Glucose-responsive polymeric micelles via boronic acid-diol

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Glucose-responsive polymeric micelles via boronic acid-diol complexation for insulin delivery at neutral pH Heba Gaballa, and Patrick Théato Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01508 • Publication Date (Web): 04 Jan 2019 Downloaded from http://pubs.acs.org on January 4, 2019

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Biomacromolecules

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Glucose-responsive polymeric micelles via boronic

2

acid-diol complexation for insulin delivery at neutral

3

pH

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Heba Gaballa 1 and Patrick Theato 1,2,3*

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1

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45, D-20146 Hamburg, Germany.

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2

8

(KIT), Engesser Strasse. 18, D-76131 Karlsruhe, Germany.

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3

Institute for Technical and Macromolecular Chemistry, University of Hamburg, Bundesstrasse

Institute for Chemical Technology and Polymer Chemistry, Karlsruhe Institute of Technology

Soft Matter Synthesis Laboratory, Institute for Biological Interfaces III, Karlsruhe Institute of

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Technology (KIT), Herrmann-von-Helmholtz-Platz 1, D-76344 Eggenstein-Leopoldshafen,

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Germany.

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13 14

Dedicated to Prof. Dr. Rudolf Zentel on the occasion of his 65th birthday.

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1 Environment ACS Paragon Plus

Biomacromolecules 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT

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In the present study, glucose-responsive polymeric complex micelles based on the complexation

19

of phenylboronic acid (PBA) based block copolymer and diol-functionalized polymers are

20

reported. The phenylboronic acid and diol-based block copolymers were successfully synthesized

21

in only two reaction steps using RAFT polymerization and post-polymerization modification of

22

the

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pentafluorophenylacrylate)] poly[(AMP-b-(AMP-co-PFPA)] reactive block copolymer. The self-

24

assembly of the complex micelles was investigated under neutral conditions using DLS and TEM.

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The interaction of the PBA block copolymer and diol-containing polymer via boronate ester bond

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was investigated by 1H-NMR and UV-Vis spectroscopy. The complex micelles enhanced the

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glucose responsiveness under physiological conditions compared to simple PBA micelles.

28

Furthermore, the successful glucose-triggered release of FITC-insulin from the polymeric micelles

29

was investigated.

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INTRODUCTION

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Diabetes mellitus (hyperglycemia) is a metabolic disorder that is characterized by high blood

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glucose levels (Type I and Type II).1,2 It requires a life-long treatment to keep glucose

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concentration at a normal level by monitoring glucose concentration and administrating insulin by

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multiple daily injections. Inconvenient and painful diabetic treatments inspired scientists to exploit

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alternative strategies for the treatment of diabetes. Consequently, glucose-responsive polymers

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have raised great interest in the past decades because of their promising applications in self-

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regulated insulin delivery.3–6 The common strategies that have been reported in this field are based

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on enzymatic reactions between glucose oxidase (GODx) and glucose or carbohydrate-binding

obtained

poly[(N-acryloylmorpholine-block-(N-acryloylmorpholine-co-


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lectin proteins, such as concanavalin A (Con A) as a complimentary binder to glucose.7–12

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However, these protein-based systems lacked stability for long-term use and showed cytotoxicity,

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which limited their use for clinical trials.13 To address the limitations of protein-based glucose

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oxidase and Con (A), enzyme-free boronic acid and derivatives have been developed as synthetic

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alternatives to these materials. For example, phenylboronic acid C6H5B(OH)2 (PBA) and

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derivatives have proven their capability to bind with cis-1,2-diols and cis-1,3-diols of common

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sugars, including glucose, through reversible ester formation offering a promising class of glucose-

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responsive

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(uncharged/hydrophobic) form and a tetrahedral (charged/hydrophilic) form in aqueous solutions

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with a reported pKa ≈8-9.19–28 It is noteworthy that PBA-based polymers have shown a notable

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glucose responsiveness only under high pH conditions (pH9) because of their relatively high pKa,

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which limits their in vivo studies and drug delivery applications.29–35 Kataoka et al.36 synthesized

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earlier chemically crosslinked hydrogels based on poly(N-isopropylacrylamide) (PNIPAm) and 3-

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acrylamidophenylboronic acid (AAPBA), and the gels were used for a glucose-regulated release

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of insulin. However, the addition of glucose increased the fraction of charged borate anions only

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at pH 9. For the past several years, ongoing research on boronic acid polymers adapted many

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strategies to enable the glucose-responsive behavior of these polymers under physiological

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conditions.37 For example, Wulff-type styrenic monomers have demonstrated a higher affinity than

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PBA to bind with monosaccharides at a neutral pH.38–40 However these benzoboroxle monomers

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are either complicated to prepare or they revealed poor yields after polymerization, both limiting

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their practical applications.41 Consequently, another strategy was adapted to lower the pKa of

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phenylboronic acid-based polymers which consisted of introducing electron-withdrawing groups,

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such as amino or carbonyl groups either into the polymer backbone or in the vicinity of the

materials.14–18

PBA

exists

in

an

equilibrium


between

a

triangular


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phenylboronic acid moiety to decrease the apparent pKa via the coordination between nitrogen or

