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Nitrilotriacetic Acid (NTA) and Phenylboronic Acid (PBA) Functionalized Nanogels for Efficient Encapsulation and Controlled Release of Insulin Chang Li, Gang Wu, Rujiang Ma, Yong Liu, Ying Liu, Juan Lv, Yingli An, and Linqi Shi ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00546 • Publication Date (Web): 25 Oct 2017 Downloaded from http://pubs.acs.org on October 31, 2017

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ACS Biomaterials Science & Engineering

Nitrilotriacetic Acid (NTA) and Phenylboronic Acid (PBA) Functionalized Nanogels for Efficient Encapsulation and Controlled Release of Insulin Chang Li,† Gang Wu,† Rujiang Ma,†,* Yong Liu,† Ying Liu,† Juan Lv,† Yingli An,† and Linqi Shi†,‡,* †

State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Functional Polymer

Materials of Ministry of Education, Institute of Polymer Chemistry, College of Chemistry, Nankai University, Tianjin 300071, China. ‡

Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin 300071, China.

Corresponding Authors *E-mail: [email protected] (R.M.). *E-mail: [email protected] (L.S.).

ABSTRACT: Protein drugs play a significant role in the treatment of many

diseases such as diabetes, cancers, and immune system diseases. Though polymeric nanocarriers have been designed to delivery protein drugs for prolonging circulation lifetime and providing stimuli-triggered release, problems are still often encountered including lower loading efficiency and capacity as well as poor circulation stability because of the weak interaction between protein drugs and nanocarriers. Herein, we described a new kind of bifunctional polymeric nanogels for efficient loading and glucose-triggered

release

of

insulin.

Biodegradable

poly(N-isopropylacrylamide) (PNIPAM) based nanogels was synthesized with nitrilotriacetic acid (NTA) and phenylboronic acid (PBA) as 1 / 37

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functional groups and ethylene glycol dimethacrylate (EGDMA) as crosslinker. The NTA groups could specifically bind imidazole-containing protein drugs such as insulin via chelated zinc ions, leading an efficient loading of insulin. The structure, morphology, and drug loading properties of the nanogels were well-characterized, and glucose-triggered insulin release was achieved based on the glucose-responsiveness of PBA groups. MTT assay and enzymatic degradation revealed good biocompatibility and biodegradability for the nanogels. This kind of bifunctional

nanogels

would

be

promising

candidates

for

glucose-responsive delivery of insulin in the future.

KEYWORDS:

nitrilotriacetic

acid,

nanogels,

insulin,

efficient

loading,

glucose-responsive insulin delivery

■

INTRODUCTION

Proteins, as the basis of life, are involved in all the important process in the body, such as enzymatic catalysis, signal transduction, gene regulation, cell survival and programmed apoptosis, etc.1 Many diseases are closely related to the deficiency or function changes of proteins in body.2 Thus, the delivery of active protein drugs to the specific organs, tissues, and cells is important for the treatment of a wide range of diseases like diabetes, cancers, and immune system diseases. Compared with gene therapeutics and conventional small molecule drugs, protein drugs have 2 / 37


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attracted ever increasing attention in recent years because of its high activity and low toxicity.3 However, proteins are susceptible to environmental changes and easy to lose its biological activity after administration. Oral administration of protein drug always encounters with the risk of enzymolysis by protease in the digestive system. For more conventional intravenous administration, the heterologous protein drugs are easy to be recognized and cleaned by the body immune system because of their immunogenicity. This leads to a short half-life and a low bioavailability of protein drugs.4 In order to overcome these problems, a variety of polymeric nanocarriers have been developed for the delivery of protein drugs to prolong circulation time, improve cellular uptake, and control drug release.5-8 Protein drugs can be loaded into polymeric nanocarriers by either physical embedding or chemical bonding method.9 Physical embedding of protein drugs usually involves in hydrophobic association, electrostatic adsorption, and liposomes encapsulation.10 For hydrophobic association, protein drugs are encapsulated into the micellar core upon the micellization of amphiphilic polymers. The loading efficiency is relatively low because of the weak interaction between the relatively hydrophilic protein drugs and the hydrophobic polymer chains and the activity of protein drugs may be affected because of the possibly irreversible unfolding of protein chains.11 As far as electrostatic 3 / 37



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adsorption is concerned, oppositely charged polymers are applied in loading charged protein drugs by forming polyion complex (PIC) micelles.12-13 Unfortunately, protein drugs are easy to leak from the PIC micelles because of the lower surface charge density of protein drugs and the electrostatic shielding effect caused by the presence of salt in the system.14 When liposomes encapsulation was used, protein drugs were encapsulated in liposomes with a water-filled compartments, but the loading capacity is relatively low especially if charge-neutral lipids are used.15 Additionally, chemical bonding strategies were also developed for the loading of protein drugs into polymeric nanocarriers.10 A typical strategy is the modification of proteins by chemical linking of polyethylene glycol (PEG) chains, i.e. PEGylation, which could prolong the residence in body, decrease the degradation by metabolic enzymes, and reduce the immunogenicity of protein drugs.16 In addition, protein drugs were also conjugated to other polymers to form pro-drugs that could self-assemble into polymeric nanoparticles. This could endow the polymeric nanocarriers with abilities of stimuli-responsive, targeting, and intracellular delivery of protein drugs. But the chemical modification may have negative impact on the activity of protein drugs because of the consumption or the incomplete recovery of reactive groups on the protein surface.15 Recently, polymeric nanocapsules were reported for the encapsulation of proteins, where monomers were adsorbed on protein 4 / 37


