Thermo- and pH-Responsive, Coacervate-Forming Hyperbranched

Feb 7, 2017 - What should be also noted is that previously, Liu et al. employed an A2+BB′B″ Michael addition approach for HPAE synthesis. Due to t...
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Thermo- and pH-Responsive, Coacervate-Forming Hyperbranched Poly(β-amino ester)s for Selective Cell Binding Dezhong Zhou,†,§ Luca Pierucci,†,§ Yongsheng Gao,†,§ Jonathan O’Keeffe Ahern,†,§ Xiaobei Huang,§,⊥ A. Sigen,§ and Wenxin Wang*,‡,§ ‡

School of Materials Science and Engineering, Tianjin University, Tianjin 300072, China Charles Institute of Dermatology, School of Medicine, University College Dublin, Dublin 4, Ireland ⊥ School of Materials Science and Engineering, Sichuan University, Chengdu 610064, China §

S Supporting Information *

ABSTRACT: We report a new type of thermo- and pH-responsive, coacervateforming highly degradable polymer-hyperbranched poly(β-amino esters) (HPAEs) and its selective cell binding behaviors. The HPAEs were synthesized from 5-amino-1-pentanol (S5) and trimethylolpropane ethoxylate triacrylate (TMPETA) via an A2+B3 type Michael addition. The existence of multiple hydrogen bond pairs as well as tertiary amines makes the S5-TMPETA polymers manifest temperature- and pH-dependent phase transition. By varying the length of the ethylene glycol (EG) spacers in the TMPETA, polymer molecular weight, concentration, and pH value, the phase transition of the S5-TMPETA can be easily tuned in aqueous and buffer solutions, as evidenced by UV−vis spectroscopy and DLS measurements. Especially, the S5-TMPETA prepared from S5 and trimethylolpropane ethoxylate triacrylate 692 (S5-TMPETA692) shows a lower critical solution temperature (LCST) around 33 °C, above which the S5TMPTEA can form coacervate particles able to encapsulate functional molecules effectively. Importantly, when incubation with HeLa cells, the S5-TMPTETA692 exhibits a temperature- and pH-responsive selective cell binding behaviors. In addition, the S5TMPETA are highly hydrolyzable and elicit negligible cytotoxicity. This new type of “smart” polymer should find use in a variety of biomedical applications. KEYWORDS: hyperbranched polymers, thermo-sensitivity, pH sensitivity, coacervate-forming, selective cell binding

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INTRODUCTION “Smart” materials are one type of the most interesting materials because they are able to respond to various external stimuli including pH, temperature, ionic strength, light, and chemical and biological stimuli.1−8 Polymers are one of the major types of “smart” materials because the building blocks are widely commercially available and readily customized.9−14 Of the various “smart” polymers, thermoresponsive polymers have attracted broad attention.9,15−22 In general, there are two types of thermoresponsive polymers. The first show a lower critical solution temperature (LCST) and the second show an upper critical solution temperature (UCST).13,23−26 LCST and UCST are the critical temperature below and above which the polymers are completely miscible with the solvents.27 This process happens because it is energetically favorable.20,24,28 The main driving force for this process is the water entropy. Once the polymer is not in solution, the water molecules are less ordered and thus have a higher entropy. This effect is also described as “the hydrophobic effect”.27,29 The responsive polymers are soluble because of the extensive hydrogen bonding interactions with the water molecules, which limited the polymer molecules’ intra- and intermolecular hydrogen bonding interactions. 20 Comparatively, there are more polymers presenting LCST than the UCST.13 Above the © XXXX American Chemical Society

LCST, the hydrogen bonding of polymers with water molecules will be disrupted, the intra- and intermolecular hydrogen bonding or hydrophobic interactions will dominate, therefore a transition in solubility ocurrs.30 The LCST is usually tuned by incorporating hydrophilic or hydrophobic components by copolymerization with corresponding comonomers or terminal groups.31,32 By increasing the hydrophilic nature of the polymers, the hydrogen bonding interactions are increased, which leads to a higher phase transition temperature. On the other hand, by increasing hydrophobic components the LCST will be lowered. The addition of hydrophobic groups can cause a disruption to the structure of water molecules around the polymers and thus enhances the interactions of hydrophobic species that are easy to aggregate.33 To date, a variety of linear and dendritic polymers exhibiting LCST have been designed and synthesized including poly(Nsubstituted (meth)acrylamide)s,34 poly(N-vinylalkylamide),35 poly(N-acryloyl-N′-alkylpiperazine),36 poly(dimethylaminoethyl methacrylate),37 poly(alkyloxide) copolymer,38 poly(phosphazene)s,39 poly(vinyl ether)s and polyReceived: November 23, 2016 Accepted: January 23, 2017