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oxygen and boron.42–47 Another reported method is the complexation of PBA-containing polymer

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and glycopolymer resulting in the formation of boronate ester bonds, which are highly responsive

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to physiological pH when compared to phenylboronic acid and thereby allow pH and glucose-

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responsive drug release under physiological conditions.48–52 Shi and coworkers53,54demonstrated

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an interesting strategy to prepare polymeric micelles based on the complexation of phenylboronic

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acid and glycopolymers. Interestingly, the results revealed that the release of insulin could be

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enhanced in the presence of glucose under physiological conditions due to the interaction of

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boronic acid moieties and glycopolymer-forming boronate ester crosslinking bonds. Recently, we

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reported on the successful synthesis of boronic acid-based polymers via RAFT polymerization and

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post-polymerization modification techniques and studied their pH- and sugar-responsive

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behaviors.55 However, the prepared PBA block copolymer showed a higher affinity to bind with

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fructose than glucose under basic conditions because of their relatively high pKa (~ 9). More

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recently, we published56 a paper on the self-assembly of a block copolymer containing

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phenylboronic acid and glycine under physiological pH where its relative pKa was significantly

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lowered due to the coordination between boronic acid moiety and amine and carbonyl groups in

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the glycine segment. Polymeric micelles formed from block copolymers showed a notable glucose

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responsiveness but only at a higher glucose concentration (~ 50 mg mL-1). Nevertheless,

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considering the ease and success of the synthetic strategy via post-polymerization modification in

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the preparation of well-defined boronic acid block copolymers, we considered it would be worthy

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to utilize and optimize this strategy. In the present work, well-defined block copolymers containing

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phenylboronic acid and diols are prepared in a facile one-pot RAFT polymerization of

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pentafluorophenyl ester containing monomer (PFPA) to prepare a reactive precursor block


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copolymer, which was then post-modified. Poly(acryloylmorpholine) (PAMP) is a promising

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hydrophilic substitute to poly(ethylene glycol) (PEG) in a wide range of biomedical applications

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because it is a water soluble, non-toxic and biocompatible polymer and can be synthesized using

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conventional polymerization techniques.57,58Glucosamine and 3-amino-1, 2-propanediol as diol-

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containing amines are utilized for the modification because they are available at low cost and allow

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the preparation of water-soluble diol-containing polymers. Therefore, we expect that the

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interaction of PBA-containing polymer and diol-functionalized block copolymers leads to the

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formation of complex micelles through boronate ester bonds between pendent boronic acids and

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diol moieties at neutral pH. The sizes of micelles in terms of polymer feed ratios are studied. Then,

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the addition of different concentrations of glucose and the corresponding change in micelle

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structure due to replacing complexation of boronic acid and diols with glucose is studied. Lastly,

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the self-regulated insulin delivery in response to 2 g L-1 glucose (hyperglycemia) is studied under

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physiological conditions (pH 7.4, 37oC).

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1. EXPERIMENTAL SECTION

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Materials

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Trioxane (99%), D-(-)-glucose (Glu, 99%), D-(+)-glucosamine hydrochloride (≥99%), (±)-3-

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amino-1,2-propanediol (97%), 1,4-dioxane (99.9%) and N,N-dimethylformamide ( DMF, 99.9%)

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were used as received from Sigma-Aldrich, unless otherwise stated. Tetrahydrofuran (THF,

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99,9%) and n-hexane (99.9%) were purchased and used as received from VWR.

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Azobisisobutyronitrile (AIBN, Sigma-Aldrich) was recrystallized twice from methanol and stored

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at 4oC. 3-Aminophenylboronic acid hydrochloride (98%) was purchased from Alfa Aesar. 4-

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Cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl] pentanoic acid (CDTPA) was prepared



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107

following an earlier published report.59 Pentafluorophenyl acrylate was synthesized by adapting

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the published procedure.60 N-Acryloylmorpholine (AMP, 98%, TCI) was passed over a short

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column of basic aluminum oxide prior to use in order to remove the inhibitor. Insulin (27.5 IU mg-

110

1

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Hydrochloric acid (HCl, 1N) was purchased from ROTH. Fluorescein 5-Isothiocyanate (FITC,

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97%) was obtained from TCI. Triethylamine (Et3N, 99%) was purchased from Grüssing.

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) was purchased from Serva. Sodium hydroxide (NaOH, 97%) was obtained from Geyer.

Synthesis of poly[(AMP-b-(AMP-co-PFPA)]-PF1

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AMP (4.19 g, 14.16 mmol), CTA (59.5 mg, 014 mmol), and AIBN (1.21 mg, 0.007 mmol) were

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placed in a Schlenk tube in a ratio of [AMP]: [CTA]: [I] = [100]: [1]: [0.05]. 1,4-dioxane (6 mL)

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and 1,3,5-trioxane (214.2 mg) as an internal standard were added. The flask was sealed with a

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rubber septum and the reaction mixture was subjected to 3 freeze-pump-thaw cycles and then was

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placed into a 70oC preheated oil bath under stirring for 30 min. AMP conversion was monitored

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by 1H-NMR spectroscopy (chloroform-d, 300 MHz). Meanwhile, PFPA (2.35 g, 8.30 mmol) was

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purged with argon in a separate flask for 30 min. When the AMP polymerization had been stirred

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for 30 min (22% monomer conversion calculated by 1H-NMR spectroscopy), the PFPA was

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cannulated into the reaction vessel. After 60 min (86% AMP conversion calculated by 1H-NMR

123

spectroscopy and 80% PFPA conversion calculated by

124

removed from the heat and opened to the atmosphere, and placed into liquid nitrogen. The solution

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was added drop-wise to cold hexane to precipitate a yellow polymer. The polymer was re-

126

precipitated into colder hexane from THF, centrifuged, and vacuum-dried, yielding a yellow

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powder (3.5 g, yield= 83.5%).

