Polymersomes with Rapid K+-Triggered Drug-Release Behaviors

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Polymersomes with Rapid K+-Triggered Drug Release Behaviors Xiang-Ru You, Xiao-Jie Ju, Fan He, Yuan Wang, Zhuang Liu, Wei Wang, Rui Xie, and Liang-Yin Chu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 17 May 2017 Downloaded from http://pubs.acs.org on May 20, 2017

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ACS Applied Materials & Interfaces

Polymersomes with Rapid K+-Triggered Drug Release Behaviors Xiang-Ru You,† Xiao-Jie Ju,*,†,‡ Fan He,† Yuan Wang,† Zhuang Liu,† Wei Wang,†,‡ Rui Xie,†,‡ and Liang-Yin Chu†,‡

†

School of Chemical Engineering, Sichuan University, Chengdu, Sichuan 610065, P. R. China

‡

State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu, Sichuan

610065, P. R. China

ACS Paragon Plus Environment

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ABSTRACT: A novel type of smart polymersomes with rapid K+-triggered drug release property is developed in this work. Block copolymers with biocompatible poly(ethylene glycol) (PEG) as the hydrophilic block and poly(N-isopropylacrylamide-co-benzo-18-crown-6acrylamide) (PNB) copolymer as the K+-responsive block are successfully synthesized. Due to the presence of 18-crown-6 units, the PEG-b-PNB block copolymers exhibit excellent K+dependent phase transition behaviors, which show hydrophilic-hydrophobic state in simulated extracellular fluid and present hydrophilic-hydrophilic state in simulated intracellular fluid. Polymersomes with regular spherical shape and good monodispersity are prepared by the selfassembly of the PEG-b-PNB block copolymers.

Both hydrophilic FITC-dextran and

hydrophobic DOX are selected as model drugs and are successfully encapsulated into the PEGb-PNB polymersomes. After being placed into the simulated intracellular fluid with high K+ concentration, the PEG-b-PNB polymersomes immediately disassemble accompanied with rapid and complete release of drugs. Such K+-responsive polymersomes with desired drug release properties provide a novel strategy for advanced intracellular drug delivery and release, which can enhance the safety and efficacy of cancer therapy. KEYWORDS:

polymersomes; responsive materials; drug delivery systems; K+-triggered

release; block copolymers


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INTRODUCTION Despite great achievements in cancer chemotherapy, enhancing the efficacy and reducing the side effects of chemotherapeutic agents to tumor cells remain great challenges. In the past decade, nanomedicine utilizing various nanoconstructs as drug carriers presents significant potential to improve cancer treatment.1-5 Among the nanocarriers, polymersomes, which are also referred to as polymeric vesicles, attract growing interests due to their versatile architectures. Due to the vesicle-like nanostructure, in which an aqueous core is enclosed by a thick bilayer membrane formed by self-assembly of amphiphilic block copolymers,6,7 polymersomes show great potential in biomedical fields as versatile nanocarriers to encapsulate various active molecules, such as diagnostic reagents,8-10 therapeutic drugs,11,12 proteins and genes.13,14 In contrast to block copolymer micelles, polymersomes can encapsulate not only hydrophobic or fatty molecules into their bilayer membranes, but also hydrophilic compounds within the aqueous core.15-17 Meanwhile, polymersomes exhibit a high stability for long blood circulation as compared to liposomes,18,19 and excellent loading capacity of drugs, robustness of membrane and unique property of stealth.20

Due to their diverse loading capacity and membrane

robustness, polymersomes are highly attractive for drug delivery applications. Particularly, the development of stimuli-responsive polymersomes to further control the release of drugs has attracted a lot of interests and been extensively studied.21-23 The smart polymersomes can release drugs by changing the stability and permeability of the bilayer membrane in response to diverse environmental stimuli at the target sites. Such controlled release behaviors can significantly improve the therapeutic effect of drugs and minimize their side effects.24 Up to now, various stimuli-responsive block copolymers have been synthesized to construct smart polymersomes. According to the type of environmental stimuli, the stimuli-