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surface and then in situ polymerized into crosslinked polymer films around proteins.17 This kind of polymeric nanocapsules could not only effectively avoid the degradation of protein drugs but also had small size (~ 20 nm) that was favorable for systemic circulation.18 But the preparation process of the protein nanocapsules was complicated and the loading efficiency was not very high because only one single protein drug was encapsulated within each nanocapsule in most cases. Therefore, it is important to develop new polymeric nanocarriers for efficient loading and delivery of protein drugs. A strategy that takes advantage of non-covalent but specific interactions between protein drugs and polymeric nanocarriers may be promising for addressing the above problems.

Recently,

the

researches

about

materials

containing

nitrilotriacetic acid (NTA) have attracted our attention. NTA is a quadridentate chelator, which can bind transition metal ions such as Zn(II) and Ni(II) through its nitrogen atom and three carboxylate oxygens. The remaining two coordination sites of metal ions can be occupied by other coordination groups such as histidine imidazole.19-22 At present, the studies

on

NTA-containing

materials

mainly

focused

on

the

immobilization and isolation of hexa-histidine-tagged proteins based on the specific interaction between NTA chelated transition metal ions and hexa-histidine. Xu et al. synthesized NTA-modified FePt magnetic nanoparticles to separate, transport, and anchor hexa-histidine-tagged 5 / 37



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proteins.23 Katzenellenbogen and co-workers reported NTA-modified silica nanoparticles for the purification of histidine-tagged proteins via chelated nickel ions.24 The bound proteins could be released quickly in a mild condition such as weak acidity, competitive molecules containing imidazolyl group, and transition metal ions.21 Inspired by these, Dowdy et al. reported the synthesis of a pH sensitive nitrilotriacetic linker (NTA3) to peptide transduction domains (PTD) to enable intracellular delivery of hexa-histidine modified peptides and proteins.25 We are enlightened that NTA-installed polymeric nanocarriers would be promising candidates for efficient loading and delivery of protein drugs. However, studies related to this aspect are very rare and the only one example reported by Dowdy and co-workers involved in pre-modification of peptides and proteins with hexa-histidine group.25 It would be more important to develop universal platform for efficient loading and delivery of unmodified protein drugs. It is well known that many proteins contain histidine residues with the imidazole ring exposed on surface. For example, insulin is an SS-linked double chain protein and has a histidine imidazolyl group on the chain B. Mature insulin is stored in the secretory vesicles of pancreatic islet beta cells in a hexamer form (a unit of six insulin molecules) with a zinc ion coordinated in the center by six histidine imidazolyl groups.26 The hexamer is an inactive form with long-term stability, which can be readily transformed into active 6 / 37


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monomer form and released into blood to play its physiological role.27 Therefore, it is very possible that NTA-installed polymeric nanocarriers could be used for efficient loading and delivery of insulin. In this study, NTA-functionalized poly(N-isopropylacrylamide) (PNIPAM) nanogels were synthesized via precipitation polymerization and used for loading as well as delivery of insulin as illustrated in Scheme 1. Phenylboronic acid (PBA) was also incorporated into the nanogels for endowing them with glucose-responsiveness.28 Insulin was efficiently loaded into the nanogels via NTA-coordinated Zn(II) and glucose-responsive release was achieved. This kind of NTA/PBA bifunctional nanogels would be promising candidates for efficient loading and glucose-responsive release of insulin in the future.

Scheme 1. Schematic illustration of loading and release of insulin by the P(NIPAM-co-AAPBA-co-AANTA) nanogels. Insulin was loaded into the nanogels via NTA-coordinated Zn(II) and released out upon the swelling of the nanogels in response to glucose. 7 / 37



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■ MATERIALS AND METHODS Materials. N-isopropylacrylamide (NIPAM, 98%) was purchased from Sigma, recrystallized from hexane, and dried under vacuum prior to use. Ammonium

persulphate

(APS),

ethylene

glycol

dimethacrylate

(EGDMA), Nε-Carbobenzoxy-L-lysine (CBZ-Lys), palladium-carbon (Pd-C), α-D(+)-glucose (97%), and (m-acrylamidophenyl)boronic acid (AAPBA) were purchased from Aldrich and used without further purification. Fluorescein isothiocyanate-labeled insulin (FITC-insulin) was synthesized according to our previous work.29 Other reagents were of analytical grade and used as received. The water (>18MΩ) used in all experiments was obtained from a Millipore Milli-Q system.

Synthesis

of

AANTA.