128 129

19

F-NMR spectroscopy) the tube was

Post-modification of poly [(AMP-b-(AMP-co-PFPA)] with 3-amino-phenylboronic acid (P1), glucosamine (P2), and 3-amino-1,2-propanediol (P3) 6 Environment ACS Paragon Plus

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In a typical modification reaction, poly[(AMP-b-(AMP-co-PFPA)] 100 mg (0.0078 mmol of

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PFPA) was dissolved in 3 mL of DMF in a small vial with a stirring bar, then 1.2 equiv. with

132

respect to PFPA of the desired amine was added into the solution to which 2 equiv. of triethylamine

133

was added. The reaction mixture was stirred at 40oC for 24 hrs. Upon completion, the solution was

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purified by dialysis against water (MWCO 6000 Da) and then lyophilized to give the desired block

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copolymers. Finally, the polymer samples were analyzed by 1H,

136

spectroscopy.

137

19

F NMR, and FT-IR

Preparations of block copolymer micelles.

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First, 20 mg of block copolymer P1 was dissolved in basic water (pH 10) and then acidic water

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(pH 2) was slowly added into the polymer solution under vigorous stirring until the appearance of

140

opalescence. This micelle solution was dialyzed against water (pH 7) with MWCO 6000 for 24

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hrs. Finally, the micelle solution was diluted to a concentration of 1 mg mL-1 by PBS buffer (pH

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7.4). For the preparation of complex micelles, P2 and P3 were dissolved in PBS buffer (pH 7.4)

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with a concentration of 1.5 mg mL-1. Then a given volume of P1 basic solution was quickly added

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into P2 and P3 under vigorous stirring. The mixed solutions became opalescent indicating the

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formation of complex micelles. The micelle solutions were dialyzed against water and finally, the

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solutions were diluted with PBS buffer (pH 7.4) to the desired concentrations.

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Boronic acid-Alizarin red S (ARS) assay

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ARS was used as a catechol model dye to study the response of the complex micelles to glucose

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under physiological conditions. The complexation of ARS and polymeric micelles was performed

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in PBS buffer solution (pH 7.4) at room temperature. Briefly, ARS solution with a concentration

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of (1.0 x 10-4 M) was prepared. Polymeric micelles of P1, P1-P2, P1-P3 (1 mg mL-1, pH 7.4) were



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prepared as reported previously. The polymeric solutions were added slowly under stirring into

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the ARS solution with a final concentration of (0.5 x 10-4 M). To study the glucose responsiveness,

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glucose (2 mg mL-1) was added into these solutions under stirring. The solutions were then kept

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for 12 hr to allow the glucose and the micelles to equilibrate. The UV-vis and fluorescence spectra

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were recorded for ARS and the polymeric micelles in the absence and presence of glucose.

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Preparation of Fluorescein-labeled insulin (FITC)

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Based on a previous report, the preparation of FITC-insulin was followed.61 Briefly, insulin (15

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mg, 0.0025 mmol) was added into 2 mL PBS buffer (pH 7.4) into a flask. Then fluorescein 5-

160

isothiocyanate (FITC) (2.1 mg, 0.005 mmol) was dissolved in dried DMSO (0.4 mL) and added

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dropwise to the solution containing insulin. The mixture was stirred at 4oC under argon and

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protected from light for 12 hrs. The solution was dialyzed against water to remove the unreacted

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FITC. FITC-insulin was precipitated through adjustment of the pH to 4 - 5 with HCl (0.1 M) and

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stored at 4oC.

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Loading and release of FITC-insulin.

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FITC-insulin (3mg) was dissolved in PBS buffer (pH 7.4) and mixed with P2 and P3 prepared

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solutions. A basic solution of polymer P1 was prepared and then added dropwise under stirring to

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the mixture of P2-FITC-Ins and P3-FITC-Ins until the solution changed from transparent to

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yellowish translucent. The micelle solution was dialyzed against water with a dialysis membrane

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(MWCO: 3500 Da) for 24 h and replaced with fresh water every 6 hrs to remove the unloaded

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FITC-insulin. At last, FITC-loaded micelle solutions were adjusted to a concentration of 1 mg mL-

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1

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mL of FITC-Ins loaded micelles were sealed in a dialysis membrane with MWCO 3500 Da and

. The release profile was studied in PBS buffer solution (pH 7.4) by dialysis method. Briefly, 10


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Biomacromolecules

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then dialyzed against PBS buffer (pH 7.4) with glucose concentration 2 g L-1 under gentle stirring

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at 37oC. At each predetermined interval, 1 mL of micelle solution was drawn from the dialysis bag

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for fluorescence measurement (Ex= 450 nm, Em= 581 nm) and replaced by 1 mL fresh solution.

