Glucose-Responsive Gene Delivery at Physiological Ph Through Tertiary-Amine Stabilized Boronate-PVA Particles Synthesized by One-Pot Reaction

pharmaceutics

Article Glucose-Responsive Gene Delivery at Physiological pH through Tertiary-Amine Stabilized Boronate-PVA Particles Synthesized by One-Pot Reaction

Mangesh Morey, Akshay Srivastava † and Abhay Pandit *

CÚRAM, SFI Research Centre for Medical Devices, National University of Ireland, H91 W2TY Galway, Ireland; [email protected] (M.M.); [email protected] (A.S.) * Correspondence: [email protected]; Tel.: +353-91-495833 † Current Address: Akshay Srivastava is currently working at Department of Medical Devices, National Institute of Pharmaceutical Education and Research, Ahmedabad, Gujarat 382355, India.

Abstract: We report a physiologically stable and cytocompatible glucose-responsive nonviral gene delivery system made up of boronate functionalized polymeric material. Herein, we utilize boronate cis-diol interactions to develop a glucose-responsive submicron particle (SMP) system. The stability of the boronate interaction at a physiological pH was achieved by copolymerization of dimethyl aminoethyl methacrylate (DMAEMA) with acrylamidophenylboronic acid (AAPBA) and the formation of a complex with polyvinylalcohol (PVA) which is governed by cis-diol interactions. The shift in hydrodynamic diameter of SMPs was observed and correlated with increasing glucose concentrations at a physiological pH. Optimal transfection was observed for a 5 µg dose of the gaussia luciferase reporter gene in NIH3T3 cells without any adverse effect on cellular viability. The destabilization of the AAPBA–PVA complex by interacting with glucose allowed the release of encapsulated bovine

serum albumin (BSA) in a glucose-responsive manner. In total, 95% of BSA was released from SMPs at a 50 mM glucose concentration after 72 h. A two-fold increase in transfection was observed in

Citation: Morey, M.; Srivastava, A.; 50 mM glucose compared to that of 10 mM glucose. Pandit, A. Glucose-Responsive Gene Delivery at Physiological pH through Keywords: glucose-responsive gene delivery; boronic acid; free radical polymerization; drug delivery Tertiary-Amine Stabilized Boronate-PVA Particles Synthesized by One-Pot Reaction. Pharmaceutics 2021, 13, 62. https://doi.org/ 1. Introduction 10.3390/pharmaceutics13010062 Nonviral gene therapy is a promising therapeutic choice for many diseases [1,2]. However, low transfection is one of the main barriers associated with nonviral gene car- Received: 30 October 2020 riers [1–3]. This challenge can be solved by the use of a stimulus-responsive delivery Accepted: 2 January 2021 system [4]. Glucose-responsive drug delivery systems represent up-and-coming appli- Published: 6 January 2021 cations in diabetes therapy and have attracted much more interest in recent years [5,6]. Glucose-responsive delivery systems are widely preferred stimulus-responsive delivery Publisher’s Note: MDPI stays neutral with regard to jurisdictional clai- systems based on Glucose oxidase enzyme (GOx), glucose binding proteins (GBPs), and ms in published maps and institutio- phenylboronic acid (PBA) [6]. The use of these glucose oxidase-based systems is minimal, nal affiliations. as enzyme activity can be reduced over time [7]. Concanavalin A is a natural GBP class that is one of the most widely used lectins for glucose-responsive insulin delivery. The problem with concanavalin-based systems is that they are inflammatory [8]. PBA is a synthetic molecule that reversibly binds to 1,2- or 1,3-cis-diols, including many kinds of sugars, to Copyright: © 2021 by the authors. Li- form cyclic esters. censee MDPI, Basel, Switzerland. Recently, boronate-based systems have been reported for their potential applications This article is an open access article as saccharide [9], optical [10], and conductivity sensors [11,12], and also in drug release distributed under the terms and con- systems [13–15]. Boronate-based systems have been widely preferred because of their ditions of the Creative Commons At- stability, efficiency, and ease of use. In aqueous media, phenylboronic acid forms a pH- tribution (CC BY) license (https:// dependent equilibrium between the nonionic trigonal boronic acid and anionic tetrahedral creativecommons.org/licenses/by/ boronate. Boronate provides a reversible interaction with cis-diol-containing carbohydrates, 4.0/).

Pharmaceutics 2021, 13, 62. https://doi.org/10.3390/pharmaceutics13010062 https://www.mdpi.com/journal/pharmaceutics Pharmaceutics 2021, 13, 62 Pharmaceutics 2021, 13, x FOR PEER REVIEW 2 of 10 2 of 10

nucleotides, nucleoside,bility, efficiency, and glycoproteins and ease of use. [16 In]. aqueous It has beenmedia, used phenylboronic to develop acid glucose-forms a pH-dependent equilibrium between the nonionic trigonal boronic acid and anionic tetrahedral responsive delivery systems for closed-loop insulin delivery [6]. However, boronate-based boronate. Boronate provides a reversible interaction with cis-diol-containing carbohy- glucose-responsivedrates, polymers nucleotides, have nucleoside, been reported and glycoprote to remainins functional[16]. It has been only used in alkalineto develop glu- media and not at physiologicalcose-responsive pH delivery [17]. Thissystems is duefor closed-loop to the high insulin pKa delivery value of [6]. the However, boronate boronate- moiety. Hence, boronate’sbased glucose-responsive optimal use in polymers biomedical have engineeringbeen reported to is remain restricted functional due to only the in alka- limitations of boronateline media groups’ and inactivity not at physiological at physiological pH [17]. pHThis (7.4) is due [17 to– the20]. high Several pKa value methods of the boro- have been developednate tomoiety. reduce Hence, the pKaboronate’s of the optimal boronate use in group biomedical to enhance engineering its functionalis restricted due to sensitivity at physiologicalthe limitations pH. Oneof boronate such approach groups’ inactivity involves at introducingphysiological anpH amino(7.4) [17–20]. group Several methods have been developed to reduce the pKa of the boronate group to enhance its in the vicinity of boronicfunctional acid sensitivity to decrease at physiological the boronate pH. One group’s such pKaapproach value involves by forming introducing a an coordination bond betweenamino group nitrogen in the vicinity and boron of boronic (Figure acid1 ).to Thedecrease effect the could boronate be increasedgroup’s pKa by value by introducing a tertiaryforming amine a coordination into the boronate bond between system nitrogen [21]. Boronate-containing and boron (Figure 1). The polymeric effect could be systems also showedincreased adverse by introducing effects on a cell tertiary metabolic amine into activity the boronate [22]. Thesesystem problems[21]. Boronate-contain- still need to be resolveding before polymeric their systems potentially also showed signiﬁcant adverse contribution effects on cell to clinicalmetabolic applications activity [22]. These can be recognized. problems still need to be resolved before their potentially significant contribution to clin- ical applications can be recognized.