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DOI: 10.1021/acsami.6b15005 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces ethersl etc.40,41 The LCST spans a broad temperature range from 0 to 100 °C and can be adjusted easily.13 The most intensively investigated polymer has been the PNIPAM,42,43 which shows a LCST at 32 °C that is close to normal physiological body temperature (37 °C).24 The intriguing thermosensitive behaviors make these polymers be widely used in controlled drug delivery, bioseparations, filtration, smart surface, regulation of enzyme activity, etc., in the forms of hydrogels, interpenetrating networks, micelles, films, particles, and polymersomes.13,44 Despite the advantages, however, the synthesis of these polymers or the monomers are usually complicated; the majority of the polymers are nondegradable or degrade slowly, whereas polymer degradation is a highly desirable quality for numerous biomedical applications.9,20 Moreover, above LCST, most of these polymers undergo a coil−globule transition that results in significant dehydration which would leads to polymer conformational changes and potentially damage the sensitive biomacromolecules encapsulated. In contrast, coacervate-type thermosensitive polymers are more attractive.45,46 Poly(β-amino ester) (PAE) is a type of polymer that can be synthesized via conjugate addition of amines to acrylates under mild reaction conditions without a catalyst, initiator or high pressure.47−50 Various commercially available amines and acrylates can be used as monomers making the chemical compositions and structures of PAEs highly flexible and tailorable.51 Importantly, the multiple ester groups in the backbone of PAEs can degrade via hydrolysis in physiological conditions, which reduces the safety concerns for in vitro and in vivo applications.52 To date, thousands of PAEs with linear or highly branched structures have been prepared and applied in nonviral gene delivery, drug delivery and hydrogels.53 In general, in neutral aqueous solution, PAEs are hydrophobic and not soluble because of the nature of the hydrocarbon and ester backbones.54 However, when decreasing the pH value, the multiple tertiary amines in the backbones can protonate and increase the hydrogen bonding interactions with the surrounding water molecules, PAEs therefore become soluble.55 Previously, Brooks et al. reported the synthesis of thermal reversible choline phosphate-based polymers as multivalent universal biomemebrane adhesive.56 Further, they developed a pH and temperature-sensitive polymeric choline phosphatebased adhesive that can be derivatized to carry drugs or other agents and can be tuned synthetically to binding to tumor cells at pH 6.8.57 The solubility transition of PAE inspires us to hypothesize that modulation of the hydrogen bonding pairs on the backbones and side chains by simply adjusting the monomer compositions, a new type PAE based thermoresponsive “smart” polymer exhibiting tunable pH- and temperature-responsive phase transition can be developed. In addition, the multiple ester and amines groups on the backbone can act as hydrogen bonding acceptors to prevent complete dehydration of PAEs above the phase transition temperature; therefore, coacervate polymer particles could be formed. Herein, a series of thermo- and pH-responsive, coacervate formation, hyperbranched PAEs (HPAEs) were synthesized via a novel A2+B3 type Michael addition. 5-Amino-1-pentanol (S5) and trimethylolpropane ethoxylate triacrylate (TMPETA) were chosen as the A2 and B3 type monomers, respectively. By gradually increasing the length of the ethylene glycol spacer in TMPTETA, S5-TMPETA displayed a pH- and temperaturedependent, tunable phase transition in aqueous and various buffer solutions. The phase transition behaviors were

characterized by UV−vis spectrum and DLS measurements. The effects of polymer molecular weight, concentration and pH value on the LCST or cloud point temperature (Tcp) were systematically explored. The formation of coaceravate particles and the ability to encapsulate small molecules were also shown. Especially, the S5-TMPETA692 showing a LCST at around 33 °C was further labeled with fluorescein isothiocyanate (FITC), the pH- and temperature-mediated selective cell binding behaviors of S5-TMPETA692 were investigated.