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The cumulative release was calculated according to the following formula:

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Cumulative release % =

I0 - It x 100 % I0

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Where I0 denotes the fluorescence intensity of FITC-insulin loaded micelles at t = 0, while It

180

denotes the fluorescence intensity at different sampling times during the release.

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Characterizations

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1

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in CDCl3 and D2O/NaOD.

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spectrometer in CDCl3 and D2O/NaOD. Chemical shifts are reported relative to CDCl3 at 7.27

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ppm or D2O at 4.79 ppm. The molecular weight and corresponding molecular weight distribution

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(Mw/Mn) of poly[(AMP-b-(AMP-co-PFPA)] was determined by gel permeation chromatography

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(GPC) using polystyrene standards at room temperature using THF as eluent with a flow rate of

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1.0 mL min-1. The instrument was equipped with an intelligent AI12 pump, an RI 101 detector,

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two pre-columns MZ-Gel SDplus 50 × 8 mm with 50 Å and 100 Å, respectively, and a column

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MZ-Gel SDplus 300 × 8 mm linear 5 µm. FT-IR spectra before and after post-polymerization

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modification were recorded using the ATR (Smart iTR) unit on a Thermo Scientific Nicolet IS10

192

FT-IR spectrometer. The self-assembly behavior of block copolymer aqueous solutions and the

193

hydrodynamic diameters (Rh) and size distributions were determined by dynamic light scattering

194

(DLS). The DLS instrument consisted of a Malvern Zetasizer Nano-ZS90 apparatus equipped with

H-NMR spectra of block copolymers were recorded on a Bruker Fourier 300 NMR spectrometer 19

F-NMR spectra were obtained on a Varian Gemini 2000BB



195

a He–Ne laser operated at 632 nm and the measurements were made at a scattering angle of 90°.

196

Transmission electron microscopy (TEM) measurement was performed using a Hitachi H600

197

electron microscope instrument, operated at an acceleration voltage of 100 kV. TEM samples

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were prepared by dropping micelle solution (10 µL) onto a carbon-coated copper grid for 2 min

199

following removal of excess by a filter paper. Absorption spectra were measured on a JASCO V-

200

630 UV/Vis-photo-spectrometer, utilizing a Starna Silica (quartz) cuvette with 10 mm path

201

lengths, two faces polished. Fluorescence spectra were measured using a Jobin-Yvon-Horiba

202

Fluoromax-4 spectrofluorometer utilising Starna Silica (quartz) cuvette with 10 mm path lengths,

203

four faces polished, and the excitation wavelength was 450 nm.

204 205 206 207 208 209 210 211 212

RESULTS AND DISCUSSION


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Biomacromolecules

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Scheme 1. Synthesis of poly [(AMP)22-b-(AMP60-co-PFPA45)] -PF1, poly[(AMP)-b-(AMP-co-

215

PBA)]-P1, poly[(AMP)-b-(AMP-co-GA)]-P2 and poly[(AMP-b-poly(AMP-co-PRD)]-P3.

216 217 218 219

Synthesis of poly [(AMP)22-b-(AMP60-co-PFPA45)]



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220

The synthetic route for the preparation of a precursor block copolymer containing reactive PFPA

221

was already reported previously by our group.62,63 However, in the present study, we modified the

222

synthesis procedure yielding a block copolymer containing PPFPA in one step to overcome any

223

unwanted side reaction of PFPA during the polymerization (Scheme 1). By this newly developed

224

methodology, poly[(AMP)-b-(AMP-co-PFPA)] was synthesized by RAFT polymerization in 1,4-

225

dioxane at 70oC by a sequential addition of PFPA. AMP monomer conversion was monitored by

226

1

227

added to the polymerization mixture for 60 more minutes. The polymerization was then quenched

228

by cooling down in liquid nitrogen and exposure to air. The calculated overall AMP conversion

229

was 86%, as calculated from 1H-NMR (DPtarget = 96, DPfirst block = 22, DPsecond block = 60), and PFPA

230

conversion had reached 80%, as calculated from

231

monomer conversion was calculated by comparing the proton signals of AMP at δ = 5.6 ppm, 6.2

232

ppm and 6.4 ppm with the proton signal of 1,3,5-trioxane at δ = 5.1 ppm. Due to overlapping

233

signals in 1H-NMR, PFPA monomer conversion was calculated by comparing the integration of

234

PFPA monomer and PPFPA polymer signals at o-(δ = −153 ppm), p-(δ = −156ppm) and m-(δ =

235

−161.8 ppm) in 19F-NMR. GPC results showed a unimodal molecular weight distribution with Mn

236

= 2.6x104 g mol-1 and a dispersity of Đ = 1.3 (See ESI S1). The yielded block copolymer was

237

purified by precipitation in n-hexane and then characterized by 1H and

238

(Figure 1) in chloroform. The characteristic peaks for both blocks could be assigned; however,

239

they were partly overlapped. Nevertheless, the chemical shifts for (-CH3) protons of the Z-end

240

group in the CTA are shown at (0.88 ppm), indicating that the CTA is still intact after

241

polymerization. In addition, the characteristics peaks of PPFPA are shown at (-153 ppm), (-156

H-NMR spectroscopy, and after 30 min 22% of AMP monomer was consumed; PFPA was then

19

F-NMR (DPtarget = 56, DP = 45). The AMP


19

F-NMR spectroscopy

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242

ppm) and (-161 ppm) in the 19F spectrum of the purified polymer, indicating the successful chain

243

extension with PFPA.