DMAEMA-Boronate

Figure 1. Schematic representation of stabilization of boronate by tertiary amine of dimethyl aminoethyl methacrylate Figure 1. Schematic representation of stabilization of boronate by tertiary amine of dimethyl (DMAEMA) in a submicron particle (SMP) system. The pKa of boronate was reduced by the addition of amino group in theaminoethyl vicinity of boronic methacrylate acid by the (DMAEMA) formation of a incoordination a submicron bond particle between (SMP)nitrogen system. and boron. The pKa of boronate was reduced by the addition of amino group in the vicinity of boronic acid by the formation of a coordination bond betweenThe nitrogen goal of the and study boron. is to develop a boronate-based glucose sensitive gene delivery system. As per our knowledge, this is the first of its kind research to report the glucose- The goal of theresponsive study is gene to developdelivery system. a boronate-based Additionally, we glucose have made sensitive boronate gene active deliv- at physio- ery system. As perlogical our pH knowledge, and made this this synthesis is the very first simple of its by kind using research a single-pot to reaction. report To the achieve this, we hypothesized that copolymerization of the boronate-containing monomer with glucose-responsivedimethyl gene delivery aminoethyl system. methacrylate Additionally, (DMAEMA) we haveand the made complexation boronate of activea boronate at moi- physiological pH andety with made polyvinyl this synthesis alcohol (PVA) very simplein a single by reaction using amixture single-pot could reaction.provide an To efficient achieve this, we hypothesizedmethod for the that fabrication copolymerization of boronate-containing of the boronate-containing particles for delivery monomer in physiological with dimethyl aminoethyl methacrylate (DMAEMA) and the complexation of a boronate moiety with polyvinyl alcohol (PVA) in a single reaction mixture could provide an efficient method for the fabrication of boronate-containing particles for delivery in physiological conditions. Herein, we fabricate boronate-PVA submicron particles (SMPs) and charac- terize the release of bovine serum albumin (BSA) as a standard biomolecule at different glucose concentrations. The SMPs were validated for glucose-responsive delivery of gaussia luciferase (GLuc) polyplexes which transfected an NIH3T3 fibroblast cell line without adversely affecting cellular viability. Pharmaceutics 2021, 13, x FOR PEER REVIEW 3 of 10

Pharmaceutics 2021, 13, 62 conditions. Herein, we fabricate boronate-PVA submicron particles (SMPs) and character- 3 of 10 ize the release of bovine serum albumin (BSA) as a standard biomolecule at different glucose concentrations. The SMPs were validated for glucose-responsive delivery of gaussia luciferase (GLuc) polyplexes which transfected an NIH3T3 fibroblast cell line without adversely affecting cellular viability. 2. Materials and Methods 2.1.2. Materials Synthesis and Methods of 3-Acrylamidophenyl Boronic Acid (AAPBA) 2.1. Synthesis of 3-Acrylamidophenyl Boronic Acid (AAPBA) AAPBAAAPBA was was synthesized synthesized from 3-aminophenylboronic from 3-aminophenylboronic acid, as previously acid, reported as previously reported [23]. The[23]. synthesisThe synthesis of of AAPBA AAPBA was carried carried out, out, as expressed as expressed by the byfollowing the following reaction reaction (Scheme1). (Scheme 1).

