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Sustained-release of exendin 4 using injectable and ionic-nano-complex forming polymer hydrogel system for long-term treatment of type 2 diabetes mellitus Bo-Bae Seo, Mi-Ran Park, and Soo-Chang Song ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b19669 • Publication Date (Web): 04 Apr 2019 Downloaded from http://pubs.acs.org on April 4, 2019

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ACS Applied Materials & Interfaces

Sustained-release of exendin 4 using injectable and ionicnano-complex forming polymer hydrogel system for longterm treatment of type 2 diabetes mellitus

Bo-Bae Seo a, Mi-Ran Park a, and Soo-Chang Songa,*

a Center

for Biomaterials, Korea Institute of Science & Technology, Seoul, 130-650, Republic

of Korea

* Corresponding author. Tel: +82-2-958-5123. Fax: +82-2-958-5089. E-mail address: [email protected].

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ABSTRACT Daily treatment of diabetes to stabilize blood glucose level poses a challenge for patients with diabetes mellitus. Diabetes is a long-term metabolic disorder, and the treatment lasts for the rest of the patient’s life after diagnosis. We presented a new injectable hydrogel depot system using exendin 4 (Ex-4) interactive and complex forming polymeric ionic-nanoparticles for long-term anti-diabetes treatment. Protamine-conjugated polymer (ProCP) was developed to form ionic-nano-complexes with Ex-4, as amino group-rich protamine and the negatively charged Ex-4 (pI: 4.86) interact with each other due to their opposite electric charges in physiological conditions. Morphologically, the ProCP were nano-particles in aqueous condition (10 wt% of ProCP in phosphate buffered solution, < 25C) and formed condensed ionic- and nano-complexes with Ex-4. The complexes formed a bulk hydrogel when exposed to body temperature. A slow release of the Ex-4/ProCP ionic-nano-complexes occurred from the hydrogel depot, followed by Ex-4 dissociation from the ionic-nanocomplex and hydrolysis of ProCP. Given that the Ex-4 release occurs after the complex release from the hydrogel, the periods of Ex-4 release and hydrogel maintenance may be similar. The present system showed a considerably prolonged Ex-4 release. Additionally, it showed potential as a long-term effective and reproducible anti-diabetes treatment.

KEYWORDS: injectable hydrogel, ionic-complex, sustain-release, exenatide, diabetes mellitus


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▪ INTRODUCTION Diabetes mellitus primarily occurs as a result of obesity, and the incidence of diabetes is increasing worldwide. Type 2 diabetes (T2D) mellitus is a long-term metabolic disorder that is characterized by high blood sugar levels, insulin resistance, and relative lack of insulin.1,

2

Pharmacological intervention with an oral antidiabetic agent (OAD) is

considered as a second step after diet and exercise. Effective and safe alternatives to insulin can be prescribed for diabetes patients who are no longer achieving good glycemic control on OADs.2, 3 Glucagon-like peptide-1 (GLP-1) receptor agonists such as exendin 4 (Ex-4) are promising options for patients with T2D that do not benefit from OAD treatment. Ex-4 is a 39-amino-acid peptide with a molecular weight of 5.2 kDa. Byetta® containing Ex-4 was the first of a new class of incretin peptide mimetics approved by the Food and Drug Administration for adjunctive glycemic control in patients with T2D who were taking metformin, a sulfonylurea, or a combination of metformin and a sulfonylurea.4-6 Several studies reported the beneficial anti-diabetes role of Ex-4 such as glucose-dependent enhancement of insulin secretion, glucose-dependent suppression of inappropriately high glucagon secretion, retard of gastric emptying, reduction in food uptake and body weight, and an increase in β-cell mass.1, 2 Moreover, Ex-4 has a longer duration of action than that of native GLP-1 when administered subcutaneously; Ex-4 has a circulating half-life of 60–90 min, while GLP-1 has 1–2 min-half-life in blood plasma. However, there is a need for a prolonged therapeutic efficacy of Ex-4, because the administration of Ex-4 is conducted by subcutaneous (SC) injection once or twice a day.7, 8 To investigate the long-term effect of GLP-1 analogs, including Ex-4, several pharmaceutical techniques such as acylation, PEGylation, microsphere, and bio-conjugation to a monoclonal antibody (mAb) and natural/synthetic polymers have been actively studied. An acylated GLP-1 analog, semaglutide, was developed by Novo Nordisk, which showed a