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EXPERIMENTAL SECTION

Materials. The monomers trimethylolpropane ethoxylate triacrylate 428 (TMPETA428, with an average molecular weight of 428), trimethylolpropane ethoxylate triacrylate 692 (TMPETA692, with an average molecular weight of 692), trimethylolpropane ethoxylate triacrylate 912 (TMPETA912, with an average molecular weight of 912), 5-amino-1-pentanol (S5), solvent dimethyl sulfoxide (DMSO), chloroform-d (99.8%; CDCl3), sodium acetate (NaOAc; 3.0 M), phosphate-buffered saline (PBS; 0.1 M), Nile Red and fluorescein isothiocyanate isomer (FITC) were purchased from Sigma-Aldrich. Solvents dimethylformamide (DMF), diethyl ether (99%) and cell viability assay Alamarblue assay kit were purchased from Fisher Chemical. Human Embryonic Kidney 293 cells (HEK293) and human cervical cancer cell line (HeLa) were obtained from ATCC, UK. Cells were cultured in DMEM media with 10% FBS and 1% penicillin/ streptomycin (P/S) at 37 °C, 5% CO2 conditions. Synthesis of S5-TMPETA. The stoichiometric ratio of the amine monomer S5 to triacrylate monomer TMPETA was set as 1:1.2. S5 and TMPETA was first dissolved in DMSO at a total monomer concentration of 50% (w/v) and then the mixture was placed into an oil bath which was preheated to 90 °C. At certain time points, 20 μL of the sample mixture was taken to measure the molecular weight using gel permeation chromatography (GPC). When the weight-average molecular weight (Mw) approached 15 kDa, the reaction was stopped by diluting with DMSO to 100 mg/mL and cooling down to room temperature, and then excessive S5 was added to end-cap the polymers and consume all the unreacted vinyl groups for another 48 h. The polymers were purified by precipitating with cold diethyl ether three times to remove the excessive amine and oligomers. The final S5TMPETA products were dried in vacuum oven for another 48 h to remove the residual solvent and stored at −20 °C for subsequent studies. Gel Permeation Chromatography (GPC). Number-average molecular weight (Mn), weight-average molecular weight (Mw) and the polydispersity index (PDI) and alpha value (α) of the Mark− Houwink plots of the polymers were determined using a GPC (Agilent Technologies, 1260 affinity) equipped with a refractive index detector (RI), a viscometer detector (VS-DP) and a dual angle light scattering detector (LS 15° and LS 90°). Polymer samples were dissolved in DMF to the concentration of 10 mg/mL. GPC columns (30 cm PLgel mixed-C, two in series) were eluted with dimethylformamide (DMF) with 0.1% LiBr at 1 mL/min at 80 °C. Poly(methyl methacrylate) (PMMA) standards were used for calibration. Nuclear Magnetic Resonance (NMR) Analysis. Chemical composition and purity of the S5-TMPETA were confirmed with 1H NMR on a 400 MHz Varian NMR spectrometer. Polymers were dissolved in chloroform-d to 5 mg/mL. Spectra were analyzed using MesReNova processing software. The chemical shifts were reported in part per million (ppm) and referenced against chloroform-d (7.26 ppm). Lower Critical Solution Temperature (LCST) or Cloud Point Temperature (Tcp) Measurements. Turbidity measurements were carried out on a SpectraMax M3 microplate photometer in a quartz cell of 1 cm diameter.20 Polymers were dissolved in ultrapure water or buffer solutions [sodium acetate (NaOAc; 0.1 M; pH = 5.2), phosphate-buffered saline (PBS; 0.1 M; pH 7.4), Dulbecco’s Modified Eagle Medium (DMEM; pH 7.4) and disodium phosphate (Na2PO4; 0.1 M; pH 9.0)] to a certain concentration and vortexed for 30 s to ensure complete dissolution and equilibration. Temperature of B