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responsive polymersomes can be divided into in-vitro-stimuli-responsive type and in-vivostimuli-responsive type. Smart polymersomes responding to the in vitro stimuli (also referred to as exogenous stimuli) such as temperature,24-29 light30,31 and magnetic field,32,33 are usually achieved with the help of external equipment such as cryoprobe, UV light, far-infrared radiation and magnetic field, which increase the cost of the therapy and dependence of specialists. In addition, the drug release triggered by the in vitro stimuli is lack of precision. On the contrary, smart polymersomes responding to the in vivo stimuli (also called endogenous stimuli) such as pH,34-39 ion,40 glucose41 and redox,38,42,43 enable the release of drugs precisely at the lesion sites. Among them, pH-responsive polymersomes have been researched the most extensively, because there exist lots of pH gradients in human body.34-39 But, the pH gradients between different pathological sites or different organelles within cells are subtle, which limits the accuracy of drug release. Intracellular release of drugs could further enhance drug effectiveness and reduce drug side effects, which is benefit for cancer therapy. Cell cytosol exhibits very high glutathione (GSH) concentration (2~10 mM) as compared to that in the extracellular compartment (2~20 µM). By utilizing such redox potential as a stimulus, redox-responsive polymersomes were developed for intracellular drug release.43 In addition to pH and GSH differences in human body, there exists a significant difference in K+ concentrations between intracellular fluid (140150 mM) and extracellular fluid (3.5-5.5 mM). Thus, polymersomes that could recognize and respond to such significant K+ stimulus, provide an alternative promising and appealing platform to realize effective and rapid intracellular drug release. In this work, we develop a novel type of polymersomes with rapid K+-triggered drug release properties. The polymersomes could switch their self-assembled bilayer structures in response to different K+ concentrations. In our previous reports44,45, poly(N-isopropylacrylamide-co-benzo-


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18-crown-6-acrylamide) (PNB) copolymers with 18-crown-6 as the K+ sensor presented obvious K+-responsive hydrophilic/hydrophobic phase transition.

To construct the K+-responsive

polymersomes, poly(ethylene glycol) (PEG) with hydrophilicity and excellent biocompatibility is used as the hydrophilic block and the PNB copolymer acts as the K+-responsive block for the synthesis of PEG-b-PNB block copolymers (Figure 1a). By controlling the content of 18-crown6 units in PEG-b-PNB, the lower critical solution temperature (LCST) of the block copolymers is adjusted to be lower than 37 oC (Ta) in extracellular fluid while be higher than 37 oC (Tb) in intracellular fluid (Figure 1b). At 37 oC between these two LCST values of Ta and Tb, the PNB block is in hydrophobic state in aqueous solution without K+, and the PEG-b-PNB block copolymers presenting amphiphilic characteristics could self-assembly into polymersomes, and thus encapsulating hydrophilic and/or hydrophobic drugs.

Similarly, the PNB block is

hydrophobic in extracellular fluid at 37 oC (B2 in Figure 1b), so that polymersomes could maintain their structural integrity and well encapsulation of drugs.

After isothermally

transferring into intracellular fluid, 18-crown-6 units immediately capture K+ to form stable complexes, leading to a positive LCST shift of PEG-b-PNB block copolymers to a higher temperature. That is, the PNB block transfers from hydrophobic state into hydrophilic state in intracellular fluid at 37 oC (B1 in Figure 1b). As a result, the amphiphilic equilibrium of PEG-bPNB block copolymers is destructed, so that polymersomes rapidly dissociate and the loaded drugs are released quickly (Figure 1c). Such smart polymersomes with desired K+-triggered rapid drug release properties provide a novel strategy for advanced intracellular drug delivery and release, which can enhance the safety and efficacy of cancer therapy.

EXPERIMENTAL SECTION


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Materials. Benzo-18-crown-6-acrylamide (BCAm) was obtaind by modifying 4’-nitro-benzo18-crown-6 (98%, TCI) according to previously reported method.46,47 N-isopropylacrylamide (NIPAAm, 98%, TCI) was recrystallized from a mixture of hexane and acetone.