once-weekly profile.1, 9 PEGylation also can prolong the duration of Ex-4 effect and reduce immunogenicity in vivo, and site-specific PEGylation techniques have been developed to enhance therapeutic activity of Ex-4.10-14 Conjugation of GLP-1 molecules to fragments of mAb also increases circulation time; Eli Lilly’s Dulaglutide was approved in 2014, and a once-weekly dosing regimen was allowed.1,

15

A controlled Ex-4 release system using

poly(lactic-co-glycolic acid) (PLGA) micro-particles is currently available in the market as a once-weekly product (Byducreon). The encapsulated Ex-4 release and entire degradation of PLGA microsphere, however, require a long period (approximately 7 weeks).16 For chronic treatment, the accumulated PLGA microsphere could be disadvantageous because of their acidic degradation products, which could augment inflammatory reactions. Moreover, Byducreon showed poor efficacy, and PLGA depot system needs 14 to 28 times higher amount of Ex-4 as compared to Byetta® for maintaining effective Ex-4 concentration.1 In addition, a complex form of GLP-1 analog and zinc showed more uniform peptide distribution with a steady release profile in the PLGA microspheres, whereas the encapsulated soluble native peptide showed an undesirable initial burst release.17 Besides, human albumin fused GLP-1 (Syncria®), human transferrin fused Ex-4, and GLP-1 analog linked to the fragment crystallizable (Fc) fragment of IgG4 (Dulaglutide) are in clinical trials.1 However, these techniques are necessarily accompanied by complicated modification processes and using an organic solvent. On the other hand, injectable hydrogel depot systems for sustained-release of Ex-4 without complicated modification processes have been reported, having a simple preparation method for Ex-4 loading.18, 19 Additionally, these injectable gel systems show a considerable extension of release-period and effective reduction of blood glucose levels. As mentioned above, hydrogel depot systems would be beneficial for Ex-4 long-term delivery with a simple preparation method and effectiveness on long-term therapeutic effects. However, many


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injectable Ex-4 depot hydrogels show discordance on periods of Ex-4 release and biodegradation of carrier hydrogels; the hydrogels remain much longer than the Ex-4 release periods, which could represent a considerable limitation for repeatable diabetes treatment. Thus, there would be a very low concentration of Ex-4 at the end of the dose cycle, even if the carriers remain longer. The reason of faster release behaviors of Ex-4 than the remaining periods of hydrogel depots may be that the Ex-4 is simply loaded into hydrogel depot without particular interaction with the delivery carriers. Moreover, repeatable anti-diabetes treatment is necessary for the diabetes patients who need to take care of their health for the rest of life. Therefore, it is important to have similar periods of Ex-4 release and carrier biodegradation as much as extending the Ex-4 release period. We developed injectable, temperature-dependent gelling, and drug depot hydrogel systems

using

poly(organophosphazenes).20-22

Especially,

these

injectable

poly(organophosphazene) hydrogel systems showed several beneficial properties as a protein delivery system such as hydrophilic nature, simple protein loading without organic solvent, high loading efficiency without any loss of protein content, and easy administration to the body with fine-gauge needle.20, 23, 24 In previous reports, poly(organophosphazenes) formed nano-sized particles in aqueous environment, and the solution state of the polymer nanoparticles with 10 wt% concentration in aqueous environment showed in situ gelling behavior based on temperature increase associated with body temperature.21, 25 Additionally, functional side-chains were easily substituted in the polymer’s backbone based on the target protein.20, 22, 25