Scheme 1. Synthesis of acrylamidophenylboronic acid (AAPBA). Scheme 1. Synthesis of acrylamidophenylboronic acid (AAPBA). Briefly, 3-aminophenylboronic acid (1.72 gm, 10 mM) was dissolved in 20 mL NaOH and cooledBriefly, in an 3-aminophenylboronic ice bath. Acryloyl chloride (1.6 acid mL) (1.72 was then gm, added, 10 mM) and the was reaction dissolved in 20 mL NaOH was stirred for one hour. Following this, the HCl (2M) was then added to adjust the solu- andtion to cooled pH 1, which in an caused ice bath. the precipitation Acryloyl of chloride the product (1.6 in the mL) form was of a white then powder. added, and the reaction was stirredThe product for was one filtered hour. on Following the sintered this, glass thefunnel HCl and (2M) washed was with then 50 mL added cold dis- to adjust the solution to pHtilled 1, water. which The causedprecipitated the product precipitation was collected of and the dissolved product in the in water the form heated of at a white powder. The product60 °C and, wasfinally, filtered the dissolved on the precipitate sintered was glass kept at funnel 4 °C overnight. and washed The monomer with 50 mL cold distilled crystals were formed and washed with cold water several times and finally dried under ◦ water.vacuum. The precipitated product was collected and dissolved in the water heated at 60 C and, finally, the dissolved precipitate was kept at 4 ◦C overnight. The monomer crystals were2.2. Synthesis formed of AAPBA-PVA and washed SMPs with by Oil coldEmulsion water Method several times and finally dried under vacuum. AAPBA-PVA SMPs were synthesized by the oil emulsion method by mixing two solutions. Solution A was prepared by mixing modified boronate monomer AAPBA (20 mg, 2.2.25 mM), Synthesis DMAEMA of AAPBA-PVASMPs(20 µl, 30 mM), 2-hydroxyethyl by Oil methacrylate Emulsion Method(HEMA), (150 µl, 300 mM), AAPBA-PVAand 10 µl N,N,N’,N SMPs’-Tetramethylethane-1,2-diamine were synthesized by (TEMED) the oil was emulsion added to this method by mixing two solutions.solution just before Solution the initiation A was of polyme preparedrization. by Solution mixing B was modified prepared by boronate adding monomer AAPBA PVA (20 mg, 50 µM) and 10 mg ammonium persulfate (APS). The homogenous oil phase (20was mg,prepared 25 mM),by adding DMAEMA 3.8 mL of paraffin (20 µl, oil 30 and mM), 50 µl 2-hydroxyethylof emulsifier Tween methacrylate20 and 150 (HEMA), (150 µl, 300µl of mM),Spam 80. and Free 10 radicalµl N polymerization,N,N’,N’-Tetramethylethane-1,2-diamine was initiated by mixing solutions A and (TEMED) B, was added to thiswhich solution were immediately just before added thedropwise initiation in the oil of phase polymerization. and incubated overnight Solution at B was prepared by room temperature. The emulsion was then centrifuged at 11,000 rpm for 15 min, and the addingpellet was PVA washed (20 twice mg, with 50 n-hexaneµM) and followed 10 mg by ammoniumdistilled water. The persulfate final product (APS). was The homogenous oil phasefreeze-dried was and prepared stored at 4 by °C. adding 3.8 mL of paraffin oil and 50 µl of emulsifier Tween 20 and 150 µl of Spam 80. Free radical polymerization was initiated by mixing solutions A and 2.3. Physiochemical Characterization B, which were immediately added dropwise in the oil phase and incubated overnight at Synthesis of AAPBA was further characterized by proton nuclear magnetic reso- roomnance (1 temperature.H NMR) and boron The nuclear emulsion magnetic was resonance then (11 centrifugedB NMR) by an Agilent-NMR5- at 11,000 rpm for 15 min, and the pelletvnmrs500 was instrument washed (Agilent twice Technologies with n-hexane Ireland followedLimited, Cork, by Ireland) distilled using water. DMSO The final product was freeze-dried and stored at 4 ◦C.

2.3. Physiochemical Characterization Synthesis of AAPBA was further characterized by proton nuclear magnetic resonance (1H NMR) and boron nuclear magnetic resonance (11B NMR) by an Agilent-NMR5- vnmrs500 instrument (Agilent Technologies Ireland Limited, Cork, Ireland) using DMSO as a solvent. The synthesis of both AAPBA and SMPs was conﬁrmed by Fourier-transform infrared spectroscopy (FTIR) analysis (FTIR—Varian 660-IR, Agilent Technologies Ireland Limited, Cork, Ireland). Freeze-dried powder of samples was used for this experiment. Scanning electron microscopy (SEM) was used for surface morphological analysis of SMPs. The gold coated samples were visualized under SEM (Hitachi S-4700 Scanning Electron Microscope, Daresbury, Warrington WA4 4AB, UK). Surface charge on the SMPs was determined by zeta sizer (Malvern, Nano-ZS90, Malvern, UK).

2.4. Functional Integrity of Boronate Curcumin forms a red complex with boronate and color formation can be observed by the thin layer chromatography (TLC) method [24]. To prepare the curcumin stain, 100 mg Pharmaceutics 2021, 13, 62 4 of 10

of curcumin was dissolved in a 100 mL solution of ethanol with 2 N HCl (99:1 v/v). Boronic acid was dissolved in methanol and a single spot of each sample was placed using a glass “spotter” onto a silica TLC plate (Sigma Aldrich, City Dublin, Ireland). The TLC plate was then inserted into the curcumin stain for ﬁve seconds and then dried using a heat gun for ﬁve seconds.

2.5. Transfection A range of different plasmid:polymer ratios, i.e., 1:2, 1:3.5, 1:7, 1:20, have been investigated to obtain an optimal complexation concentration of polymer for transfection. Agarose gel electrophoresis was run to determine the optimal concentration for complete complexation. The transfection experiment was carried out using NIH3T3 cells to conﬁrm the results further. The widely used transfection reagent polyethylenimine (PEI) was used as positive control while naked plasmid was used as the negative control. Transfection of cells was analyzed after 48 h by luciferase assays using a BioLux® luciferase assay kit (Biolab, Dublin, Ireland).

2.6. Polyplex Dose Optimization of GLuc polyplex concentration for higher transfection was analyzed using NIH3T3 cells. In a 12-well plate, 25,000 cells/well were seeded and incubated overnight for the attachment. A few different polyplex concentrations, i.e., 0, 1, 2, 5, 25 µg were added to the cells and incubated for four hours. After four hours, the polyplex solution was replaced with regular growth medium, and the transfected cells were incubated for 48 h. After incubation, transfection was determined by luciferase assay as outlined above.

2.7. Glucose-Responsive Behavior The glucose-responsiveness of the fabricated boronate SMPs was investigated by using the following techniques. Swelling of the SMPs in the presence of different glucose concentrations was analyzed with zeta sizer (Malvern, Nano-ZS90, Malvern, UK), whereas glucose-responsive release of BSA was estimated by a spectrophotometer. Further- more, glucose-responsive gene delivery was conﬁrmed by luciferase assay using a gaussia luciferase assay kit.