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Figure 1. Hyperbranched poly(β-amino esters) (HPAEs) are synthesized via A2+B3Michael addition reaction. (a) Amine monomer S5 was conjugated to the triacrylate monomer TMPETA in DMSO at 90 °C to produce the HPAEs. (b) GPC traces of S5-TMPETA after end-capping and purification. (c) 1HNMR spectrum of S5-TMPETA. polymer samples was increased gradually from 25 to 50 °C. Transmittance was recorded as a function of temperature and measured at a wavelength of 350 nm. The LCST or Tcp was defined as the temperature at which transparency was 50%. Ultrapure water or buffer solution without polymers were used as the references. DLS Measurements. Polymer solutions were prepared as mentioned above and then transferred to a cuvette and equilibrated for 30 s, size and zeta potential measurement were performed on a Malvern Instruments Zetasizer (Nano-2590) with a scattering angle of 173°. For each sample, at least four measurements were conducted and the results were presented as mean ± SD (standard deviation). Coacervate Analysis. The polymer was dissolved in ultrapure water to 10 mg/mL. And then 1 mL of the sample was extracted to a centrifuge tube and heated up to ∼40 °C to form the coacervate particles immediately. The solution was then centrifuged at 3600 rpm for 5 min at 40 °C.20 The supernatant was removed carefully utilizing a micropipette. The polymer-rich coacervate phase at the bottom of the tube was then weighted before and after freeze-drying. The coacervate concentration was determined by comparing the weight of the dried polymer with the weight of the coacervate particles. This procedure was repeated three times and the average concentration of coacervate was calculated. Encapsulation of Nile Red by S5-TMPETA. To 1.0 mL of the S5-TMPETA solution (5 mg/mL) was added 50 μL of Nile Red solution (1 mg/mL in DMSO) and it was vortexed for 30 s. Fifty microliters of the mixture was then positioned on a glass slip. Temperature was increased gradually from 25 to above 37 °C. Nile Red encapsulated coacervate particles were observed under a green fluorescence channel using an inverted fluorescent microscope (Olympus CKX41). Degradation Analysis. The polymers were dissolved in PBS at a concentration of 10 mg/mL and stirred with a magnetic stirring bar at 37 °C. At certain time points, 1 mL of the mixture was extracted and freeze-dried immediately. And then the degradation products were dissolved in 1 mL of DMF and the molecular weights were analyzed using GPC as mentioned before.

Cytotoxicity Assay. The cytotoxicity of S5-TMPETA polymers was evaluated by Alamarblue assay. HEK293 cells were seeded on a 96 well plate at a density of 2 × 104 cells per well. After 24 h of incubation, polymers were dissolved in DMSO to 100 mg/mL and then added into the supernatants in the wells to a final polymer concentration of 10, 50, 100, 200, 500, or 1000 μL/mL, respectively. The cells were cultured for another 24 h. After the cell culture media was removed, Alamarblue reagent (10% in PBS) was added. The cells were incubated for another 2 h, and absorbance of the media was then measured at 570 nm using a multiple reader (SpectraMax M3). Cells without any treatments were used as positive controls. Polymers Labeled with FITC. The S5-TMPETA polymers and FITC were dissolved in DMSO to 1 mg/mL, respectively. After that, to 10 mL of polymer solution 50 μL of FITC solution was added. The mixture was stirred in dark for another 24 h. And then the mixture was dialyzed with DMSO for 2 days to remove the unreacted FITC. The final products were freeze-dried to remove the DMSO. Cell Binding of S5-TMPETA at Different Temperature and pH values. S5-TMPETA labeled with FITC were dissolved in DMSO to 100 mg/mL. HeLa cells were seeded on a 96 well plate at a density of 1 × 104 cells per well. After 24 h incubation, the cell culture media was removed. One μL of S5-TMPETA solution diluted with 100 μL of PBS (pH 7.4) or sodium acetate (pH 5.2) was then added into the well. The cells were incubated either at 25 or 37 °C for 10 min. And then the supernatants in the wells were removed and the cells were washed with PBS six times to remove the unbinded S5-TMPETA. The cells were observed and imaged with an inverted fluorescent microscope immediately.

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RESULTS AND DISCUSSION Synthesis of HPAE via an A2+B3Michael Addition Polycondensation. Dendritic polymers displaying a threedimensional architecture with multiple terminal groups have demonstrated their unique advantages in comparison with the linear counterparts.58 Thermosensitive linear structured polyC

DOI: 10.1021/acsami.6b15005 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 2. Phase transition of S5-TMPETA in water. (a) UV−vis spectrum of S5-TMPETA with different EG spacer length at the temperature range between 25 and 50 °C, polymer concentration is 10 mg/mL. (b) Optical photograph of S5-TMPETA692 polymer in water before and after heating to 37 °C, polymer concentration is 10 mg/mL. (c) Sizes of S5-TMPETA692 solution at 25 and 37 °C measured by DLS, polymer concentration is 10 mg/mL.