Nα,Nα-bis(carboxymethyl)-Nε-benzyloxycarbonyl-lysine (CBZ-NTA) and Nα,Nα-bis(carboxymethyl)-lysine hydrate (NTA-Lys) were synthesized according to literature procedures.30 Briefly, 6 g CBZ-Lys and 5.96 g bromoacetic acid were dissolved in 40 mL 1 M NaOH solution and 21.5 mL 1 M NaOH solution respectively, and then the CBZ-Lys solution was slowly added into the bromoacetic acid solution under ice bath and the mixed solution was stirred at 70 oC for 4 h. CBZ-NTA was precipitated out by addition of an appropriate amount of 2 M HCl into the reaction mixture under room temperature and dried under vacuum to obtain a white solid. For the preparation of NTA-Lys, 2 g CBZ-NTA and 0.2 g 8 / 37


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Pd-C was dispersed in 50 mL HPLC grade methanol under ice bath and the solution was left to stir under H2 gas at 25 oC for 10 h. Then the precipitates of NTA-Lys and Pd-C were collected and washed 10 times with 200 mL pure water to obtain the NTA-Lys solution. A white solid of NTA-Lys

was

obtained

by

freeze-drying.

Nε-acrylamido,Nα-bis(carboxymethyl)-lysine (AANTA) used in this study as a co-monomer was synthesized by NTA-Lys reacting with acryloyl chloride. 1 g NTA-Lys and 0.6 g sodium bicarbonate was dissolved in 5 mL pure water and the solution was incubated in an ice bath, to which 0.96 mL of acryloyl chloride was added very slowly. The mixed solution was stirred for 15 h under room temperature and then crude AANTA was collected by rotary evaporation. The crude product was dispersed in HPLC grade methanol and solid impurities were removed by filtration. A white solid of AANTA was finally obtained by rotary evaporation.

Synthesis

of

the

polymer

P(NIPAM-co-AAPBA-co-AANTA)

nanogels

nanogels. were

synthesized

The by

precipitation polymerization28 of NIPAM, AANTA, and AAPBA with EGDMA as the cross linker. The nanogel sample (named Nanogel-1) used throughout this study was synthesized as follows. Briefly, 0.340 g NIPAM, 0.063 g AAPBA, 0.158 g AANTA, and 0.008 g EGDMA were dissolved in 100 mL water. The solution was filtered to remove any precipitates and transferred to a three-necked round bottom flask 9 / 37



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equipped with a condenser. Polymerization was carried out by heating the solution mixture to 70 °C under nitrogen atmosphere for 8 h with the addition of 0.01 g of APS as the initiator. The nanogels were purified by dialysis (cutoff 12 000 Da) against pure water for one week. Other two nanogel samples (Nanogel-2 and Nanogel-3) were also synthesized with varying monomer feed ratios.

Monitoring the fluorescence of ARS in the nanogel solutions. The glucose-responsiveness of the nanogels in PBS 7.4 was characterized by monitoring the fluorescence of Alizarin Red S (ARS) in the nanogel solutions according to our previous work.31 ARS was added to the nanogel solutions and stirred over 30 min for combining with the nanogels. Then a given volume of concentrated glucose solution was added to the mixed solution to obtain final glucose concentrations of 2, 5, and 10 g L−1 respectively. The fluorescence spectra of ARS were recorded as a function of time.

Loading and controlled release of FITC-insulin. The FITC-labeled insulin were prepared as our previous work.29 For the loading of FITC-insulin, to the PBS solution of nanogels

with a concentration of

0.125 mg/mL a given volume of zinc acetate solution was added and stirred overnight under room temperature. The free zinc ions were removed by dialysis against PBS buffer. Then, a given volume of FITC-insulin solution was added into the solution of nanogels with zinc 10 / 37


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ions coordinated. The solution mixture was magnetically stirred overnight and the free FITC-insulin was removed by using a dialysis bag with a molecular weight cutoff of 12 kDa. The same amount of FITC-insulin was used for the preparation of drug-loaded nanogel solutions with different Zn(II) concentrations. The fluorescence emission intensity of the FITC-insulin loaded nanogels was measured at 529 nm upon excitation at 494 nm to determine the loading content and encapsulation efficiency. The release of FITC-insulin from the nanogels was evaluated by immersing a dialysis bag that contained drug-loaded nanogels in solution of PBS 7.4 with varying glucose concentrations at 37 °C. Briefly, 2 mL of FITC-insulin loaded nanogels solution was injected in a dialysis bag with a molecular weight cutoff of 12–14 kDa and immersed in 20 mL of PBS 7.4 with glucose concentrations of 0, 5, 10, and 20 g/L respectively. At certain time intervals, 1 mL of buffer solution was sampled to measure the FITC-insulin concentration and 1 mL of fresh buffer was added. As for the on–off controlled release of insulin, the dialysis bag was immersed in PBS 7.4 with or without 10 g/L glucose alternately for 1 h at 37 oC. Then, 1 mL of buffer solution was taken to measure the insulin concentration and 1 mL of fresh buffer was added.

Measurement of absorption constant of FITC-insulin by the nanogels via Langmuir isotherm. The experiment of absorption equilibrium of FITC-insulin by the nanogels was carried out as follows. 11 / 37



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Three series of PBS stock solutions of glucose (0, 2, 5 g/L), FITC-insulin (0, 0.2, 0.4 … 1.2 mg/mL), and nanogel/Zn(II) (0.125 mg/mL/75 µM) were prepared. Then, 5 mL of glucose PBS solution, 10 mL of FITC-insulin PBS solution, and 10 mL of nanogel/Zn(II) solution were mixed and left in a shaker operating at 150 times/min for 24 h to reach equilibrium. Free FITC-insulin was removed by using a dialysis bag (cutoff 12 kDa). The fluorescence emission intensity of FITC-insulin at 529 nm was measured upon excitation at 494 nm to determine the content of FITC-insulin absorbed by nanogel/Zn(II). The absorption equilibrium of FITC-insulin by nanogel/Zn(II) was

studied under glucose

concentrations of 0, 2, and 5 g/L with varying FITC-insulin concentrations.