We used ionic- and nano-complex-forming techniques for controlled release of target

proteins, resulting in its drastically enhanced bioavailability.20-22 The ionic-interactions could provide a favorable condition for the controlled release of the target proteins without loss of protein activities. We have used different ionic groups according to the isoelectric points of the target proteins (pI). For bone morphogenetic protein-2 (BMP-2) (pI: 8.2) delivery, we



synthesized negatively charged polymer that has carboxylic acid terminus.21,22 For human growth hormone (hGH) (pI: 5.12) delivery, positively charged polymer that has polyethylenimine (PEI) terminus was used.20 In this study, we developed an injectable sustained-Ex-4 release system using protamine conjugated poly(organophosphazene) (ProCP). In order to ensure the controlled release behavior of Ex-4, a 39-amino-acid peptide with less negatively charged parts, a protamine moiety was conjugated to the polymer for strong ionic-interactions with Ex-4 because as protamine has a higher pKa value than PEI. The protamine group-tailed ProCP existed in a nano-particle state in aqueous environment because of its amphiphilicity and formed an ionic-nano-complex with carboxylic-group rich Ex-4, because protamine had many arginine sequences and high density of positive charge. We confirmed that the nano-sized ionic-nano-complexes were composed of ProCP and Ex-4. These ionic-nano-complexes formed a bulk hydrogel with temperature increase, and they were then released from the hydrogel timely. Long-term Ex-4 release and enhanced bioavailability of Ex-4 were confirmed. The system’s anti-diabetes roles in diabetes (db/db) mice model with lowering blood glucose, reducing food uptake, and losing body weight effects were verified.

▪ RESULTS AND DISCUSSION Synthesis and characterization of ProCP. ProCP was designed to interact with Ex-4. ProCP has positively charged side-chains to interact with the negatively charged Ex-4 by their opposite electric charges. For strong ionic-interactions with Ex-4, a peptide with less negatively charged parts, the ratio of the protamine group was increased (1.67-times) compared to the previously studied protamine conjugated polymer.26 The synthesis procedure of the ProCP consisted of four steps as shown in Scheme 1. Poly(dichlorophosphazene) (Ι)


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was substituted with hydrophobic IleOEt and hydrophilic AMPEG to induce amphiphilicity and thermosensitive gelling behaviors. GlyGlyOAll group was substituted for further generation of the carboxylic acid group by removal of allyl group. Then, protamine was conjugated to the carboxylic acid termini of the poly(organophosphazene) (III) by amide linkage. Finally, two ProCPs (IV) were synthesized to have different amounts of protamine groups via different activation ratios of carboxylic acid termini. The synthesized ProCPs were characterized by the measurement of side-chain ratios and viscosity changes with temperature increase (Table 1). The molar ratios of substitutes were calculated from the integration ratios of 1H NMR (Figure S1). ProCP-1 showed less amount of protamine group than that of ProCP-2; ProCP-1 and ProCP-2 showed 1 and 2.5% protamine group among the whole side-chains, respectively. As a control, a polymer having no ionic-charged group (ContP), which could not form ionic-complex with Ex-4, was prepared. These three polymers were dissolved in PBS solution to be 15 wt% concentration of polymer solutions, and their viscosity changes were measured according to temperature changes (Table 1 and Figure S2). All polymer solutions showed 0 Pa·s until the starting point of viscosity increase (Tass). Approximately at 37C, the three polymer solutions showed hydrogel state with viscosities of 113 to 425 Pa·s. In addition, the molar ratio of the conjugated protamine group was controlled from 1.5% to 2.5% using the same pro-ProCP-2. The temperature-dependent sol-gel transition behaviors of the three different ProCP-2 were found to be different (Table S1 and Figure S3). In detail, Tass and Tmax increased, whereas V37C and Vmax decreased with the addition of the protamine group due to the increased hydrophilicity of the polymer. Evaluation of cytotoxicity of ProCP-2 was performed in NIH3T3 cells. The cells were incubated with cell culture media containing ProCP-2 (ProCP-2 concentration, 0–1 mg/mL) for 24 h. The incubated cells demonstrated high viability with all concentrations of



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the polymer (Figure S4).

Scheme 1. Synthesis scheme of protamine conjugated poly(organophosphazene) (ProCP).