mers have been intensively studied.58 With the advances in polymer chemistry, thermosensitive dendritic poly(amidoamine) (PAMAM), poly(propyleneimine) (PPI), poly(ethylene imine) (PEI), and polyethers have also been developed by Thayumanavan et al. in recent years.59−62 To date, PAEs have been widely used in gene/drug delivery and hydrogel fabrication.47,63,64 However, the thermoresponsiveness of PAEs has never been investigated, although the existing thermosensitive polymers have the limitations such as complicated synthesis procedure, high cytotoxicity, low degradation rate, etc., which severely limit their broad applications and safety in various biomedical fields.9,20 “A2+B3” type polycondensations have attracted broad interest in the synthesis of hyperbranched polymers since the pioneering work by Kakimoto and Frechet et al. in 1999.65 Jikei et al. reported the synthesis of hyperbranched aromatic polyamides using aromatic diamines (A2) and trimesic acid (B3).66 Later, Voit et al. studied this type of hyperbranched polymers in more detail.67 Kinetic calculations further predicted that the first condensation reaction of A2 with B3 was faster than subsequent propagation, resulting in an accumulation of A-ab-(B)2 intermediates. Thus, the later stage of polymerization resembled the more common AB2 polycondensation, and the final products exhibited comparable structures to the products generated from the polymerization of AB2 monomers.68 Here, commercially available amine S5 and triacrylates TMPETA428, TMPETA692, or TMPETA912 were used as the A2 type and B3 type monomers, respectively, three HPAEs, termed S5-TMPETA428, S5-TMPETA692, and S5-TMEPTA912, were first synthesized via the A2+B3Michael addition (Figure 1a). To effectively control the molecular weight, we set the stoichiometric ratio of A2 to B3 at 1:1.2 (Table S1), the reaction was conducted in DMSO at 90 °C with a 50% monomer concentration. GPC was used to monitor the

evolution of molecular weight, the reaction was stopped by end-capping with excessive S5 when the weight-average molecular weight (Mw) approaching the target value (∼15 kDa). The polymers were purified by precipitation with diethyl ether to remove the excessive monomers. The final products were characterized with GPC (Figure 1b) and NMR (Figure 1c). The three HPAEs have a close molecular weight around 15 kDa (Table S2) and are highly soluble in DMSO, DMF, methanol, etc., at high concentrations (500 mg/mL). The absence of vinyl group signal peaks in NMR indicates the successful end-capping and complete consume of the triacrylates. Actually, we originally used excessive stoichiometric ratio of S5 to TMPETA (1.2:1 and 1.5:1) to synthesize S5TMPETA under similar conditions, the molecular weights of polymers were difficult to control: the polymerization either gelled or only low molecular weight was achieved. What should be also noted is that previously, Liu et al. employed an A2+BB′B″ Michael addition approach for HPAE synthesis. Due to the unequal reactivity of the multiple functional groups in the BB′B″ monomers, gelation was suppressed successfully.69 However, the need for BB′B″ type monomers greatly limited the diversity and functionality of the HPAEs developed. All these results demonstrated that our two-step A2+B3Michael addition strategy is a reliable as well as generalizable approach for HPAE synthesis. EG Spacer Length Determines the Thermoresponsiveness of S5-TMPETA. We hypothesized that by gradually increasing the EG spacer length of the hydrophobic monomer TMPETA, temperature-dependent phase transition behaviors would observed, therefore the Tcp of the three S5-TMPETA in deionized water were first measured using UV−vis spectrum. Unexpectedly, even the concentration as low as 1 mg/mL concentration, the S5-TMPETA428 was not soluble at 25 °C. In contrast, the S5-TMPETA692 was dissolved very well at 10 D

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Figure 3. Phase transition of S5-TMPETA692 with different molecular weight and at different concentrations in water. (a) Transmittance of S5TMPETA692 with molecular weights of 8.4, 15.4, 32.4, and 49.4 kDa in the temperature range of 25−50 °C (10 mg/mL). (b) Tcp of S5TMPETA692 with different molecular weights. (c) Transmittance of S5-TMPETA692 (15.4 kDa) at the concentration 1, 5, and 10 mg/mL. (d) Tcp of S5-TMPETA692 (15.4 kDa) at different concentrations.