244 245

Figure 1. 1H- and 19F-NMR of poly[(AMP)-b-(AMP-co-PFPA)].

246 247 248

Poly[(AMP)-b-(AMP-co-PBA)]-P1,

poly[(AMP)-b-(AMP-co-GA)]-P2

poly[(AMP-b-(AMP-co-PRD)]-P3


and


249

Synthesis of polymers featuring boronic acid groups is often challenging due to difficult synthesis

250

of both monomers and (co)polymers, their poor solubility in solvents, possible crosslinking during

251

polymerization and their complicated purification and characterization procedures.64 We

252

developed a straightforward route to prepare PBA-based polymers by post-polymerization

253

modification of PFPA-reactive pendant groups. Here lies the diversity and versatility of our

254

strategy as we prepared block copolymers containing PBA and diol functionalities avoiding any

255

complicated synthetic steps. All the reactions were conducted in DMF at 40oC for 24 hrs with 1.2

256

equiv. of amines. Figure 2 illustrates the successful installation of functional amines with

257

quantitative yield characterized by 1H-NMR spectroscopy. The reaction was conducted with 3-

258

aminophenyl boronic acid, glucosamine, and 3-amino-1,2-propanediol, yielding P1, P2, and P3,

259

respectively. In Figure 3, the peaks in the FT-IR spectra corresponding to PFPA ester at 1780 and

260

1540 cm-1 completely disappeared after modification in P1, P2 and P3, confirming the successful

261

substitution.


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Figure 2. 1H-NMR of modified polymers: P1 (black), P2 (blue) and P3 (fuchsia).


Biomacromolecules

D

C

Transmitance (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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B

A

4000

3500

3000

2500

2000

1500

1000

Wavenumber (cm -1) 264 265

Figure 3. FT-IR spectra (A): Block copolymer PAMP-b-(AMP-co-PFPA) (PF1) before

266

modification, (B) polymer modified with 3-aminophenyl boronic acid (P1), (C) polymer modified

267

with glucosamine (P2), and (D) polymer modified with 3-amino-1,2-propanediol (P3).

268 269

Self-assembly and the formation of complex micelles

270

Boronic acids are known to form reversible covalent bonds with cis 1,2- and cis 1,3 diol containing

271

molecules in aqueous media. In this study, the amphiphilic block copolymer P1 and the water-

272

soluble block copolymers P2 and P3 micellar solutions were prepared. We hypothesized that PBA

273

and diol-containing block copolymers could form a cross-linking via boronic ester formation with

274

the boronic acid-diol segments (Scheme 2).

275

concentration of 1 mg mL-1 at pH 7.4 below its pKa, where the PBA is in its uncharged/hydrophobic

Self-assembly of P1 was investigated at a


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276

form. DLS analysis revealed particle sizes with an average size (Dh) of ~ 171 nm, and TEM

277

imaging showed nearly spherical particles in the range of 100 – 150 nm (Figure 4) with a proposed

278

structure of hydrophobic PBA in the core and hydrophilic AMP as the outer shell. P2,P3

P1 HO

OH B OH

OH Diol

O

B

OH O

O

B

OH O

Glucose

Diol

Complexation with glucose

Complexation with diol Self-assembly

Disassembly

O N

279

O

280

Scheme 2. Schematic illustration of the formation of complex micelles of poly[(AMP)-b-(AMP-

281

co-PBA)]-P1 and poly[(AMP)-b-(AMP-co-GA)]-P2 or poly[(AMP-b-poly(AMP-co-PRD)]-P3.

(b)

(a) Number (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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10

100

1000

Size (d.nm)

282 283

Figure 4. (a) DLS data showing the particle size distribution and (b) TEM image of P1 polymeric

284

micelle solution (1 mg mL-1).

285 286

Moreover, crosslinked complexes of P1-P2 and P1-P3 were characterized by 1H-NMR

287

spectroscopy in D2O (see Figure 5). The aromatic protons of PBA between 7.2-7.5 ppm of P1



Page 18 of 36

288

clearly diminished upon complexation with P2 and P3, demonstrating the formation of ester

289

bonding between PBA and diol-containing polymers. What is noteworthy is the appearance of

290

characteristic peaks of glucosamine (α,β C1H1-) at 5.3 ppm and 1, 2-propanediol (-CH2-CH-CH2)

291

at 3.4 ppm.

(a)

(b)

(c)

292 293

Figure 5. 1H NMR spectra of (a) P1 and (b) complex micelle P1-P2 and (c) complex micelle P1-

294

P3. ∗ denotes the residual solvent peak from D2O-d.