2.7.1. Hydrodynamic Diameter Change by DLS Measurement Changes in the hydrodynamic diameter of SMPs in the presence of glucose were determined by zeta sizer (Malvern, Nano-ZS90). The SMPs (2 mg) were incubated with 5, 10, 20, and 50 mM of glucose in PBS for 30 min and the change in hydrodynamic diameter was investigated. The same amount of SMPs was also incubated with PBS only as the negative control, and the hydrodynamic diameter was calculated. All the samples were run in triplicate.

2.7.2. Glucose-Responsive Release of Encapsulated BSA To assess the glucose-responsive delivery from SMPs, BSA was encapsulated within the SMPs during their fabrication. Release of BSA in the presence of 10, 20 and 50 mM glucose concentrations in PBS over 168 h was determined. The released BSA was quantified using the Bradford assay, and the absorbance reading was measured using a varioskanflash plate reader (Thermo Scientific, Dublin, Ireland).

2.7.3. Glucose-responsive Transfection In a 12-well plate, 25,000 NIH3T3 cells/well were seeded and incubated overnight for attachment. Glucose-responsive SMPs loaded with GLuc polyplexes were incubated with the cells in a range of glucose concentrations—i.e., 0, 10, 20 and 50 mM for 24 h. The expression of the excreted luciferase protein 48 h post-transfection was quantiﬁed using the luciferase assay using a gaussia luciferase assay kit. Pharmaceutics 2021, 13, 62 5 of 10

2.8. Cytotoxicity 2.8.1. Metabolic Activity The influence of the SMPs on the metabolic activity of NIH3T3 fibroblasts was quantified using the alamarBlueTM cell metabolic activity assay. NIH3T3 cells were grown in a 96-well plate and treated with 10, 20, 50, and 100 µg of SMPs per well and incubated for 48 h. After 48 h, 10% alamarBlueTM solution in the media was added to cells, and the plates were incubated for a further four hours at 37 ◦C. The supernatant’s fluorescence was measured by using a varioskan flash microplate reader (Thermo Fisher Scientific, MA, USA). The used excitation and emission wavelengths were 530–560 nm and 590 nm, respectively. The following formula was used to determine the percentage of viable cells—fluorescence of treated cells/fluorescence of untreated cells × 100.

2.8.2. Live Dead Assay In an eight-chamber slide, 10,000 NIH3T3 cells/chamber were seeded and grown to 70% confluency. AAPBA-PVA SMPs (100 µg) were added to each of the chambers for 48 h. Then, cells were washed with PBS and incubated with 10 mM calcein-AM green (Life Technologies, Dublin, Ireland) and 1 mM ethidium homodimer-1 (Life Technologies, Ireland) for 30 min. The samples were imaged by using a fluorescence microscope (Olympus BX51, Dublin, Ireland).

3. Results and Discussion 3.1. Chemical and Morphological Characterization One-pot synthesis of AAPBA–PVA complex with DMAEMA stabilization was performed in this study. In the first step, 3-aminophenylboronic acid was modified to AAPBA through reaction with acryloyl chloride. This modification of the monomer was necessary to add an allyl moiety for free radical polymerization. The synthesis of AAPBA was confirmed by FTIR, 1H NMR and 11B NMR (Figure S1). FTIR analysis showed that the characteristic peaks at 1666 and 1636 cm−1 are attributable to C=O and C=C bond stretching vibrations, respectively. A typical amide band appears in the spectrum of AAPBA at 1557 cm−1. The absorption band at 1433 cm−1 is ascribed to benzene stretching vibrations (Figure S1C). The characteristic absorption of boronate took place at 1356 cm−1 (21). The synthesis of AAPBA was confirmed by 1HNMR (400 MHz, DMSO-d6) δ = 5.75 (1H, CH2=CH–), 6.27 (1H, CH2=CH), 6.40 (1H, CH2=CH–), 7.28 (1H, phenyl), 7.49 (1H, phenyl), 7.82 (1H, phenyl), 7.88 (1H, phenyl), 8.03 (2H, –B(OH)2, 10.07 (1H, –NH–) (Figure S1A) and 11B NMR 28.7(11B) (Figure S1B). The synthesis of boronate-PVA SMPs was achieved by using the oil emulsion method. The fabrication of boronate-PVA SMPs also involved a free radical polymerization of AAPBA and DMAEMA by using APS/TEMED. Boronate forms a complex with PVA by cis-diol interactions and the polymeric form of boronate with DMAEMA was formed by free radical polymerization. We anticipated that the binding of boronate with PVA and copolymerization of AAPBA with DMAEMA and HEMA would simultaneously take place. This provides a one-step fabrication method to synthesize boronate-containing particles. The addition of DMAMEA as a tertiary amine protects the boronate group from nucleophilic attack by water molecules. The amino group provided by DMAEMA acts as the lewis base, which donates an electron pair to the vacant p-orbital of boron and forms an N→B coordination bond (21). This prevents the surrounding water molecules from forming the tetrahedron boronate configuration. Hence, in the presence of a tertiary amine, the tetrahedral anionic form of boronate becomes active at a neutral pH and governs boronate and cis-diol interactions. The size of glucose-responsive SMPs synthesized by this method was 825–875 nm in diameter (Figure2B,C) . The particles had a negative charge, which was −12.2 mV as determined by a zeta sizer. The significant peaks present from both reactants in the FTIR spectra −1 −1 −1 −1 were 3270 cm /O–H 2905 cm , and 2940 cm /C–H alkyl groups, 1413 cm /CH2, 1143 cm−1/C–O or C–O–C, 1085 cm−1/C–O–C (from PVA), and 1665 cm−1/C=O, 1270 cm−1/C–H, 1350 cm−1/B–OH, 701 cm−1/phenyl group (from AAPBA confirms the Pharmaceutics 2021, 13, x FOR PEER REVIEW 6 of 10