Enzymatic degradation and MTT assay of nanogels. Proteinase K solution with a concentration of 0.4 g/L was prepared by dissolving it into a PBS 7.4 solution. Then it was added to nanogel solutions at different temperatures for starting the degradation of the nanogels. It was characterized by monitoring the variations of light scattering intensity and hydrodynamic diameter distribution f(Dh) of the nanogel solution as a function of time. The cytotoxicity of the nanogels was evaluated by MTT assay according to our previous work.32-33 NIH 3T3 mouse fibroblast cells were seeded into a 96-well plate at a density of 104 cells per well in 100 µL RPMI1640 complete media containing 10% FBS and incubated at 37 12 / 37


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o

C in humidified 5% CO2 atmosphere for 24 h. The culture medium of

each well was replaced by 100 µL fresh medium containing nanogels with concentrations from 0.01 to 1 g L-1. After incubation for 24 h, 25 µL of MTT solution (1 g L-1) were used to replace the mixture in each well. The cells were incubated for another 4 h and the MTT solution was replaced by 150 µL of DMSO and the plates were slightly shaken for 10 min. The optical absorbance was measured at 492 nm using a microplate reader (Labsystem, Multiskan, Ascent, Finland). The NIH 3T3 mouse fibroblast cells without any treatment were used as the control.

Characterizations. 1H NMR spectra of CBZ-Lys, CBZ-NTA, NTA, AANTA, and nanogels were recorded on a Varian UNITY-plus 400 M NMR spectrometer at room temperature using DMSO and D2O as solvents respectively. Dynamic light scattering (DLS) measurement was performed on a laser light scattering spectrometer (BI-200SM) equipped with a digital correlator (BI-9000AT) at 636 nm. All samples were prepared by filtering a certain volume of nanogel solutions through a Millipore filter into a clean scintillation vial. Transmission electron microscopy (TEM) measurement was performed on a JEM-100CXII electron microscope at an acceleration voltage of 100 kV. Samples for TEM were prepared by depositing a drop of diluted nanogel solution (0.05 g/L) onto a carbon-coated copper EM grid and vacuum dried under room temperature. UV-vis absorption spectra were recorded on a 13 / 37



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TU-1810 UV-vis spectrophotometer (Purkinje General, China). FT-IR spectra were collected on a Bio-Rad FTS 6000 FT-IR instrument using 128 scans at an 8 cm−1 resolution. Fluorescence emission spectra were recorded on a Hitachi F-4600 fluorescence spectrophotometer under room temperature.

■ RESULTS AND DISCUSSION To introduce functional groups of NTA and PBA into the nanogels, their acryl derivatives were used as co-monomers for free radical polymerization. AAPBA was commercially available while AANTA was synthesized via a sequential three-step reaction from CBZ-Lys. Firstly, CBZ-Lys, of which the ε-amino was protected by carboxybenzyl group, was used to couple bromoacetic acid, resulting in CBZ-NTA, the nitrilotriacetic

acid

derivative

with

the

ε-amino

protected

by

carboxybenzyl group. Then, CBZ-NTA was deprotected by reduction under H2/Pd-C, leading to NTA-Lys, the nitrilotriacetic acid derivative. Finally, AANTA, the acryl co-monomer, was obtained by acylation of the ε-amino of NTA-Lys with acryloyl chloride. 1H NMR spectra of CBZ-Lys, CBZ-NTA, NTA-Lys, and AANTA were shown in Figure 1. In spectra I for CBZ-Lys, a small peak b at 3.1 ppm was attributed to the α-methenyl of adjacent carbonyl, while in spectra II for CBZ-NTA, the peak b became stronger and moved to 3.5 ppm, indicating the introduction of 14 / 37


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carboxymethyl to the α-amino of CBZ-Lys by coupling bromoacetic acid. In spectra III for NTA-Lys, signals from the benzyl group (d, 5.2 ppm and e, 7.2 ppm) disappeared after deprotection compared with spectra II, indicating the complete removal of the CBZ block. After the reaction of NTA-Lys with acryloyl chloride, sharp peaks for double bond (d, 5.7– 6.2ppm) were observed in spectra IV compared with spectra III, which indicated the NTA-Lys was acylated successfully and the co-monomer AANTA was obtained finally.

Figure 1. 1H NMR spectra of CBZ-Lys in D2O (I), CBZ-NTA in DMSO (II), NTA-Lys in D2O (III), and AANTA in D2O (IV).