2,2’-

azoisobutyronitrile (AIBN, 98%, Aladdin) was used after recrystallization with ethanol. Poly(ethylene glycol) methyl ether amine (mPEG-NH2, 2000 Da, Aladdin), fluorescein isothiocyanate-dextran

(FITC-dextran,

4000

Da,

Sigma-Aldrich),

2-

dodecylsulfanylthiocarbonylsulfanyl-2-methyl propionic acid (DMP, 98%, Sigma-Aldrich), phosphotungstic acid (2% w/w, Beijing Zhongjingkeyi Technology) and doxorubicin hydrochloride (DOX·HCl, 98%, Meilun biochemical) were used without further purification. All solvents and other chemicals with analytical grade were used as received (Chengdu Kelong Chemical Reagents Co., Ltd.).

Deionized water (18.2 MΩ, 25 oC) from Millipore water

purification system was used in whole experments. Synthesis of Amphiphilic Block Copolymers.

Firstly, carboxyl-terminated poly(N-

isopropylacrylamide) (PN) and PNB polymers were synthesized by the reversible additionfragmentation chain transfer polymerization (RAFT) method with DMP as the chain transfer agent and AIBN as the initiator according to reported work.48

The molecular weights of

polymers can be flexibly controlled by adjusting the mole ratios of monomer, chain transfer agent and initiator during the synthesis process.49 As shown in Table 1, carboxyl-terminated PN polymers with different molecular weights labeled as PN1, PN2 and PN3 were synthesized. Briefly, NIPAAm, DMP and AIBN were dissolved in 1,4-dioxane, and the NIPAAm concentration was 0.3 mol L-1. Then the mixed solution was reacted under nitrogen atmosphere at 70 oC for 24 hours.

After polymerization completed, the solution was cooled to room

temperature to stop the reaction. The reaction solution was diluted with tetrahydrofuran (THF)


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and then precipitated by anhydrous ether. This process was repeated for three times to remove the unreacted reactants. After drying under vacuum at 25 oC for 12 hours, carboxyl-terminated PN polymers were obtained. Similarly, carboxyl-terminated PNB copolymers with different mole ratios of BCAm were synthesized (Figure 2). The feed mole ratios of BCAm in total monomers were 10%, 15% and 20%, which were labeled as PNB1, PNB2 and PNB3, respectively. PEG-b-PN and PEG-b-PNB block copolymers were synthesized by a condensation reaction with EDC as condensation agent. PEG-b-PN block copolymers with different molecular weight ratios of hydrophilic block to total block copolymers were synthesized. Briefly, carboxylterminated PN, EDC and NHS were dissolved in deionized water, which was cooled down to 3 o

C and purged with nitrogen for 20 minutes to remove the dissolved oxygen. mPEG-NH2

aqueous solution was then dropwise added into the mixed solution, and the reaction was carried out for 24 hours. The synthetized block copolymers were purified by dialysis against deionized water for one week, and the water was refreshed every 8 hours. Finally, PEG-b-PN block copolymer was obtained by freeze-drying.

Similarly, to synthesize PEG-b-PNB block

copolymers, carboxyl-terminated PNB copolymers were used instead of carboxyl-terminated PN polymers in the condensation reaction (Figure 2). Three kinds of PEG-b-PNB block copolymers with different mole ratios of BCAm were synthesized, and labeled as PENB1, PENB2 and PENB3, respectively. Composition Characterizations of the Synthesized Polymers.

The composition of the

synthesized polymers was characterized by Fourier transform infrared spectrometry (FT-IR; IR Prestige-21, Shimadzu) and nuclear magnetic resonance spectrometry (1H NMR; Bruker-400, Bruker). FT-IR samples were prepared by KBr disc technique and 1H NMR samples were


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prepared as 1% (w/v) solutions in D2O (HOD internal standard). The molecular weights of the synthesized polymers were determined by gel permeation chromatography (GPC; Waters-2410, Waters). The actual mole ratios of BCAm units in PNB copolymers (Cn) were determined from 1

H NMR results. The calculated method is shown in the Supporting Information. The critical

micelle concentration (CMC) values of the block copolymers were determined using a fluorescence spectrometer (RF5301PC, Shimadzu) and pyrene was used as a fluorescent probe. Study of the K+-Responsive Properties of PNB Copolymers and PEG-b-PNB Block Copolymers.