PVA and copolymerization of AAPBA with DMAEMA and HEMA would simultaneously take place. This provides a one-step fabrication method to synthesize boronate-containing particles. The addition of DMAMEA as a tertiary amine protects the boronate group from nucleophilic attack by water molecules. The amino group provided by DMAEMA acts as the lewis base, which donates an electron pair to the vacant p-orbital of boron and forms an N→B coordination bond (21). This prevents the surrounding water molecules from forming the tetrahedron boronate configuration. Pharmaceutics 2021, 13, 62 Hence, in the presence of a tertiary amine, the tetrahedral anionic form of boronate 6 of 10 becomes active at a neutral pH and governs boronate and cis-diol interactions. The size of glucose-responsive SMPs synthesized by this method was 825–875 nm in diameter (Figure 2B,C). The particles had a negative charge, which was −12.2 mV as determined by a zeta sizer. The significant peaks present from both reactants in the FTIR spectra were 3270 synthesiscm−1 of/O–H the 2905 AAPBA-PVA cm−1, and 2940 complexcm−1/C–H alkyl (Figure groups,2A). 1413 Curcumin cm−1/CH2, 1143 reacts cm−1/C–O with or several boron- containingC–O–C, species 1085 cm to−1 give/C–O–C a colored(from PVA), complex and 1665 called cm−1/C=O, a rosocyanine 1270 cm−1/C–H, complex. 1350 cm−1/B- This color is due OH, 701 cm−1/phenyl group (from AAPBA confirms the synthesis of the AAPBA-PVA to complexcomplex formation (Figure 2A). between Curcumin the reacts boron with several species boron-containing and curcumin species (24). to give In our a col- study, curcumin replacedored the complex PVA andcalled interacted a rosocyanine with complex. boronate This color to produceis due to complex a visible formation red complex be- confirmed by TLCtween (Figure the boron2D). species Hence, and thecurcumin functional (24). In our integrity study, curcumin of boronate replaced the was PVA maintained and in the complexedinteracted form. with boronate to produce a visible red complex confirmed by TLC (Figure 2D). Hence, the functional integrity of boronate was maintained in the complexed form.

Figure 2. Characterization of the synthesized SMPs. (A) Confirmation of the synthesis of AAPBA-polyvinylalcohol (PVA) Figure 2. Characterizationby Fourier-transform of the infrared synthesized spectroscopy SMPs. (FTIR). ( AThe) Confirmationidentified peaks of ofthe the chemical synthesis groups as of follows: AAPBA-polyvinylalcohol 1665 cm−1/C=O, (PVA) by Fourier-transform1636 cm− infrared1/C=C, Amide spectroscopy II/1555 cm−1, Benzene/1429 (FTIR). The cm identified−1, Boronate/1347 peaks cm−1 of, OH/3200–3500 the chemical cm− groups1. (B) Size asmeasurement follows: of 1665 cm−1/C=O, −1 AAPBA-PVA SMPs by dynamic−1 light scattering (DLS). (C)− SEM1 image of AAPBA-PVA SMPs.−1 (D) Functional integrity −of1 1636 cm /C=C,boronate Amide confirmed II/1555 by the cm development, Benzene/1429 of a red-colored cm spot, in Boronate/1347 curcumin thin layer cm chromatography, OH/3200–3500 (TLC). cm .(B) Size measurement of AAPBA-PVA SMPs by dynamic light scattering (DLS). (C) SEM image of AAPBA-PVA SMPs. (D) Functional 3.2. Transfection Studies integrity of boronate confirmed by the development of a red-colored spot in curcumin thin layer chromatography (TLC). 3.2.1. Optimization of the Plasmid:Polymer Ratio A different range of plasmid:polymer ratios was used to form polyplexes. At 1:7 and 3.2. Transfection1:20 plasmid:polymer Studies ratios, the complete complexation of GLuc was observed by agarose gel electrophoresis (Figure 3A). However, transfection of the cells occurred only with the 3.2.1. Optimization1:20 ratio (Figure of3B). the Therefore, Plasmid:Polymer this was the ratioRatio that was used for all further transfection Aexperiments. different range The commonly of plasmid:polymer used transfection re ratiosagent PEI was was used used as to a form positive polyplexes. control, At 1:7 and and naked plasmid was used as a negative control. 1:20 plasmid:polymer ratios, the complete complexation of GLuc was observed by agarose gel electrophoresis (Figure3A). However, transfection of the cells occurred only with the 1:20 ratio (Figure3B). Therefore, this was the ratio that was used for all further transfection experiments. The commonly used transfection reagent PEI was used as a positive control, Pharmaceutics 2021, 13, x FOR PEER REVIEW 7 of 10 and naked plasmid was used as a negative control.