The P(NIPAM-co-AAPBA-co-AANTA) nanogels were synthesized by precipitation polymerization of NIPAM with AANTA and AAPBA as functional co-monomers, EGDMA as cross linker, and APS as initiator as illustrated in Scheme 2. In order to prove the existence of functional 15 / 37



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groups in the synthesized nanogels, UV-vis, IR, and 1H NMR spectra were recorded for the nanogels and shown in Figure 2. The UV-vis spectrum of AAPBA was also provided together with nanogels in Figure 2A. The absorption peak at 265 nm was ascribed to the phenyl ring of AAPBA. When it was incorporated in nanogels, an absorption peak at 250 nm was observed, indicating the presence of PBA in the nanogels. A slight blue shift of the absorption peak may be attributed to the change of chemical environment of phenyl ring after polymerization. The IR spectrum of the nanogels in Figure 2B displayed characteristic absorption bands of carboxylate at 1460 cm-1 and 1650 cm-1, suggesting the successful incorporation of NTA into the nanogels. More direct evidence for the presence of PBA and NTA in the nanogels was found from 1

HNMR analysis. As shown in Figure 2C, peaks a (1.0 ppm), b (3.7 ppm),

and c (7.0 ~ 7.9 ppm) were attributed to methyl of NIPAM, α-methenyl of adjacent carbonyl of AANTA, and phenyl ring of AAPBA respectively. The characteristic peaks from AAPBA and AANTA confirmed the successful introduction of these functional groups into the nanogels. The composition of the nanogels was accordingly determined by the relative intensities (integral areas) of these characteristic peaks. As shown in Table 1, the molar ratios of AAPBA and AANTA to NIPAM in Nanogel-1 were calculated to be 8.3% and 10.0% respectively, which were relatively lower than the feed ratios of 10% and 17%. When different feed ratios 16 / 37


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were used, decreased molar ratios of AAPBA and AANTA to NIPAM were still observed for Nanogel-2 and Nanogel-3, indicating that AAPBA and AANTA were not completely included into the nanogels during polymerization.

Scheme 2. Synthesis of the P(NIPAM-co-AAPBA-co-AANTA) nanogels via precipitation polymerization of NIPAM, AAPBA, and AANTA with EGDMA as crosslinker.

Figure 2. (A) UV-vis spectra of AAPBA and the nanogels in PBS 7.4. (B) IR spectrum of the nanogels. (C) 1H NMR spectrum of the nanogels in DMSO.

17 / 37



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Table 1. Characterization of three nanogel samples with varying monomer ratios at 25oC. NIPAM/AAPBA/AANTA (n/n/n) Sample

Dh (nm)b

Feed ratio

Calculated ratio

Nanogel-1

100:10:17

100:8.3:10

277

Nanogel-2

100:11:6

100:8.3:3.3

155

100:5:16

100:3.3:10

180

Nanogel-3 a

a

1

b

Calculated from HNMR spectrum; Determined by DLS.

The morphology and size of the nanogels were characterized by TEM and DLS and the results were shown in Figure 3. TEM images in Figure 3A and 3B showed that the nanogels had well-defined spherical morphology and highly uniform particle size of about 230 nm. Besides, the nanoparticles were evenly distributed in the field of view and aggregation or fusion was not observed, indicating proper rigidity and good integrity for the nanogels. Figure 3C and 3D showed hydrodynamic diameter distributions f(Dh) of the nanogel under different conditions. The black square lines indicated very narrow and monodisperse size distributions for the nanogels in the absence of glucose, which were consistent with the TEM results. The average sizes of the nanogels were 277 and 246 nm respectively under 25 and 37 oC, both were larger than that from TEM measurements, which were reasonable for the nanogels in a hydrated state. The smaller size for the nanogel under 37 oC was attributed to the volume phase transition (VPT) of PNIPAM-based nanoparticles above the LCST.28 The size tunability of the nanogels depending upon the change of the monomeric ratios was studied by DLS 18 / 37


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and the results were listed in Table 1. With the decreasing feed ratios of AAPBA or AANTA, the diameters of Nanogel-2 and Nanogel-3 decreased to 155 and 180 nm respectively at 25 oC, indicating an apparent effect of monomer feed ratio on the size of the nanogels.

Figure 3. (A and B) TEM images of the nanogels. (C and D) DLS results and glucose-responsiveness of the nanogels in aqueous solutions of PBS 7.4 at 25oC and 37oC respectively.

In this study, PBA was incorporated to endow the nanogels with glucose responsiveness. PBA could bind glucose and transform from a neutral, hydrophobic form to a negatively charged, hydrophilic form.34-37 Thus the nanogels would undergo volume transition upon the stimuli of 19 / 37



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glucose because of the change from hydrophobicity to hydrophilicity. This enabled us to observe the glucose-responsiveness of the nanogels by monitoring the hydrodynamic diameter distributions f(Dh). As shown in Figure 3C, the nanogels swelled with increased glucose concentration at 25 oC and the average hydrodynamic diameters Dh were 322, 384, and 456 nm under the glucose concentrations of 2, 5, and 10 g/L respectively, larger than that of 277 nm in the absence of glucose. Smaller sizes were obtained under different glucose concentrations at 37 oC as shown in Figure 3D, indicating a thermo-sensitive contraction of the nanogels above the LCST of PNIPAM. The glucose-responsiveness of the nanogels was also characterized by monitoring the fluorescence of ARS (Alizarin Red S) in the nanogel solutions. It is well known that ARS, which has a catechol moiety, will become fluorescent when it combines PBA group and the competitive replacement of ARS by glucose will lead to fluorescence quenching.31 The ARS had high and constant fluorescence intensity in the nanogel solution without glucose. When glucose was added, the fluorescence intensity of ARS decreased as a function of time because of the replacement of ARS by glucose. The fluorescence intensities of ARS in the nanogel solutions with 2, 5, and 10 g/L glucose decreased to 78%, 69%, and 62% respectively in 1 h, which indicated the nanogels were glucose-responsive

under

physiological 20 / 37


conditions.