295 296

To prepare stable complex micelles, different feed ratios of P1, P2, and P3 were prepared in order

297

to study the appropriate conditions for the complexation (see Table 1). It should be noted that a

298

too high content of glucosamine in polymer P2 compared to P1 was unfavorable because unstable

299

micelles were formed with an average size ca. 50 nm (CM1_G) and could be disintegrated with

300

time. This might be due to the increase in the total hydrophilic content by diffusing more


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Biomacromolecules

301

glucosamine chains into the micelle core and subsequently, the micelles would fall apart. However,

302

when the feed ratio was 1:1 (w/w) (CM5_G), the average size increased to ca. 200 nm and the

303

formed micelles were more stable. Nevertheless, it was observed that the size of the complex

304

micelles increased when increasing the ratio of 1, 2-propanediol content P3 with respect to P1. For

305

example, the complex feed ratio of 1:1 (w/w) (CM5_P) showed aggregation with an average size

306

of ca. 900 nm which might be attributed to the increase in the crosslinking points forming the

307

micelle core through PBA-diol complexation.

308

Table 1. Complex micelles with different compositions, all micelle solutions had a final

309

concentration of 0.5 mg mL-1.

Code

Composition (w/w)

PBA/diol(mol/mol)

Dh (nm)

CM1_G

1.0:0.2

1:6.9

50±10

CM2_G

1.0:0.4

1:3.4

95±20

CM3_G

1.0:0.6

1:2.3

80±20

CM4_G

1.0:0.8

1:1.7

130±20

CM5_G

1.0:1.0

1:1.3

200±30

CM1_P

1.0:0.2

1:2.4

50±20

CM2_P

1.0:0.4

1:1.2

80±20

CM3_P

1.0:0.6

1:0.8

400±50

CM4_P

1.0:0.8

1:0.6

1000±20

CM5_P

1.0:1.0

1:0.4

900±20

310 311

Glucose responsiveness of the complex polymeric micelles



312

The most efficient conditions in micelle complex formation yielding stable micelles and less

313

polydispersity was selected for further investigations. The complex micelles formed upon the

314

complexation of P1 and P2 or P1 and P3 were monitored by DLS in terms of the hydrodynamic

315

diameter (Dh) (Figure 6 a,b). The results revealed particles with average size ca. 230 nm upon

316

complexation between P1 and P2 suggesting that the incorporated glucosamine-containing

317

polymer would form not only a cross-linked core between PBA and glucosamine units but also a

318

swollen core increasing the size of the complex micelles. Contrarily, the complex micelles of P1

319

and P3 showed a particle size of ca. 80 nm, which could be possibly due to the significant increase

320

in the hydrophilicity of the cross-linked micelles and the decrease in the hydrophobic core. The

321

observed size changes in DLS were in good agreement with the TEM images, demonstrating

322

spherical particles with a significant increase in the size of P1-P2 micelles and a decrease in the

323

size of P1-P3 micelles (Figure 6 c,d). To evaluate the glucose responsiveness of the complex

324

micelles, the particle size change was monitored by DLS upon the addition of glucose. When 2

325

mg mL-1 of glucose was added, the particle size increased to ca. 530 nm for P1-P2 and to ca. 190

326

nm for P1-P3 which could be attributed to the swelling of the hydrophobic core due to the

327

formation of boronate ester bonds between boronic acid and glucose replacing the complexation

328

between PBA and diol units. The increase in glucose concentration to 5 mg mL-1 further increased

329

the size of P1-P2 micelles to ca. 730 nm, revealing the increase in swelling degree caused by the

330

increase in the amount of boronate ester, however P1-P3 complex showed a size of ≈ 6 nm,

331

confirming the disintegration of the micelles into unimers suggesting that 5 mg mL-1 of glucose

332

was sufficient for replacing the complexation between P1-P3 by glucose inducing the cross-linked

333

core to collapse. However, the complete disintegration of P1-P2 micelles was only observed in the

334

presence of glucose with a concentration of 10 mg mL-1, leading to unimers with an average size


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Biomacromolecules

335

of ~ 10 nm, indicating the full disintegration of the polymeric micelles. The results revealed the

336

increased degree of complexation between P1-P2 more than P1-P3 and thus higher glucose

337

concentration was needed to allow the micelles to be fully disintegrated.


Biomacromolecules

P1+P2+ 0 mg/mL Glu P1+P2+ 2 mg/mL Glu P1+P2+ 5 mg/mL Glu P1+P2+10 mg/mL Glu

Intensity (%)

40 30 20 10

1

10

100

Dh (nm)

1000

10000

P1+P3+ 0 mg/mL Glu P1+P3+ 2 mg/mL Glu P1+P3+ 5 mg/mL Glu

40

Intensity (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 36

30 20 10

1

10

100

Dh (nm)

1000

10000

338 339 340

Figure 6. DLS data in the absence and presence of glucose a) complex micelles of P1-P2, b)

341

complex micelles of P1-P3, and TEM images of c) P1-P2 and d) P1-P3.