The K+-responsive behaviors of PNB copolymers with different contents of

BCAm and corresponding PEG-b-PNB block copolymers were investigated by evaluating their LCST values in different K+ solutions. PN and PEG-b-PN polymers were used as the control groups. The LCST values of the polymers were dermined by testing the temperature-dependent optical transmittances of polymer solutions using a temperature-controlled UV−vis spectrometer (UV-1700, Shimadzu).

The LCST was defined as the temperature at which the optical

transmittance changing to half of the initial value. As is known, although there are many cations in human body, 99% of them are K+ and Na+. Therefore, the aqueous solution containing 5 mM K+ was used as the simulated extracellular fluid, and the aqueous solution with 150 mM K+ was used as the simulated intracellular fluid. A certain amount of Na+ was added into K+ solutions to avoid the interference of the ionic strength. The polymer concentrations in the solutions were fixed at 5 mg mL-1. Preparation of Polymersomes. Polymersomes assembled from PEG-b-PNB were prepared by a dialysis method.50-52 Briefly, PEG-b-PNB block copolymers were dissolved in 1,4-dioxane to reach a concentration of 0.5% (w/w). To induce the self-assembly of the block copolymers to form polymersomes, deionized water was added into the 1,4-dioxane solution under vigorous


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stirring at an adding speed of 0.1 wt% per minute by an injection pump (LSP01-2A, Baoding Longer Precision Pump). Based on previous reported work, the added water amount was 50 wt% in total solution.52 After two more hours stirring, the suspension was dialyzed against deionized water for one week to remove organic solvent. The water was refreshed every 8 hours. The whole preparation processes were carried out at 45 oC, and the resultant polymersomescontaining solution was also kept in an incubator of 45 oC. DOX was used as hydrophobic model drug and FITC-dextran was used as the hydrophilic model drug for fabrication of drugloaded polymersomes. The preparation processes of DOX-loaded polymersomes and FITCdextran-loaded polymersomes were different. 1 mg mL-1 FITC-dextran was dissolved in the deionized water, while 1 mg mL-1 DOX was dissolved in the 1,4-dioxane.

Drug-loaded

polymersomes self-assembled by PEG-b-PN block copolymers were used as the control groups. The drug entrapment efficiency (EE) and loading content (DL) were calculated using the following equations: 𝐸𝐸 = 𝐷𝐿 =

#$ #%

#$ #,

×100%

× 100%

(1) (2)

Where ma means the mass of drug loaded into polymersomes, mb means the mass of drug fed initially and mc means the mass of drug-loaded polymersome. Morphology Characterization of Polymersomes. The morphology of polymersomes was investigated by transmission electron microscope (TEM; Tecnai G2 F20 S-TWIN, FEI). TEM specimens were prepared by dropping 20 µL polymersomes-containing solution onto a copper grid. A few minutes later, the specimens were stained with phosphotungstic acid solution for 30 seconds and then dried in air. The hydrodynamic diameter of polymersomes was measured by dynamic light scattering (DLS) technique using a laser nanoparticle sizer (ZEN3690, Malvern).


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Study of the K+-Triggered Drug Release Behaviors of Polymersomes. The K+-triggered disassembly of polymersomes was studied by evaluating the optical transmittance change of the polymersomes-containing solution at 37 oC by UV-vis spectrometer at regular intervals. During the test process, the ambient solution of polymersomes was changed from the simulated extracellular fluid to the simulated intracellular fluid. For investigating the K+-triggered release behaviors of FITC-dextran and DOX from polymersomes, the absorbance changes of FITCdextran and DOX in the ambient mediums were monitored by UV-vis spectrometer at 493.5 nm and 495.5 nm respectively.