The

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glucose-responsiveness laid the foundation for controlled release of insulin by the nanogels.

Figure 4. (A) Fluorescence emission spectra of ARS (λext = 468 nm) in the nanogels solution as function of time with 5g/L glucose in PBS 7.4. (B) Time-dependent fluorescence emission intensity of ARS at 565 nm in the presence of glucose with concentrations of 2, 5 and 10 g/L respectively.

Polymeric nanogels have been widely employed as reservoir in drug delivery,38-39 but the loading efficiency is relatively low because of the weak interaction between nanogels and drugs. In this study, and the NTA-installed nanogels were employed to load insulin efficiently via the specific interaction between the NTA-chelated Zn(II) and the histidine imidazole of insulin. Zinc ions are electron-deficient Lewis acid, which can coordinately bind electron-rich atoms such as nitrogen and oxygen with the maximum number of six. After chelating by the quadridentate NTA group, zinc ions can further coordinately bind other electron-rich atoms by the remaining two coordination sites. As mentioned in the introduction, mature insulin is stored in the secretory vesicles of pancreatic islet beta cells in a hexamer form (a unit of six insulin 21 / 37



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molecules) with a zinc ion coordinated in the center by six histidine imidazolyl groups.26, 40 Additionally, insulin could also form a hexamer with zinc ions in vitro such as in the insulin-containing microcapsules prepared

by

DL-lactic/glycolic

acid

(PLGA).41

As

for

other

imidazole-containing protein, recombinant human growth hormone (rhGH) was likely to be stabilized with zinc because rhGH formed a dimmer with zinc.42 It was believed that there was a strong interaction between zinc ions and imidazole-containing proteins such as insulin and rhGH. In our system, Zn(II) was immobilized in the nanogels by the quadridentate chelator NTA. It was reasonable that the remaining two coordination sites of Zn(II) could further bind insulin via interaction with the

imidazolyls

of

histidine.

This

paved

the

way

for

our

NTA-functionalized nanogels to efficiently load insulin which had coordinating residue of histidine imidazolyl. For the loading of FITC-insulin, zinc ions were firstly incorporated into the nanogels by chelating to NTA. In this work, nanogel solution with a concentration of 0.125 mg/mL was used, in which the theoretical NTA concentration was calculated to be 75 µM based on the monomer feed ratios in synthesis. 37 and 75 µM of zinc ions were selected as one half and equal to the theoretical NTA concentration respectively. The chelation of Zn(II) by NTA group in the nanogels was assessed by the intensity of the UV−vis absorption band of Zn(II)-coordinated carboxyl at about 220 nm.43 As 22 / 37


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shown in Figure 5A, the UV-vis absorbance of the nanogel solutions was relatively weak in the absence of Zn(II). When zinc ions were chelated, the absorbance band at 220 nm became stronger and the intensity increased with increasing amount of Zn(II) used. The absorbance intensity reached the maximum in the case of using 75 µM of Zn(II), which suggested the coordinating saturation of NTA. When the concentration of zinc ions was increased to 100 µM, the UV-vis spectrum nearly overlapped with that of 75 µM of Zn(II) as shown in Figure 5A, which further indicated the saturation of NTA groups in the nanogels.

Figure 5. (A) UV-vis spectra of the nanogels in PBS solutions with different concentrations of Zn(II). (B) Fluorescence emission spectra of the nanogel solutions with different concentrations of Zn(II). The same amount of FITC-insulin was used for the preparation of the drug-loaded nanogels except the case of nanogels-PBS without FITC-insulin.

In this study, FITC-insulin was loaded into the nanogels with Zn(II) coordinated by NTA and the free FITC-insulin was removed by dialysis. Successful loading of insulin into the nanogels was confirmed by the fluorescence emission spectra of FITC excited at 494 nm as shown in 23 / 37



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Figure 5B. For the NTA-functionalized nanogels in PBS without FITC-insulin, any fluorescence was not observed. In the case of nanogels without Zn(II), an FITC fluorescence at 529 nm was observed, indicating that insulin was absorbed into the nanogels based on diffusion. When zinc ions of 37 µM were used, the FITC fluorescence increased apparently with the same amount of FITC-insulin added as above, which definitely demonstrated the key role of the NTA-chelated Zn(II) in promoting the loading of insulin inyo the nanogels. With the increasing of zinc ions, the intensity of FITC fluorescence continued to increase and reached the maximum at 75 µM of Zn(II). When excessive amount of FITC-insulin compared with that of NTA in the nanogels was used for loading, the fluorescence emission intensities of FITC at 529 nm increased with concentrations of Zn(II) in the range of 0 - 75 µM and reached a maximum at 75 and 100 µM as shown in Figure 5B, which suggested the saturation of NTA-chelated zinc ions by FITC-insulin above 75 µM of Zn(II). The amount of FITC-insulin loaded in the nanogels could be quantitatively calculated from the intensity of the FITC fluorescence emission spectra at 529 nm in Figure 5B. It increased from 816 to 1355 with the increasing of Zn(II) from 0 to 75 µM, indicating a 66% increase in the loading capacity of the nanogels with the aid of NTA-chelated Zn(II) compared with that in the absence of Zn(II). The loading efficiency of insulin by the nanogels was increased accordingly from 11.0 to 18.3%, 24 / 37