342 343

It is also known that boronic acid can form a complex with Alizarin Red S (ARS), as shown in

344

Scheme 3. This complex could be perturbed by the addition of glucose, setting up a second


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Biomacromolecules

345

complex between the boronic acid and the glucose-forming complex (4). Here, the complexation

346

of the phenylboronic acid-based polymer P1 and the diol-containing polymers P2 and P3 were

347

investigated by probing the spectral changes upon reaction with ARS. In a buffer solution (pH

348

7.4), ARS existed in its neutral form (1), exhibiting a maximum absorption band at ~520 nm and

349

another peak at ~ 334 nm. Upon the addition of ARS to P1, a blue shift could be observed with a

350

maximum absorption band at ~ 489 nm, confirming the binding interaction between P1 and ARS.

351

When glucose was added (2 mg mL-1), the maximum absorption band did not change from 489

352

nm, which might be attributed to the lower pH of the solution (pH 7.4) which is less than the

353

reported pKa of PBA-based polymers. Interestingly, there was a slight shift of the maximum

354

absorption band to ~ 494 nm (Figure 7A) when the pH of the solution increased to pH 9 which is,

355

indeed, enhanced by the binding affinity of glucose with PBA, leaving ARS in form (1). It has

356

been reported that the complexation of PBA with diols could significantly lower the pKa of PBA

357

based polymers through the formation of boronate ester bonding, which would enhance the

358

glucose-responsiveness under neutral conditions.14 To further investigate the influence of

359

complexation of P1 with P2 and P3, we studied their UV-Vis spectra with ARS at pH 7.4. As

360

shown in Figure 7B, the absorption band of ARS with the P1-P2 complex was recorded at ~ 486

361

nm indicating the formation of an ester bond between ARS and boronic acid, resulting in the

362

absorption shift. After the addition of glucose, a maximum absorption showed at ~ 492 nm with a

363

similar shift compared to P1 at pH 9. The results demonstrated that the addition of glucose altered

364

the interaction between boronic acid and diol units in P2 and formed a second binding between

365

the boronic acid and glucose through the reversible boronate ester formation. The spectrum of

366

ARS with the P1-P3 complex showed an absorption band at ~ 498 nm with a ~13 nm shift

367

compared with P1-P2 (Figure 7C) and a significant shift to ~ 524 nm upon the addition of glucose.



368

The results revealed the higher binding affinity of P1 with P3 than ARS, not allowing PBA to

369

freely bind with ARS. Nevertheless, glucose could competitively replace the complexation of P1-

370

P3 than ARS leading to a significant shift in the spectrum. Another reason could be that the P1-

371

P2 complex micelles were entirely intact and more stable even upon the addition of 2 mg mL-1

372

glucose, while the P1-P3 complex was completely replaced upon the addition of glucose shifting

373

the absorption band of ARS. This enabled us to observe the glucose responsiveness of the complex

374

micelles under neutral conditions with an enhanced binding affinity of glucose with P1-P3

375

complex rather with P1-P2 complex. To further probe the responsiveness of complex micelles to

376

glucose, we performed fluorescence spectroscopy studies on the same solutions of ARS and

377

polymers. Interestingly, the fluorescence spectroscopic results are in a good agreement with the

378

UV-Vis results. The results in Figure S2 showed that the fluorescence intensity of ARS-P1 only

379

decreased by 22% upon the addition of glucose at pH 9 where the boronate ester can form between

380

glucose and PBA as in form 4, however in the case of P1-P2 and P1-P3 the intensity decreased by

381

12% and 51%, respectively under the physiological pH, suggesting lowering the apparent pKa of

382

the crosslinked micelles. Moreover, the results revealed the significant response of P1-P3 complex

383

to glucose which is observed by the significant decrease in the fluorescence intensity to overlap

384

with the ARS spectrum with weak fluorescent properties.


Page 24 of 36

Page 25 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

O

O

OH OH

O

1

3

O S O O Na

Glucose HO

Non-fluorescence

OH B OH

OH

OH B 4 O O

O OH B O

O

O S O Na O

2

Fluorescence

diol

385 386

Scheme 3. Schematic illustration of ARS and boronic acid complex and the interaction upon the

387

addition of glucose.


Biomacromolecules

388 (A)

(B) 0.6

0.6

ARS ARS_P1 ARS_P1_Glu_pH 7.4 ARS_P1_Glu_pH 9

0.4

520 nm

0.3

400

520 nm

0.3

0.2

486 nm

494 nm

0.1

0.0 300

0.4

489 nm

0.2

ARS ARS_P1_P2 ARS_P1_P2_Glu

0.5

Abs

0.5

Abs

500

492 nm

0.1

600

0.0 300

700

350

400

450

500

550

600

650

700

Wavelength (nm)

Wavelength (nm)

(C) 0.6

ARS ARS_P1_P3 ARS_P1_P3_Glu

0.5

0.4

Abs

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 36

520 nm

0.3

0.2

498 nm 0.1

524 nm 0.0 300

350

400

450

500

550

600

650

700

Wavelength (nm)

389 390

Figure 7. UV-vis absorption spectra of ARS upon complexation with polymeric micelle solutions

391

of (A) P1, (B) P1-P2, and (C) P1-P3. The measurements were conducted in the absence of glucose

392

and upon addition of (2 mg mL-1) glucose.