RESULTS AND DISCUSSION Compositions of PNB Copolymers and PEG-b-PNB Block Copolymers. The structure and morphology of aggregates self-assembled from the block copolymers depends on many factors such as the composition and initial concentration of the block copolymers, the nature of the cosolvent and the content of water, etc.53,54 Generally, the block copolymers with the ratio of hydrophilic block to hydrophobic block higher than 1:1 tend to form micelles; while the ratio lower than 1:2 favors the formation of polymersomes.55,56 Therefore, in this work, molecular weight ratio of the hydrophilic block to the total block copolymers is expected to be less than 30%. By controlling the mole ratio of NIPAAm, DMP and AIBN during the synthesis process, the molecular weight of PN polymers can be flexibly adjusted (Table 1). When the mole ratio of NIPAAm, DMP and AIBN is 150:1:0.2, the molecular weight of PN3 is about 12,000 Da. The FT-IR spectrum of PN3 is shown in Figure S1b in the Supporting Information. After combined with PEG with molecular weight of 2000 Da, the molecular weight ratio of hydrophilic block in PEG-b-PN block copolymer is about 14%, which is suitable for fabricating polymersomes.52,53


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The molecular weights of BCAm-based copolymers cannot be precisely measured by GPC method. The reason may be that there exists certain absorption effect between crown ether and currently-existed chromatographic column, so the copolymers containing crown ether units are difficult to be washed off during the measurements with the currently available instrument, which causes that the measured data are much lower than the true values. The molecular weights of PN and PNB polymers are both mainly determined by the mole ratio of monomer, chain transfer agent and initiator during the synthesis process. So, to synthesize PNB copolymers with suitable molecular weight for fabricating PEG-b-PNB polymersomes, the preparation recipes of PNB copolymers are similar to that of PN polymers. To ensure the polymersomes developed in this work are ideal drug delivery systems for human body, which are expected to release drug at 37 oC, it is essential for the LCST values of PEG-bPNB block copolymer to be lower than 37 oC in simulated extracellular fluid and higher than 37 o

C in simulated intracellular fluid. The adjustment of the LCST values could be achieved by

controlling the content of BCAm in PEG-b-PNB. The operation temperature for K+-triggered drug release would be around 37 oC between these two LCST values.

As shown in the

Supporting Information, the chemical compositions of PNB copolymers are characterized by FTIR and 1H NMR (Figure S1c-e and Figure S3). Calculated from the 1H NMR results, the Cn values of PNB1, PNB2 and PNB3 are 8.2%, 13.8% and 18.1%, respectively. The chemical compositions of PENB1, PENB2 and PENB3 block copolymers are also characterized by FT-IR (Figure S2 in the Supporting Information) and 1H NMR (Figure 3). The 1H chemical shifts at around 1.19, 7.06 and 3.70 ppm are the characteristic peaks of protons on the isopropyl, benzene ring and methylene group, respectively.

FT-IR results are analyzed and discussed in the


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Supporting Information. The characteristic peaks in the 1H NMR spectra and FT-IR spectra indicate the successful synthesis of PEG-b-PNB block copolymers. K+-Responsive Properties of PNB Copolymers and PEG-b-PNB Block Copolymers. The effects of cations on the stability of polymersomes result from the their influences on the hydrophilic-hydrophobic equilibrium states of the block copolymers. In our previous study, the effects of K+, Na+, Mg2+ and Ca2+ ions, which are the main cations in human body, on the phase transitions of PNB copolymers have been systematically investigated.44 The results indicated that, among those cations, the PNB copolymers show good selectivity and sensitivity only towards K+. Similarly, the Na+, Mg2+ and Ca2+ ions should show little effects on the hydrophilichydrophobic equilibrium states of the PEG-b-PNB block copolymers. Therefore, only the effects of K+ concentrations on the LCST values of the PEG-b-PNB block copolymers are investigated. Temperature-dependent optical transmittance changes of PNB1, PNB2 and PNB3 copolymer solutions with different K+ concentrations are shown in Figure 4a-c. Due to the presence of 18crown-6, all PNB copolymers exhibit obvious K+-responsive properties. For the copolymers, the LCST values increase with changing K+ concentration from 5 mM to 150 mM. For PNB1, PNB2 and PNB3, the LCST values are 33.8, 33.5 and 34.7 oC in simulated extracellular fluid, while change to 36.9, 38.7 and 44.8 oC respectively in simulated intracellular fluid. The BCAm units in PNB copolymers can capture K+ to form stable inclusion complexes, which could increase the hydrophilicity of PNB copolymers, and thus lead to the increase of the corresponding LCST values. With increasing the mole ratio of BCAm in PNB copolymers, the difference of LCST values in simulated extracellular and intracellular fluids increases (Figure 4d). PENB1, PENB2 and PENB3 solutions with different K+ concentrations exhibit similar LCST shift (Figure 5). The LCST values of PENB1, PENB2 and PENB3 block copolymers in