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which was much higher than that of 13.6% in our previous work with hydrophilic complex micelles.29 Besides, zeta potentials of the nanogels were measured to study the effect of coordinated Zn(II) on the net negative charge of the nanogels. The zeta potential of the nanogels was measured to be -4.03±0.30 mV in the presence of 75 µM of Zn(II), which was very closed to that without zinc ions, -5.37±0.42 mV. This suggested that coordination of zinc ions would not have prominent effect on the net negative charge of the nanogels and thus would not affect the loading of insulin by the nanogels. From the results above, the chelation of zinc ions in the NTA-functionalized nanogels could significantly improve the loading capacity and efficiency for insulin. In this study, the release of FITC-insulin was evaluated by immersing a dialysis bag that contained drug-loaded nanogels in solutions of PBS 7.4 with different glucose concentrations at 37 °C. The cumulative release of FITC-insulin was calculated by monitoring the changes of the FITC fluorescence intensity in the external fluid. As shown in Figure 6A, only about 13% insulin was released from the nanogels in 12 h in the absence of glucose. When glucose was added, the release of insulin was accelerated obviously and the cumulants of released insulin were 30%, 42%, and 53% in 12 h for glucose concentrations of 5, 10, and 20 g/L respectively, indicating a larger cumulative amount of insulin released under a higher glucose concentration. The release of insulin nearly 25 / 37



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sustained for 60 h and the final cumulants of insulin released were 26%, 45%, 63%, and 75% for 0, 5, 10, and 20 g/L glucose respectively. The glucose-responsive release behavior of the nanogels can be explained as follows. The main body of nanogels was composed of PNIPAM that was thermosensitive and would become hydrophobic above its LCST (~32 oC). The nanogels would be on the whole hydrophobic and in a contracted state at physiological 37 oC, which would retard the release of insulin to a large extent. The introduction of PBA endowed the nanogels with glucose-responsiveness because of the transition of PBA from a neutral, hydrophobic form to a negatively charged, hydrophilic form after binding glucose. This would result in the swelling as well as increased permeability of the nanogels and thus facilitate the release of insulin.38

26 / 37


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Figure 6. (A) Glucose-responsive release of insulin from the nanogels with 75 µM Zn(II) under varying concentrations of glucose; (B) On-off release of insulin from the nanogels with 75 µM Zn(II) triggered by 10 g/L glucose. All the experiments were carried out at 37 oC. (C) Langmuir isotherms for equilibrium absorption of FITC-insulin by the nanogels with NTA chelated Zn(II) under different glucose concentrations.

Pulsed release of insulin in response to the fluctuation of glucose concentration is necessary for in vivo application on the treatment of diabetes. In this study, “on–off” insulin release experiment was carried out for the nanogels in the presence of 75 µM Zn(II) with 10 g/L glucose a trigger as shown in Figure 6B. During the incubation of the drug-loaded nanogels in PBS 7.4 without glucose for 1 h, the release of insulin was relatively slow and it could be considered as the “off” state. When the nanogel solution was subsequently incubated in PBS 7.4 with 10 g/L glucose for 1 h, the release of insulin was apparently accelerated and it could be defined as the “on” state. The “on–off” regulation of insulin release showed four apparent circles in 8 h, which suggested that the nanogels offered an effective strategy for self-regulated insulin delivery in response to glucose. Therefore, this kind of NTA/PBA-functionalized nanogels with the advantages of efficient loading and glucose-responsive release may be promising candidates for in vivo insulin delivery. Though the swelling of the nanogels in response to glucose could facilitate the diffusion of insulin out, it would be better to understand the real mechanism for the accelerated release of insulin in the presence of 27 / 37



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Page 28 of 37

glucose. The Langmuir isotherm of FITC-insulin absorbed by the nanogels with Zn(II) chelated by NTA was measured to study the apparent binding constant for Zn-insulin interaction. The absorption equilibrium of FITC-insulin by nanogel/Zn(II) was studied under glucose concentrations of 0, 2, and 5 g/L with varying FITC-insulin concentrations and the results are shown in Figure 6C. The Langmuir model was used to fit the experimental data and the Langmuir equation could be expressed as

ܳ=

ொ೘ ௄ೌ ஼ ∗ ଵା௄ೌ ஼ ∗

(1)

where Q (mg/g) and C* (mg/mL) are the absorbed FITC-insulin in the nanogels at equilibrium and the concentration of FITC-insulin in the PBS solution, respectively. Qm is the maximum absorption amount, and Ka (mL/mg) is the absorption constant. The experimental data were fitted to the Langmuir model through nonlinear regression analysis. The fitted parameters Qm, Ka, and R2 were summarized in Table 2. High R2 values close to 1 indicated that the Langmuir model well predicted the absorption behavior. Results in Table 2 showed that the maximum absorption amount Qm and the absorption constant Ka significantly decreased with increasing glucose concentration, indicating it had apparent influence on the absorption behavior of FITC-insulin by the nanogels. 28 / 37


