1). The ζ-potentials of the vesicles and DOX·HCl in acidic aqueous solution (pH 4) are about 32 and 13 mV, respectively (Figures 7Aa and Ab), so electrostatic repulsion between the vesicles and the DOX·HCl is responsible for inhibition of the transmembrane traffic of DOX·HCl. The ζ-potential of sodium fluorescein in aqueous solution at pH 4.6 is about −22 mV, so the transmembrane traffic of sodium fluorescein is allowed, owing to electrostatic attraction of fluorescein with the vesicle. G

DOI: 10.1021/acsami.7b02966 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

the China Postdoctoral Science Foundation (Grant BH2060000011) is gratefully acknowledged.

the dialysis process, protonation of the vesicle membrane and encapsulation of DOX occurred. The ζ-potential of the protonated DOX-loaded vesicles (UV 120 min) was about 25 mV, as shown in Figure 7Ag. Although the pores in the vesicle membrane are open and large enough for the DOX·HCl to escape in this acidic medium, the transmembrane traffic of DOX·HCl is inhibited due to the electrostatic repulsion. For the fluorescein-loaded vesicles, when they were dialyzed against acetate buffer at pH 4.6 (20 mmol) for 7 h, nearly 80% of the sodium fluorescein was released (Figure 7B). In the acidic solution at pH 4.6, the pores in the vesicle membrane are large enough for the fluorescein to escape, and there is no electrostatic repulsion between the vesicles and fluorescein, so release of the fluorescein is observed. In PBS buffer at pH 7.4 (20 mmol), release of the fluorescein is greatly inhibited due to the closure of the pores at this condition, and only about 5% of the fluorescein was released in 7 h (Figure 7B).

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4. CONCLUSIONS In summary, the artificially smart vesicles herein have superior morphological stability and multiple selectivity for the transmembrane traffic of substances. The vesicles exhibit intelligent pH-regulated membrane permeability due to the tertiary amine groups in the membrane and superior structural stability due to the light-triggered cross-linking of the membrane. The pore size range for transmembrane traffic of substances can be controlled by adjusting the cross-linking density of the membrane. Due to the tertiary amine in the membrane, the vesicles are positively charged in acidic aqueous solution. Transmembrane traffic is inhibited for the electropositive substances but is allowed for the electronegative substances. Furthermore, it is the first time vesicles have been fabricated with selective membrane permeability via the approach of PISR, which allows highly efficient preparation of the intelligent vesicles at up to 30% solid content simultaneously with the polymerization. Given the efficiency and the potential scalability, this superior selfassembly method may well ultimately prove to be the preferred route for fabrication of intelligent and robust vesicles used for nanoreactors, artificial organelles, nanocarriers, and so on.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b02966. 1 H NMR of the CMA, PHPMA, and PHPMAP(DIPEMA-co-CMA); GPC traces; TEM images (Figure S1−S8); and Table S1 (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Cai-Yuan Pan: 0000-0003-2859-3621 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the National Natural Science Foundation of China (Grants 21525420 and 21374107) and H

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