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simulated extracellular and intracellular fluids are almost the same as the corresponding PNB1, PNB2, and PNB3 copolymers. That is, the combination of PEG block does not affect the K+responsive behavior of PNB copolymers. To successfully achieve intracellular drug release in vivo, the LCST values of PEG-b-PNB block copolymers need to be lower than 37 oC in simulated extracellular fluid, while be higher than 37 oC in simulated intracellular fluid, which allows a phase transition of PNB block from hydrophobic state (in simulated extracellular fluid) to hydrophilic state (in simulated intracellular fluid) at 37 oC.

Theoretically, these block

copolymers are all suitable for the application in human body. However, the LCST values of PENB1 and PENB2 in simulated intracellular fluid are close to 37 oC, which means the temperature range for polymersome disassembly is narrow. The temperature of diseased regions sometimes is higher than normal body temperature, that the polymersomes may not disassemble in intracellular fluid. Therefore, PENB3 is chosen to prepare polymersomes for drug release in this work due to its appropriate LCST values in simulated intracellular and extracellular fluids as well as the large LCST shift value. Therefore, PENB3 is chosen to prepare polymersomes for drug release in this work due to its appropriate LCST values in simulated intracellular and extracellular fluids as well as the large LCST shift value. As shown in Figure S6 in the Supporting Information, the CMC value of PENB3 is 32 mg·L-1. Unlike PNB and PEG-b-PNB, PN3 and PEG-b-PN polymers show little change in LCST with increasing K+ concentration from 5 mM to 150 mM (Figure S4), indicating PN3 and PEG-b-PN polymers without 18-crown-6 units do not have K+-responsive characteristics. Morphology Characterization of Polymersomes.

The morphologies of the resultant

polymersomes self-assembled from PENB3 block copolymers are analyzed by TEM and DLS. Blank, FITC-dextran-loaded and DOX-loaded polymersomes all exhibit regular spherical shapes


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and typical hollow structures as evidenced by their TEM images of higher transmission in the center compared to the periphery (Figure 6a-c). Meanwhile, the prepared polymersomes show good monodispersity (Figure 6e-f). The average diameters of blank, FITC-dextran-loaded and DOX-loaded polymersomes are 255, 396 and 342 nm respectively, with polydispersity index (PDI) values of 0.107, 0.114 and 0.119 respectively (Figure 6e-f), which are calculated by DLS method using a variant of the so-called cumulants method57. Due to the encapsulation of drugs, the diameters of drug-loaded polymersomes (Figure 6b and 6c) are larger than that of the blank polymersomes (Figure 6a). In addition, hydrophilic FITC-dextran loaded in the core of the polymersomes can cause a higher osmotic pressure, which results in more water diffusing into the polymersomes and a larger size of the polymersomes.

It should be noted that the

polymersome size measured by DLS is larger than that observed by TEM. The reason is that DLS reflects the hydrodynamic diameter of polymersomes while TEM shows the images of polymersomes in dried state. K+-Triggered Disassembly and In Vitro Drug Release of Polymersomes. The K+-triggered disassembly behavior of PEG-b-PNB polymersomes can be evaluated from the optical transmittance change of the polymersomes-containing solution. Aqueous solution containing polymersomes is an opaque suspension, thus its optical transmittance is low. While, when the polymersome structure is disassembled and the block copolymers are dissolved in water, the aqueous solution becomes transparent and its optical transmittance is high. As expected, for polymersomes constructed from the PEG-b-PN block copolymer, the polymersomes-containing solutions are both turbid and their optical transmittance exhibits nearly no change when changing the ambient K+ concentration from 5 mM to 150 mM at 37 oC (Figure 7a). Namely, the polymersome structure of PEG-b-PN could maintain intact upon the stimulus of large K+