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Table 2. Langmuir model parameters for isotherms of FITC-insulin absorbed by the nanogels with NTA chelated Zn(II) under different glucose concentrations. Cglucose (g/L)

0

2

5

Qm (mg/g)

460

307

251

Ka (mL/mg)

45

28

14

R2

0.98

0.98

0.95

Biodegradability and biocompatibility of drug delivery systems are very important properties for their in vivo applications. It is necessary for the polymeric nanocarriers being degraded and eliminated from body after mission completed. In this work, the nanogels were synthesized by free radical polymerization of acrylamide monomers with EGDMA as the crosslinker. The ester bond in EGDMA could endow the nanogels with biodegradability. Herein, Proteinase K, which can degrade ester bond,44-45 was used for enzymatic degradation of the nanogels and the experiment was monitored by measuring the change of the light scattering intensity of the nanogel solution as a function of time as our previous work.46 As shown in Figure 7A, the relative light scattering intensity of the sample invariably changed in the absence of Proteinase K, indicating the stability of the nanogels under 25 oC. When Proteinase K was used, the relative light scattering intensity of the nanogel solutions decreased quickly, which may be attributed to the decrease in the number of the nanogels in solution upon enzymatic degradation. The light scattering intensities of the nanogel solutions with Proteinase K decreased to about 0.05 in 120 29 / 37



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and 70 min at 25 and 37 oC respectively, indicating the nanogels were completely degraded by Proteinase K. A faster rate of enzymatic degradation of the nanogels at 37 oC was ascribed to a higher activity of Proteinase K at the physiological temperature. Variation of size distribution was also monitored to characterize the degradation of the nanogels in the presence of Proteinase K. Figure 7B showed the size distributions of the nanogels before and after addition of Proteinase K at different temperatures. The nanogels were originally narrowly distributed in the absence of Proteinase K under 25 and 37 oC with the average diameters of 277 and 246 nm respectively. After incubation with Proteinase K for 60 min under 25 and 37 oC, though the nanogels were not completely degraded, their size distributions broadened apparently and the average diameters increased from 277 to 430 nm and 246 to 360 nm respectively. These changes may be attributed to the swelling of the nanogels upon enzymatic degradation because of the decrease of crosslinking density. Considering the possible toxicity of the components, it is necessary to evaluate the biocompatibility of our nanogels. In this study, the cytotoxicity of the nanogels was assessed by MTT assay of the viability profiles of NIH 3T3 mouse fibroblast cells after incubation with the nanogel solutions for 24 h. As shown in Figure 7C, High cell viabilities (> 90%) were observed for all cell samples containing the nanogels with 30 / 37


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varying concentrations from 1 to 1000 µg/mL, which indicated that the PNIPAM-based nanogels were generally biocompatible. These above results demonstrated that this kind of nanogels had favorable biodegradability and biocompatibility and could be promising candidates for in vivo drug delivery.

Figure 7. (A) Light scattering intensities of nanogel solutions as a function of time upon enzymatic degradation under 25 and 37 oC. (B) Variation of size distributions of the nanogels upon enzymatic degradation under 25 and 37 oC for 60 min. (C) MTT assay of the NIH 3T3 cells under varying concentrations of the nanogels.

■ CONCLUSIONS In conclusion, we developed NTA and PBA functionalized nanogels by precipitation polymerization of NIPAM, AANTA, and AAPBA with 31 / 37



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EGDMA as the cross linker. IR and UV-vis and 1HNMR spectra confirmed the introduction of NTA and PBA in the nanogels. TEM and DLS measurements showed well-defined spherical morphology and very narrow size distributions for the nanogels. Incorporation of PBA endowed the nanogels with glucose-responsiveness as characterized by DLS and fluorescence spectrometry with ARS as the probe. Zinc ions could be substantially absorbed into the nanogels based on the chelation by NTA. The nanogels with NTA-chelated Zn(II) could effectively encapsulate insulin with a 66% increase in loading capacity compared with those in the absence of Zn(II) and the loading efficiency was increased accordingly from 11.0 to 18.3%. Glucose-responsive and on-off controlled release of insulin was successfully achieved. Enzymatic degradation and MTT assay revealed good biodegradability and biocompatibility for the nanogels. This kind of bifunctional nanogels with the features of efficient loading of insulin, glucose-responsive drug release as well as good biodegradability and biocompatibility would be promising candidates for insulin delivery.

■ AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (R.M.). *E-mail: [email protected] (L.S.). 32 / 37


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ORCID Rujiang Ma: 0000-0002-2021-3850 Linqi Shi: 0000-0002-9534-795X

Notes The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS This study was financially supported by the National Natural Science Foundation

of

China

(Nos.

51773099,

51390483,

91527306,

21620102005, and 51603105), the Natural Science Foundation of Tianjin, China (No. 15JCYBJC29700), and the PCSIRT (IRT1257).

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