Table 1. Characteristics of poly(organophosphazenes). Polymer

Structure a

Tass (C)b

Tmax (C)c

V37C (Pa.s)d

Vmax (Pa.s)e

ContP

[NP(IleOEt)57.0(GlyLacOEt)1.5(AMPEG)45.5]n

11.8

44.8

156

175

ProCP-1

[NP(IleOEt)58.0(GlyGlyOH)7.5(protamine)1.0(AMPEG)33.5]n

20.8

40.9

113

156

ProCP-2

[NP(IleOEt)60.5(GlyGlyOH)9.0(protamine)2.5(AMPEG)28.0]n

11.8

34.8

425

437.5

a The

substituted ratios were determined by 1H-NMR. The association temperature at which the viscosity start to increase. Viscosity was measured at 15 wt% of polymer concentration in PBS (pH 7.4). c The temperature at which viscosity reaches the maximum value. d Viscosities at 37 C. e Maximum viscosity. b


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Ionic- and nano-complexes composed of Ex-4 and ProCP. Ionic-complex formation was the main driving force of sustained-Ex-4 release in this system. In order to confirm the strong ionic interactions between Ex-4 and ProCP, gel retardation was investigated with nativePAGE (Figure 1). Ex-4 was added to ContP, ProCP-1, or ProCP-2 nano-particle solutions. Every Ex-4-loaded polymer nano-particle was prepared by mixing and incubation method. After electrophoresis, different degrees of Ex-4 retardation were observed between the three groups, reflecting the different strengths of ionic interactions. In detail, there was no Ex-4 retardation in the Ex-4/ContP particle group, whereas clear evidence of Ex-4 retardation was shown in the Ex-4/ProCP-1 and Ex-4/ProCP-2 complex groups. Therefore, it was confirmed that the loaded Ex-4 was trapped by the protamine group of ProCP via forming ioniccomplexes. In addition, with the increase in the concentration of ProCP at a fixed concentration of Ex-4, the trapped Ex-4 was not easily dissociated from the complexes of Ex4/ProCP. When the ionic-complex formed with ProCP-1, the Ex-4 band was weakened when the concentration ratio of Ex-4 versus ProCP-1 was 1:50 (Ex-4: ProCP-1), whereas the Ex-4 band was weakened when the concentration ratio of Ex-4 versus ProCP-2 was 1:10 (Ex-4: ProCP-2). The ionic-complexes were strongly formed when ProCP had more protamine termini; higher amount of protamine termini in the ProCP-2 than that of the ProCP-1 interacted with the Ex-4 stronger than that of the ProCP-1. From these retardation behaviors, we confirmed that the ionic-interaction force was strong enough to capture the Ex-4, and the strength of ionic-interaction could be controlled by adjusting the density of the protamine termini on the ProCPs. As additional characterization of the formation of ionic-complexes of Ex-4 and ProCP, morphological analysis, size distribution, and zeta-potentials were compared to those of Ex-4 unloaded ProCP nano-particles in aqueous condition. ProCP-2 exists around 85-nm sized nano-particles (a) in an aqueous environment, and Ex-4 loaded Ex4/ProCP-2 nano-complex showed smaller size (b) as shown in Figure 2A. The ProCP-2 nano-



particles showed size decrease after Ex-4 loading, and this change was caused by the formation of strong ionic-complex of Ex-4 and ProCP-2. With the DLS method, the ProCP-2 nano-particles showed 84.20 ± 19.6 nm and polydispersity index (PDI) value of 0.57 and decreased to 35.29 ± 7.82 with decreased PDI value of 0.32 after formation of ioniccomplexes with Ex-4 (Figure 2B). These decreases in size and PDI values were regarded as evidence of strong ionic-complexation. Previously, we reported the ionic-complex formation of ionic functionalized poly(organophosphazenes) and their opposite charged short interfering RNAs (siRNA) or proteins. 21, 25 After the ionic-complex formation, significant differences on size and zeta-potentials were measured compared to the condition before ionic-complex formation. In the measurement of zeta-potentials, Ex-4 and ProCP-2 showed values of − 11.58 ± 1.60 and 20.74 ± 1.09 mV, respectively. However, the Ex-4/ProCP-2 nano-complexes showed values of 10.14 ± 1.29 mV (Figure 2C).