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concentration. On the contrary, for polymersomes constructed from PENB3 block copolymers, as soon as the ambient solution is changed from simulated extracellular fluid to simulated intracellular fluid, the optical transmittance shows a dramatic increase from 3% to 99% immediately, and the solution change from turbid to transparent (Figure 7b), which is caused by the disassembly of the polymersomes. As discussed above, when increasing the ambient K+ concentration from 5 mM to 150 mM at 37 oC, the PNB block in PENB3 block copolymers immediately changes from hydrophobic state to hydrophilic state, which leads to the phase transition of PENB3 from hydrophilic-hydrophobic amphiphilic state to hydrophilic-hydrophilic state. As a result, the PENB3 polymersomes could dissemble rapidly in simulated intracellular fluid with high K+ concentration, thus hydrophilic PENB3 could dissolve in water and its aqueous solution becomes transparent. The results indicate that the PENB3 polymersomes exhibit excellent and rapid K+-triggered disassembly characteristics and show great potential as intracellular drug delivery system. The K+-triggered drug release behaviors of PENB3 polymersomes are investigated using FITC-dextran and DOX as the hydrophilic and hydrophobic model drugs respectively. The EE values of FITC-dextran-loaded and DOX-loaded PENB3 polymersomes are 36% and 1% respectively, and their DL values are 7.2% and 0.2% respectively. Different drug loading positions result in such distinct difference of the drug loading capacities. Hydrophilic FITCdextran is loaded in the aqueous core of polymersomes with a large volume, while hydrophobic DOX is loaded in the thin bilayer membrane of the polymersomes. The drug concentrations in the ambient solutions of drug-loaded polymersomes are real-timely monitored. For PEG-b-PN polymersomes, neither FITC-dextran nor DOX is released in both simulated extracellular and intracellular fluids at 37 oC (Figure 8a and 9a), because the PEG-b-PN block copolymers without


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K+-responsive property could maintain the intact polymersome structures in both simulated extracellular and simulated intracellular fluids at 37 oC, which keep the well-encapsulation of the drugs. While for K+-responsive PENB3 polymersomes, rapid and complete releases of FITCdextran and DOX, which are caused by the rapid disassembly of the polymersomes, are both observed within 1 minute upon turning the simulated extracellular fluids into simulated intracellular fluids at 37 oC (Figure 8b and 9b). The results demonstrate that the proposed PENB3 polymersomes show flexible drug loading capacity for both hydrophilic and hydrophobic drugs, and present a rapid and desirable K+-triggered drug release behaviors. Such K+-responsive characteristics ensure the complete and in-time release of drugs only in the intracellular environment, which is benefit to the improvement of the efficiency of cancer therapy and reduction of the side effects.

CONCLUSIONS In summary, a novel type of polymersomes with rapid K+-triggered drug release behavior is developed as an advanced intracellular drug delivery system.

PEG with excellent

biocompatibility is used as the hydrophilic block and PNB copolymer containing 18-crown-6 is acted as K+-responsive block for the synthesis of K+-responsive PEG-b-PNB block copolymer. In response to the signal of increased K+ concentration, the LCST of PEG-b-PNB block shifts to a high temperature. Polymersomes with regular spherical shape and good monodispersity are prepared by the self-assembly of PEG-b-PNB block copolymers, and exhibit flexible loading capacity of both hydrophilic and hydrophobic model drugs.

Due to the K+-triggered

hydrophobic-to-hydrophilic transition of PNB block, the PEG-b-PNB polymersomes immediately disassemble at 37

o

C when the ambient solution changes from simulated


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extracellular fluid to simulated intracellular fluid with high K+ concentration. Accompanied with the disassembly of polymersome structure, the encapsulated drugs are rapidly and completely released.