Figure 3. Optimization of transfection efficacy of GLuc polyplex complexation. (A) The complexation of plasmid-polymer Figure 3. Optimization of transfection efficacy of GLuc polyplex complexation. (A) The complexation of plasmid-polymer investigated by agarose gel electrophoresis using different plasmid:polymer ratios; (Lanes 1: Ladder, 2: Naked plasmid, investigated3: PEI, by 4: agarose Xfect 1:0.7, gel 5: electrophoresis Xfect 1:2, 6: Xfect using1:3.5, 7: different Xfect 1:7, plasmid:polymer8: Xfect 1:20). (B) Analysis ratios; of (Lanes transfection 1: Ladder, in NIH3T3 2: Naked cells at plasmid, 3: PEI, 4:different Xfect 1:0.7, plasmid:polymer 5: Xfect 1:2, (plasmid/polymer) 6: Xfect 1:3.5, 7: ratios Xfect by 1:7, luciferase 8: Xfect assay 1:20). (Bars ( B1:) Naked Analysis plasmid, of transfection 2: PEI, 3: Xfect in 1:0.35, NIH3T3 4: cells at differentXfect plasmid:polymer 1:0.7, 5: Xfect 1:2, (plasmid/polymer) 6: Xfect 1:3.5, 7: Xfect 1:7, ratios 8: Xfect by 1:20, luciferase 9: Only cells). assay (C (Bars) Dose 1:optimization Naked plasmid, in three different 2: PEI, assays, 3: Xfect 1:0.35, i.e., luciferase assay for cellular transfection, alamarBlue™assay for metabolic activity, picogreen® assay for DNA quanti- 4: Xfect 1:0.7,fication, 5: was Xfect performed. 1:2, 6: Xfect Results 1:3.5, indicate 7: Xfect that Xfect™ 1:7, 8: Xfectshowed 1:20, its maximum 9: Only cells).transfection (C) Dosewith minimal optimization toxicity inat 5 three µg, * different assays, i.e.,represents luciferase statistical assay significance for cellular by transfection, one-way ANOVA, alamarBlue n = 3, SD,™ andassay p < 0.05. for metabolic activity, picogreen® assay for DNA quantification, was performed. Results indicate that Xfect™ showed its maximum transfection with minimal toxicity at 3.2.2. Optimization of Polyplex Dose in NIH3T3 Cells 5 µg, * represents statistical significance by one-way ANOVA, n = 3, SD, and p < 0.05. Three different concentrations of polyplex, i.e., 1, 5 and 25 µg/well, were studied for transfection capability. At 1 µg concentration, the cell viability was good; however, transfection was less (1.4 × 107 relative light units (RLUs)). In total, 25 µg of polyplex was toxic to the cells, and thus minimal transfection was observed at this concentration. A dose of 5 µg of GLuc was optimal for NIH3T3 cells where the highest transfection (1.8 × 108 RLU) was observed (Figure 3C).

3.3. Glucose-Responsive Behavior 3.3.1. Hydrodynamic Diameter To assess the influence of glucose on the AAPBA–PVA complex, the hydrodynamic diameter shift of SMPs was observed at different glucose concentrations ranging from 5 to 50 mM (Figure 4A) DLS. It was found that the increase in the hydrodynamic diameter of SMPs correlated with increasing glucose concentrations. The SMPs swelled from a diameter of 800 nm to a diameter of 2000 nm in the presence of 10 mM glucose concentration at a physiological pH. This increase in diameter is due to a slow deterioration of boronate- PVA. The glucose molecule replaces PVA and allows the inward movement of water into the SMPs, resulting in the degradation of the SMPs. The degradation of SMPs occurred when the glucose concentration increased more than 10 mM. Due to the degradation of the SMPs, the hydrodynamic diameter was reduced at 20 and 50 mM glucose concentrations. This demonstrates that the boronate remains active at a physiological pH which allows the weakening of the boronate–PVA complex in the presence of glucose and, in reverse, allows the formation of the boronate–glucose complex.

B C A * *

(%) *

* 140 (nm)

120 2500 * released

100

2000

BSA 80

diameter RLU 1500 ve 60 1000 40 500 20 Cumula 0 0

Hydrodynamic 0 10 20 50 0 5 10 20 50 Time (hr) Glucose concentra4on (mM) Glucose concentra7on (mM)

Figure 4. Glucose-responsive behavior of the synthesized SMPs. (A) The increase in the hydrodynamic diameter of AAPBA-PVA SMPs corresponded with glucose concentration up to a value of 10mM glucose; after this, the integrity of the SMPs was destabilized because of the high water pressure developed inside them. (B) Release of encapsulated bovine

Pharmaceutics 2021, 13, x FOR PEER REVIEW 7 of 10

Figure 3. Optimization of transfection efficacy of GLuc polyplex complexation. (A) The complexation of plasmid-polymer investigated by agarose gel electrophoresis using different plasmid:polymer ratios; (Lanes 1: Ladder, 2: Naked plasmid, 3: PEI, 4: Xfect 1:0.7, 5: Xfect 1:2, 6: Xfect 1:3.5, 7: Xfect 1:7, 8: Xfect 1:20). (B) Analysis of transfection in NIH3T3 cells at different plasmid:polymer (plasmid/polymer) ratios by luciferase assay (Bars 1: Naked plasmid, 2: PEI, 3: Xfect 1:0.35, 4: Xfect 1:0.7, 5: Xfect 1:2, 6: Xfect 1:3.5, 7: Xfect 1:7, 8: Xfect 1:20, 9: Only cells). (C) Dose optimization in three different assays, i.e., luciferase assay for cellular transfection, alamarBlue™assay for metabolic activity, picogreen® assay for DNA quanti- Pharmaceuticsfication,2021 was, 13 performed., 62 Results indicate that Xfect™ showed its maximum transfection with minimal toxicity at 5 µg, *7 of 10 represents statistical significance by one-way ANOVA, n = 3, SD, and p < 0.05.

3.2.2. Optimization of Polyplex Dose in NIH3T3 Cells 3.2.2. Optimization of Polyplex Dose in NIH3T3 Cells Three different concentrations of polyplex, i.e., 1, 5 and 25 µg/well, were studied for Three different concentrations of polyplex, i.e., 1, 5 and 25 µg/well, were studied transfection capability. At 1 µg concentration, the cell viability was good; however, trans- for transfection capability. At 1 µg concentration, the cell viability was good; however, fection was less (1.4 × 107 relative light units (RLUs)). In total, 25 µg of polyplex was toxic transfection was less (1.4 × 107 relative light units (RLUs)). In total, 25 µg of polyplex to the cells, and thus minimal transfection was observed at this concentration. A dose of 5 was toxic to the cells, and thus minimal transfection was observed at this concentration. µg of GLuc was optimal for NIH3T3 cells where the highest transfection (1.8 × 108 RLU) A dose of 5 µg of GLuc was optimal for NIH3T3 cells where the highest transfection was observed (Figure 3C). (1.8 × 108 RLU) was observed (Figure3C).