synthesized using the A2+B3 Michael addition approach and characterized (Figure S1 and Table S3). At a concentration of 10 mg/mL, transmittance of the polymer series in the temperature range from 25 to 50 °C was shown in Figure 3a. A Tcp of 33, 32, 29, and 28 °Cwas determined for the S5TMPETA692 with molecular weights of 8.4, 15.4, 32.4, and 49.2 kDa, respectively (Figure 3b). The increased molecular weight leading to a decrease in Tcp is due to the decreased polymer solubility.20 We also tested the effects of polymer concentration on the Tcp of S5-TMPETA692 (Mw = 15.4 kDa) in water. As expected, increased polymer concentration leads to a sharp decrease in the Tcp. At the concentrations of 1, 5, and 10 mg/mL, the Tcp of S5-TMPETA692 was 51, 36, and 33 °C, respectively. The dependence of Tcp on polymer concentration is because a higher polymer concentration would increase the hydrogen bonding pair density in the solution and thus enhances the intra- and intermolecular hydrogen bonding interactions and promotes the formation of hydrophobic particles, similar molecular weight−Tcp relationship was also observed with other thermosensitive polymers.70 The polymer molecular weight and concentration dependence of Tcp make the S5-TMPETA692 able to be used for various biomedical applications, for example, in situ formed injectable hydrogel, controlled release of drugs, and genes. Effects of pH Value and Solute on the Tcp of S5TMPETA692. The multiple tertiary amines on the S5TMPTETA692 are able to protonate and thus enhance the interactions with water molecules,51 the dependence of Tcp on media pH was further explored. Four most commonly used buffer solutions with pH values of 5.2, 7.0, 7.4 and 9.0, respectively, were tested. At the pH 5.2, there was no phase transition observed for the S5-TMPETA692 at a concentration of 10 mg/mL (Figure 4a). With the increase of pH value, the Tcp of S5-TMPETA692 decreased gradually. Tcp of 33, 32.5, and 28 °C were determined at the pH values of 7.0, 7.4, and

mg/mL. When the temperature was increased gradually from 25 to 50 °C, a sharp phase transition was observed, the transmittance of the solution decreased from over 95% at 25 °C to lower than 6% at 50 °C, and a cloudy solution was observed (Figure 2a, b), the Tcp was measured to be around 33 °C. Surprisingly, the S5-TMPETA912 was highly water-soluble, even at the concentration of 100 mg/mL, thus in the tested temperature range, no substantial decrease in transmittance was observed. The phase transition of the S5-TMPETA692 was further confirmed using DLS. It clearly shows that at 25 °C, there is almost no particle size measured because the solution is homogeneous. When it comes to 37 °C, cloudy particles with an average size of 1.4 μm were formed (Figure 2c). These results indicate as the increase of the EG spacer length in TMPETA, enhanced hydrogen bond interactions with water molecules make the S5-TMPETA692 undergo a partial dehydration and separate into polymer-rich droplets within a polymer-deficient liquid phase.30 However, further increase of the PEG spacer length, the too-extensive hydrogen bonding interactions with water molecules and limited intra- and intermolecular hydrogen-bonding interactions between polymer molecules make the S5-TMPETA912 well soluble even at high concentrations and low temperature, and therefore no phase transition would occur. Interestingly, the three monomers TMPETA are not soluble in water at the concentrations tested, which further indicates the generation of the tertiary amines by Michael addition significantly enhances the hydrogen bonding interactions of S5-TMPETA with water molecules. Effects of Polymer Molecular Weight and Polymer Concentration on the T cp of S5-TMPETA692. S5TMPETA692 shows a Tcp at around 33 °C in water. The effects of polymer molecular weight on the Tcp was further investigated. To this end, four S5-TMPETA692 with weightaverage molecular weights of 8.4, 15.4, 32.4, and 49.2 kDa were E

DOI: 10.1021/acsami.6b15005 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 4. pH Value and solute dependence of Tcp of the S5-TMPETA692 (15.4 kDa). (a) Transmittance of S5-TMPETA692 in buffer solutions with pH values of 5.2, 7.0, 7.4, and 9.0 in the temperature range between 25 and 50 °C; (b) Tcp of S5-TMPETA692 in different buffer solutions; (c) zeta potentials of S5-TMPETA692 buffer solutions with different pH values; (d) particle size of S5-TMPETA692 in buffer solutions with different pH values; (e) transmittance of S5-TMPETA692 in different media in the temperature range between 25 and 50 °C; (f) Tcp of S5-TMPETA692 in different media.