For further application for cancer therapy, suitable targeted aptamers could be

introduced on the surface of these polymersomes to made them positively aggregate at tumor sites.58,59 The polymersomes with desired K+-triggered rapid drug release properties proposed in this work provide a brand-new strategy for designing novel intracellular targeted drug delivery systems.

ASSOCIATED CONTENT: Supporting Information Available: FT-IR spectra of PN, PNB and PEG-b-PNB polymers, 1H NMR spectra of PNB copolymers, temperature-dependent optical transmittance changes of PN polymers and PEG-b-PN block copolymers, TEM image and size distribution of PEG-b-PN polymersomes, and the measurement of the CMC value of PENB3. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors * E-mail: [email protected] (X.-J. Ju) Author Contributions The manuscript was written through contributions of all authors.

All authors have given

approval to the final version of the manuscript. Notes


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The authors declare no competing financial interest.

ACKNOWLEDGMENTS The authors gratefully acknowledge support from the National Natural Science Foundation of China (91434202), Fok Ying-Tung Education Foundation for Young Teachers in the Higher Education Institutions of China (151070), Sichuan Provincial Youth Science and Technology Foundation (2017JQ0027), and the Program for Changjiang Scholars and Innovative Research Team in University (IRT15R48).

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TABLES Table 1 Mole ratios of the reactants used for the synthesis of PN polymers and resultant molecular weights and PDI values of PN polymers Polymer

NIPAAm : DMP : AIBN

Mn (Da)

PDI

PN1

100 : 1 : 0.2

6721

1.36

PN2

120 : 1 : 0.2

8031

1.43

PN3

150 : 1 : 0.2

11991

1.28


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Figures

Figure 1. Schematic illustration of the formation and K+-triggered drug release behavior of PEGb-PNB polymersomes. (a) Structural formula of PEG-b-PNB block copolymers. (b) Phase transition of PEG-b-PNB block copolymer versus temperature in extracellular and intracellular fluids, in which B1 and B2 show the phase states of the block copolymers complexed with (B1) and without (B2) K+. (c) Preparation and K+-triggered release process of hydrophilic drugloaded polymersomes.


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Figure 2. Synthesis route of PEG-b-PNB block copolymers.


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Figure 3. 1H NMR spectra of PENB3 (a), PENB2 (b) and PENB1 (c) block copolymers with different mole ratios of BCAm.


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Figure 4. K+-responsive property of PNB copolymers. (a-c) Temperature-dependent optical transmittance changes of PNB1 (a), PNB2 (b) and PNB3 (c) in different aqueous solutions. (d) LCST values of PNB1, PNB2 and PNB3 in different aqueous solutions.


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Figure 5. K+-responsive property of PEG-b-PNB block copolymers. (a-c) Temperaturedependent optical transmittance changes of PENB1 (a), PENB2 (b) and PENB3 (c) in different aqueous solutions. (d) LCST values of PENB1, PENB2 and PENB3 in different aqueous solutions.


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Figure 6. Morphology characterizations of PENB3 polymersomes. (a-c) TEM images of blank polymersomes (a), FITC-dextran-loaded polymersomes (b) and DOX-loaded polymersomes (c). (d-f) Size distributions of blank polymersomes (d), FITC-dextran loaded polymersomes (e) and DOX loaded polymersomes (f).


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Figure 7. Optical transmittance changes of aqueous solutions containing PEG-b-PN polymersomes (a) and PENB3 polymersomes (b) upon changing the simulated extracellular fluids into simulated intracellular fluids at 37 oC.


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Figure 8. K+-triggered hydrophilic FITC-dextran release behaviors from of PEG-b-PN polymersomes (a) and PENB3 polymersomes (b) upon changing the simulated extracellular fluids into simulated intracellular fluids at 37 oC.


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Figure 9. K+-triggered hydrophobic DOX release behaviors of PEG-b-PN polymersomes (a) and PENB3 polymersomes (b) upon changing the simulated extracellular fluids into simulated intracellular fluids at 37 oC.


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Graphic for TOC


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Polymersomes with Rapid K+-Triggered Drug-Release Behaviors

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