393 394

The glucose-responsive release of FITC-insulin

395

The normal glucose concentration level is (100 mg/dL, 1 g L-1), however, the typical diabetic

396

glucose level is (200 mg/dL, 2 g L-1) in hyperglycemia.54 Constant administration of insulin can

397

cause pain, inconvenience, infections, and hypoglycemia which is induced when blood glucose

398

decreases below normal levels. Thus, self-regulated insulin delivery systems are beneficial to 26 Environment ACS Paragon Plus

Page 27 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

399

overcome the inconvenience caused by injections as well as a better-controlled release and

400

enhanced bioavailability. Therefore, glucose-responsive polymeric micelles have a special

401

superiority as self-regulated insulin delivery systems, including a high encapsulation efficiency

402

and a better-controlled release of insulin in response to a change in glucose concentration.

403

Therefore, we investigated the FITC-insulin release profile of the complex micelles in response to

404

normal glucose level (1 g L-1) and a hyperglycemic level (2 g L-1) of glucose at pH 7.4 and 37oC.

405

As shown in Figure 8, in the presence of glucose with a concentration of 1 g L-1 the release profile

406

was very slow from P1-P3 complex micelles and only about 30% insulin was released in 24 h,

407

however when the glucose concentration increased to 2 g L-1, the amount of insulin released

408

significantly increased to about 90 % indicating the stability of complex micelles under normal

409

glucose concentration and better insulin release enhanced under diabetic glucose concentration.

410

As for P1-P2 complex micelles, the insulin release profile was relatively fast and not stable with

411

about 91% and 100% at glucose concentrations of 1 and 2 g L-1, respectively. This might be due

412

to the complete disintegration of the crosslinked core of P1-P2 in the presence of glucose and as a

413

consequence the fast release of insulin occurred. The results demonstrated that the release of

414

insulin from P1-P3 complex micelles was dependent on glucose concentration with better stability

415

under normal glucose concentration while the instability of P1-P2 complex micelles showed

416

similar insulin release at normal and hyperglycemic level.


100

100

80

80

Released FTIC_Insulin (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Released FTIC_Insulin (%)

Biomacromolecules

60

40

20

P1_P2_Glu 2 g/L P1_P2_Glu 1 g/L

0 0

5

10

15

20

Page 28 of 36

60

40

20

P1_P3_Glu 2 g/L P1_P3_Glu 1 g/L

0

25

0

5

10

15

20

25

Release Time (h)

Release Time (h)

417 418

Figure 8. Glucose-responsive release of FITC-insulin from complex micelles (A) P1-P2, (B) P1-

419

P3. All experiments were carried out under 1 and 2 g L-1 glucose at, pH 7.4, and 37oC.

420 421

CONCLUSIONS

422

In this study, well-defined phenylboronic acid and diol-based block copolymers were successfully

423

prepared from a precursor PPFPA block copolymer. The self-assembly and complex micelles of

424

PBA and diol polymers via boronate ester bonding were investigated. It is interesting to note, that

425

the complexation of the propanediol-based block copolymer with PBA (P1-P3) displayed a notable

426

glucose responsiveness compared to the glucosamine-containing block copolymer (P1-P2) in

427

terms of self-assembly, morphology, and binding with ARS dye under physiological pH. The

428

cumulative release of FITC-insulin from P1-P3 complex micelles was successfully triggered under

429

physiological conditions with 2 g L-1 glucose (hyperglycemia) with better stability with 1 g L-1

430

(normoglycemia). Thus, we believe that resultant glucose-responsive complex micelles with better

431

sensitivity to glucose under physiological conditions may be potentially used as a promising

432

candidate for a self-regulated insulin delivery system.

433


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Biomacromolecules

434

ASSOCIATED CONTENT

435

Supporting Information

436

Additional data on polymer characterizations (GPC analysis of poly[(N-acryloylmorpholine-

437

block-(N-acryloylmorpholine-co-pentafluorophenylacrylate)]

438

PFPA45)]) as a function of elution time and molecular weight) and Emission spectra (λex = 470

439

nm) of the ARS– polymers solutions of (A) P1, (B) P1-P2, and (C) P1-P3. The measurements were

440

conducted in the absence of glucose and upon addition of (2 mg mL-1) glucose.

(Poly[(AMP)22-b-(AMP60-co-

441 442

AUTHOR INFORMATION

443

Corresponding Author

444

* E-mail: [email protected].

445

ORCID

446

Patrick Theato: 0000-0002-4562-9254

447

Heba Gaballa: 0000-0002-1444-947X

448

Notes

449

The authors declare no competing financial interest.

450

Author Contributions

451

All authors have given approval to the final version of the manuscript.



452

ACKNOWLEDGMENT

453

We thank Hamburg University for financial support during the study.

454 455

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456

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457 458

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Table of Contents graphic

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Glucose-responsive polymeric micelles via boronic acid-diol complexation for insulin

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delivery at neutral pH

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Heba Gaballa and Patrick Theato

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Released Insulin (%)

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P1-P2 P1-P3

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Release Time (h)

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Complex glucose responsive polymeric micelles containing phenylboronic acid and diols were

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prepared for self-regulated insulin delivery application.

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KEYWORDS

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Diabetes – Glucose responsive polymers – Phenylboronic acid polymers – Complex micelles

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Glucose-responsive polymeric micelles via boronic acid-diol

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