model pH values −5.2, 7.0, 7.4, and 9.0, the pH and solute gradient between the pathological and healthy tissues have been well explored, therefore the pH and solute dependence of the Tcp would make S5-TMPETA692 find a variety of applications in target drug and gene delivery. Coacervation of S5-TMPETA692 above the Tcp. The majority of the existing thermoresponsive polymers, such as PNIPAM, undergoes a coil−globule transition resulting in complete dehydration, which would limit their application in encapsulating sensitive physiologically active biomolecules or fabrication of tissue engineering hydrogel scaffolds.9,20 In contrast, polymers such as the elastin-like polypeptides (ELPs) and polyesters displaying thermoresponsive coacervation are particularly attractive.46 The coacervation behavior of S5-TMPETA692 (15.4 kDa) above the Tcp was investigated. The polymer was dissolved in pure water at 10 mg/mL and then heated up to 40 °C. A fast centrifuge would separate the coacervates from the media. The weight loss after freeze-drying of the coacervates was calculated to be around 94.3% (Figure 5a), which indicates the high content of water in the coacervates and demonstrates that the S5-TMPETA692 is a coacevate-type temperature- and pH-responsive polymer.20 To

9.0, respectively (Figure 4b). We speculate that the pH dependence of Tcp is because the tertiary amines show different protonation ability under different pH values: at lower pH values, more tertiary amines would protonate and thus stronger hydrogen bonding interactions would formed between the polymers and the water molecules. To confirm this, zeta potentials of the S5-TMPETA solution at the tested pH values were further measured. As shown in Figure 4c, at the pH 5.2, a + 34 mV zeta potential was determined, in comparison with +4.7, + 2.6, and +0.8 mV at the pH values of 7.0, 7.4, and 9.0, respectively. Correspondingly, the average size of the hydrophobic particles increased from 37.9 nm to 1.55, 1.78, and 1.96 μm (Figure 4d). We also explored the effects of solutes on the Tcp of S5-TMPETA. Pure water, PBS and DMEM cell culture media with close pH value but increased solute concentration were tested. In agreement with previous reports, increasing the concentration of hydrogen bonding disrupting inorganic salts promotes the collapse of the S5-TMPETA692 through the “salting out effect”.71 Therefore, a decrease Tcp was observed in PBS (30.5 °C) and DMEM (31 °C) compared to that in the pure water (33 °C) (Figure 4e, f). Although here we used the F

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Figure 5. S5-TMPETA692 can form coacervate particles above the Tcp. (a) Weight of S5-TMPETA692 coacervate before and after freezedrying. (b) Optical photographs of S5-TMPETA692/Nile Red mixture at 25 and 40 °C.

Figure 6. S5-TMPETA692 is highly degradable and noncytotoxic. (a) Degradation profile of S5-TMPETA692 with different molecular weight in PBS at 37 °C. (b) Viability of HEK293 cells after incubation with S5-TMPETA692 for 24 h at different concentrations.

further demonstrate the feasibility of S5-TMPETA692 coavervates for temperature triggered encapsulation of molecules, the hydrophobic fluorescent dye Nile Red was added into the S5-TMPETA692 solution and heated up to 40 °C. Clearly, red fluorescence was observed only in the coacervate particles, indicating the successful encapsulation of Nile Red. The coacervate nature and thermo induced high encapsulation ability of S5-TMPETA692 make it a good candidate for transportation and controlled release of various desired compounds. Biodegradability of S5-TMPETA692. One of the major disadvantages of existing thermoresponsive polymers is the nondegradability or slow degradation rate, which would limit the application in vitro and in vivo, especially when a high polymer concentration is required.9 The degradations profiles of S5-TMPETA692 with a molecular weight of 15.4, 32.4, or 49.4 kDa in PBS were shown in Figure 6a. It can be seen that all the three polymers are highly degradable: after 1 h of incubation at 37 °C, the molecular weight decreased from 15.4, 32.4, and 49.4 kDa to 5.4, 9.2, and 10.1 kDa, respectively. After 6 h of incubation, all the polymers degraded to oligomers with molecular weights less than 2 kDa. Because of the high degradability, S5-TMPETA692 induced negligible cytotoxicity. As shown in Figure 6b, more than 70% viability of HEK293 cell was preserved after 24 h of incubation when a 1000 μg/mL polymer concentration was applied. It should be noted that, at the same concentration, cells treated with branched polyethylene imine (PEI, Mw = 25 kDa) showed no viability. Interestingly, at low polymer concentrations (e.g., 10, 50, and100 μg/mL), the cell viability after treatment with S5TMPETA692 was even higher than that of the untreated cells, this is because the degraded byproducts of PAE based polymers are amino acids, which would probably promote the growth of cells. pH- and Temperature-Mediated Selective Cell Binding of S5-TMPETA692. Controlled binding of biomaterials to cellular membrane is the prerequirement for various biological