3.3.3.3. Glucose-Responsive Glucose-Responsive Behavior Behavior 3.3.1.3.3.1. Hydrodynamic Hydrodynamic Diameter Diameter ToTo assess the influence inﬂuence of glucose on the AAPBA–PVA complex, the hydrodynamic diameterdiameter shift of SMPsSMPs waswas observedobserved atat different different glucose glucose concentrations concentrations ranging ranging from from 5 to5 to50 50 mM mM (Figure (Figure4A) 4A) DLS. DLS. It was It was found found that that the the increase increase in thein the hydrodynamic hydrodynamic diameter diameter of ofSMPs SMPs correlated correlated with with increasing increasing glucose glucose concentrations. concentrations. The SMPsThe SMPs swelled swelled from afrom diameter a di- ameterof 800 nmof 800 to anm diameter to a diameter of 2000 of nm 2000 in nm the in presence the presence of 10 of mM 10 mM glucose glucose concentration concentration at a atphysiological a physiological pH. pH. This This increase increase in diameter in diameter is due is due to a to slow a slow deterioration deterioration of boronate-PVA. of boronate- PVA.The glucose The glucose molecule molecule replaces replaces PVA PVA and allowsand allows the inward the inward movement movement of water of water into into the theSMPs, SMPs, resulting resulting in the in degradationthe degradation of the of SMPs. the SMPs. The degradationThe degradation of SMPs of SMPs occurred occurred when whenthe glucose the glucose concentration concentration increased increased more than more 10 mM.than Due10 mM. to the Due degradation to the degradation of the SMPs, of thethe SMPs, hydrodynamic the hydrodynamic diameter wasdiameter reduced was at reduced 20 and 50at 20 mM and glucose 50 mM concentrations. glucose concentra- This tions.demonstrates This demonstrates that the boronate that the remains boronate active remains at a active physiological at a physiological pH which allowspH which the allowsweakening the weakening of the boronate–PVA of the boronate–PVA complex in the complex presence in ofthe glucose presence and, of in glucose reverse, and, allows in reverse,the formation allows of the the formation boronate–glucose of the boronate–glucose complex. complex.

B C A * *

(%) *

* 140 (nm)

120 2500 * released

100

2000

BSA 80

diameter RLU 1500 ve 60 1000 40 500 20 Cumula 0 0

Hydrodynamic 0 10 20 50 0 5 10 20 50 Time (hr) Glucose concentra4on (mM) Glucose concentra7on (mM)

Figure 4. Glucose-responsiveGlucose-responsive behavior behavior of of the the synthesized SMPs. ( (AA)) The The increase increase in in the hydrodynamic diameter of AAPBA-PVA SMPs corresponded with glucose concentration up to a value of 10mM glucose; after this, the integrity of the SMPs was destabilized because of the high water pressure developed inside them. (B) Release of encapsulated bovine the SMPs was destabilized because of the high water pressure developed inside them. (B) Release of encapsulated bovine serum albumin (BSA) from the synthesized AAPBA-PVA SMPs was depending on the concentration of glucose present at the site. (C) The highest relative light unit (RLU) corresponds to highest transfection was observed at the highest glucose concentration—i.e., 50 mM; n = 3, SD, p < 0.05, and * represents statistical signiﬁcance by one-Way ANOVA.

3.3.2. Glucose-responsive BSA Release Different glucose concentrations (0, 10, 50, 100 mM) were studied to validate BSA release from the BSA encapsulated SMPs (Figure4B). The amount of BSA released was directly proportional to the increase in glucose concentration. The cumulative percentage of BSA released was plotted as a factor of time. It was observed that 75%, 85%, and 95% BSA was released from SMPs at 10, 20 and 50 mM glucose concentrations after 72 h.

3.3.3. Glucose-Responsive Transfection The GLuc polyplexes were successfully loaded within boronate-PVA SMPs. The release of GLuc polyplexes from the boronate-PVA SMPs correlated with the increasing glucose concentrations. A two-fold increase in transfection corresponding RLUs was Pharmaceutics 2021, 13, x FOR PEER REVIEW 8 of 10

serum albumin (BSA) from the synthesized AAPBA-PVA SMPs was depending on the concentration of glucose present at the site. (C) The highest relative light unit (RLU) corresponds to highest transfection was observed at the highest glucose concentration—i.e., 50 mM; n = 3, SD, p < 0.05, and * represents statistical significance by one-Way ANOVA.

3.3.2. Glucose-responsive BSA Release Different glucose concentrations (0, 10, 50, 100 mM) were studied to validate BSA release from the BSA encapsulated SMPs (Figure 4B). The amount of BSA released was directly proportional to the increase in glucose concentration. The cumulative percentage of BSA released was plotted as a factor of time. It was observed that 75%, 85%, and 95% BSA was released from SMPs at 10, 20 and 50 mM glucose concentrations after 72 h.

3.3.3. Glucose-Responsive Transfection Pharmaceutics 2021, 13, 62 The GLuc polyplexes were successfully loaded within boronate-PVA SMPs. The8 of re- 10 lease of GLuc polyplexes from the boronate-PVA SMPs correlated with the increasing glucose concentrations. A two-fold increase in transfection corresponding RLUs was observedobserved for for 50 50mM mM glucose-containing glucose-containing media media compared compared to to 10 10 or or 20 20 mM mM glucose-containing glucose-containing mediamedia (Figure 44C).C).

3.4.3.4. Cytocompatibilty Cytocompatibilty 3.4.1.3.4.1. Metabolic Metabolic Activity Activity WeWe assessed thethe cytotoxicitycytotoxicity ofof SMPs SMPs in in the the glucose-containing glucose-containing media media on on NIH3T3 NIH3T3 ﬁ- fibroblastbroblast monolayer monolayer culture. culture. The The cytocompatibility cytocompatibility of AAPBA-PVAof AAPBA-PVA SMPs SMPs was assessedwas assessed with withalamarBlue alamarBlue™™ assay. assay. It was It foundwas found that thethat metabolic the metabolic activity activity of the of cells the wascells notwas affected not af- fectedby the by presence the presence of SMPs of even SMPs after even 48 after h at the48 highesth at the concentration highest concentration used (100 usedµg/well) (100 µg/well)(Figure5A). (Figure This demonstrates5A). This demonstrates that the SMPs that and the theirSMPs degraded and their polymer degraded by-products polymer by- do productsnot cause do any not short-term cause any cytotoxicity. short-term cytotoxicity.

* A B C

140 (%) 120 100

ac: vity 80

60 40 20 Metabolic 0 5 10 20 50 100 DMSO

SMPs (µg) Figure 5. Cytocompatibility of the synthesized SMPs on NIH3T3 cells. ( A) The cellular metabolic activity in the presence TM of SMPs was was assessed assessed by by alamarBlue alamarBlueTM assay,assay, and and the the assays assays showed showed that that the the me metabolictabolic activity activity was was not notaffected affected in the in presence of SMPs. (B) Merged fluorescence image of live cells (green channel) and dead cells (red channel) without SMPs. the presence of SMPs. (B) Merged fluorescence image of live cells (green channel) and dead cells (red channel) without (C) Merged fluorescence image of live cells (green channel) and dead cells (red channel) in the presence of SMPs. Scale bar SMPs. (C) Merged fluorescence image of live cells (green channel) and dead cells (red channel) in the presence of SMPs. = 100 µm. n = 3, SD, p < 0.05, and * represents statistical significance by one-Way ANOVA. Scale bar = 100 µm. n = 3, SD, p < 0.05, and * represents statistical significance by one-Way ANOVA. 3.4.2. Live Dead Assay 3.4.2.The Live live Dead dead Assay assay confirmed that no cell death was seen in the presence of AAPBA- PVA TheSMPs live after dead 48 assayh at 100 confirmed µg concentration that no cell (Figure death 5B,C). was seen in the presence of AAPBA- PVA SMPs after 48 h at 100 µg concentration (Figure5B,C). 4. Conclusions 4. ConclusionsBy utilizing boronate cis-diol interactions, a glucose-responsive SMPs system was developed.By utilizingThe stability boronate of thecis boronate-diol interactions, interaction a at glucose-responsive a physiological pH SMPs was achieved system was by copolymerizationdeveloped. The stability of DMAEMA of the boronatewith AAPBA, interaction and cis at-diol a physiological interactions governed pH was achieved the for- mationby copolymerization of a complex ofwith DMAEMA PVA. The with complete AAPBA, fabrication and cis-diol of boronate-containing interactions governed SMPs the formation of a complex with PVA. The complete fabrication of boronate-containing SMPs was achieved in a single-pot synthesis. The synthesized AAPBA-PVA SMPs exhibited glucose-responsive behavior at higher glucose concentrations in physiological pH conditions. The hydrodynamic diameter of SMPs increased up to 2000 nm in 10 mM glucose, and after that degradation was initiated. This suggests the slow and responsive behavior of SMPs with reversible binding to external glucose molecules. The destabilization of the AAPBA–PVA complex by interacting with glucose allows the release of the encapsulated BSA in a glucose-responsive manner. AAPBA-PVA SMP loaded GLuc genes were delivered to the NIH3T3 cells depending on the glucose concentration present at the delivery site. Finally, the cellular metabolic activity of NIH3T3 cells was not altered in the presence of the SMPs at a concentration of 100 µg/well, as shown in alamarBlueTM assay. Herein, we have successfully presented a fabrication method of glucose-responsive SMPs for gene delivery. However, investigating these SNPs in an in vivo hyperglycemic disease model is necessary to ensure its safety and therapeutic potential.

Supplementary Materials: The following are available online at https://www.mdpi.com/1999-492 3/13/1/62/s1, Figure S1: Conﬁrmation of AAPBA synthesis by NMR, FTIR analysis. Pharmaceutics 2021, 13, 62 9 of 10

Author Contributions: Conceptualization: A.P.; Experimental Plans: A.P., M.M., A.S.; Data Curation: M.M.; Writing: M.M., A.S.; Editing: A.P.; Supervision: A.P.; Funding Acquisition: A.P. All authors have read and agreed to the published version of the manuscript. Funding: Government of Maharashtra (India), Postgraduate Scholarship, EBC-2012/C.No.164/ Edn-1 and European Regional Development Fund and Science Foundation Ireland under Ireland’s European Structural and Investment Fund (Grant Number 13/RC/2073) provided financial support. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data can be made available on request. Acknowledgments: The authors would like to thank Oliver Carroll for his technical guidance in the project, Raghvendra Bohara for the editorial and critical assessment of the manuscript, and Anthony Sloan for his editorial assistance in finalizing the manuscript. The authors would also like to thank Roisin A. Doohan, NMR Spectroscopist, School of Chemistry and Éadaoin Timmins, NCBES, NUI, Galway for assistance in the SEM facility. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations SMPs—Submicronic particles, AAPBA—Acrylamidophenylboronic acid, PVA—Polyvinyl alcohol, DEMAEMA—N,N-Dimethylaminoethyl Methacrylate, HEMA—2-Hydroxyethyl methacrylate, APS— Ammonium persulfate, TEMED—N,N,N’,N’-Tetramethylethane-1,2-diamine, BSA—Bovine serum albumin, GLuc—Gaussia luciferase, 1H NMR—Proton nuclear magnetic resonance, 11B NMR—Boron nuclear magnetic resonance, FTIR—Fourier-transform infrared spectroscopy, SEM—Scanning electron microscopy, TLC—Thin layer chromatography, PEI—Polyethylenimine, DLS—Dynamic light scattering, and RLU-Relative light units.

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