processes, such as cell imaging and drug delivery, etc.72 We hypothesize that by modulating the pH value and temperature, the pH- and thermosensitive S5-TMPETA692 would bind to the negatively charged cell membrane in a controlled manner. To validate this process, we labeled S5-TMPETA692 and the control polymer S5-TMPETA912 with FITC, whereas the polymer binds to the cell membrane effectively, green fluorescence of FITC would be observed. We first incubated the HeLa cells in PBS at pH 7.4 and 25 °C for 10 min, and then the cells were washed six times to remove the unbinded polymers. As outlined in Figure 7a, the cells treated with S5TMPETA692 exhibited strong green fluorescence. However, when increasing the temperature to 37 °C, only faint

Figure 7. S5-TMPETA692 showed temperature- and pH-mediated selective binding to cells. Fluorescent images of HeLa cells after incubation with S5-TMPETA692 at (a) pH 7.4 and 25 °C, (b) pH 7.4 and 37 °C, (c) pH 5.2 and 37 °C. G

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ACS Applied Materials & Interfaces fluorescence was observed (Figure 7b). In contrast, at the both temperatures, the cells treated with S5-TMPETA912 always exhibited strong green fluorescence (Figure S2). The different cell binding ability of the S5-TMPETA692 and S5-TMPETA912 can be explained by the different phase transition behaviors of the two polymers. At 25 °C, both of the polymers are highly soluble, the positively charged polymer chains are well-stretched and can contact the negatively charge cell membrane freely. The Tcp of S5-TMPETA692 in PBS is around 30.5 °C (Figure 4f), above which (e.g., at 37 °C), the S5TMPETA692 would collapse and most of the positive charge would be shielded (Figure 4c) from contacting the cell membrane, the electrostatic interactions that drive the polymer to bind to the cell membrane would be weakened, therefore low bind efficiency was observed. In contrast, at the both temperatures, the S5-TMPETA912 was well-dissolved and the positively charged polymer chains can bind to the cell membrane via electrostatic interactions easily. We further incubated the cells at 37 °C but decreasing the pH to 5.2 to explore the effect of pH on the binding of polymers to cells. As expected, at pH 5.2, the S5-TMPETA692 showed strong binding to the cells (Figure 7c). This is because at this pH value, the polymers are highly protonated, favoring the binding of polymers to the negatively charged cell membrane. Again, the S5-TMPETA912 exhibited very strong cell binding ability (Figure S3). All these results demonstrate that by modulating the pH and temperature, the binding of polymers to cells can be adjusted easily in a controlled manner.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Dr. Dezhong Zhou received funding from Science Foundation Ireland (SFI) Industry Fellowship (15/IFA/3037), Dr. Wenxin Wang received funding from SFI Principal Investigator Program (13/IA/1962), Investigator Award (12/IP/1688), and Health Research Board of Ireland (HRA-POR-2013-412).

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CONCLUSIONS For the first time, the thermoresponsiveness of Hyperbranched poly(β-amino esters) (HPAEs) were investigated. S5-TMPETA were synthesized by A2+B3Michael addition. The spacer length in TMPETA significantly affect the phase transition of S5TMPETA. The Tcp of S5-TMPETA692 can be readily tailored by polymer molecular weight and concentration. In water, S5TMPETA692 showed a Tcp at around 33 °C, above which, coacervate particles can form and are able to encapsulate desired molecules. By varying the temperature and pH, S5TMPETA692 can selective bind to cells. In addition, S5TMPETA are highly degradable and noncytotoxic. The unique thermo- and pH-responsiveness as well as high safety would make this type of HPAEs to find use in a variety of biomedical applications.

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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.6b15005. Recipe for the synthesis of S5-TMPETA, GPC results of S5-TMPETA, GPC traces of S5-TMPETA, and fluorescent images of cells after incubation with S5TMPETA (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Wenxin Wang: 0000-0002-5053-0611 Author Contributions †

D.Z., L.P., Y.G., and J.O.K.A contributed equally. H

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