Figure 1. Complex dissociation study of exendin 4 (Ex-4) loaded control polymer (ContP), protamine-conjugated polymer 1 (ProCP-1), and ProCP-2 nano-particles. (A) The gel retardation phenomenon was confirmed by native polyacrylamide gel electrophoresis (PAGE). Lane numbers represent weight ratios of Ex-4:polymer such as Lane 1: native Ex-4


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only, Lane 2: Ex-4:polymer = 1:5, Lane 3: Ex-4:polymer = 1:10, Lane 4: Ex-4:polymer = 1:20, Lane 5: Ex-4:polymer = 1:50, Lane 6: Ex-4:polymer = 1:100, Lane 7: Ex-4:polymer = 1:150, Lane 8: Ex-4:polymer = 1:200, and Lane 9: Ex-4:polymer = 1:300. (B) Illustrated explanation of strong complex formation of Ex-4 and ProCP-2.

Figure 2. Characterization of protamine-conjugated polymer (ProCP) nano-particles and exendin 4 (Ex-4)/ProCP nano-complexes. (A) Size-distribution and transmission electron microscopy (TEM) images of the ProCP-2 nano-particles and Ex-4/ProCP-2 nano-complexes. (B) Size changes in ProCP-2 nano-particles to Ex-4/ProCP-2 nano-complexes demonstrated compact nano-complex formation (*: p < 0.05 vs ProCP). (C) Zeta-potential changes in ProCP-2 nano-particles to Ex-4/ProCP-2 nano-complexes (*: p < 0.05 vs ProCP-2).

In vitro release of Ex-4 from Ex-4/ProCP ionic-nano-complex hydrogels. Effectiveness of the ionic-complex forming the ProCP nano-particle system on controlled Ex-4 release was examined by in vitro Ex-4 release analysis. Ex-4 was loaded to freshly prepared solutions of



ContP, ProCP-1, and ProCP-2 nano-particles. Then, each of the polymer nano-particle solutions was incubated for 20 min with Ex-4 for formation of ionic-complexes. Solutionstates of Ex-4/ContP and Ex-4/ProCP nano-particles or nano-complexes were poured into millicelles and warmed to form bulk hydrogels at 37°C. The transformed hydrogels were soaked into PBS solution and incubated in a water bath at 37°C under mild shaking motion (50 rpm) until next transfer to freshly prepared PBS solution. As shown in Figure 3A, the soaked Ex-4/ContP nano-particle and Ex-4/ProCP nano-complex hydrogels were monitored to investigate their degradation behaviors. The swelling and degradation behaviors of the three hydrogels were different: Ex-4/ContP nano-particle hydrogel swelled more but remained longer than Ex-4/ProCP nano-complex hydrogels. These slow degradation behaviors of Ex-4/ContP hydrogel may be caused by the absence of the ionic-charged termini, which can affect hydrophilicity of the polymer nano-particles. The hydrophilicity of nanoparticles effects their release from the bulk hydrogel (dissolution of nano-particles prior to degradation of polymer chains). Ex-4/ProCP nano-complex hydrogel groups showed gradual degradation for 21 days, and slightly faster degradation behaviors were observed with ProCP2 compared to ProCP-1 at the same periods. These different degradation behaviors may originate from the different amount of protamine termini. ProCP-2 had more hydrophilic protamine groups than ProCP-1, and this affected the degradation speed. However, the Ex-4 release rates showed a correlation with the presence and amounts of protamine groups (Figure 3B). The Ex-4/ContP nano-particle hydrogel maintained its maximum volume and rarely degraded until day 21; however, almost 100% of loaded Ex-4 was released during 6 days from the initial day. The loaded Ex-4 in the Ex-4/ContP nano-particle hydrogel was rapidly released from the hydrogel because there were no ionic-interacting forces to capture the Ex-4. On the other hand, suppressed initial burst release and long-term Ex-4 release were observed in the Ex-4/ProCP-1 and Ex-4/ProCP-2 nano-complex hydrogel groups. From the


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Ex-4/ProCP-1 nano-complex hydrogel, 70% of loaded Ex-4 was detected in the release buffer during 4 days from the initial day, and the sustained-release continued for 3 weeks. The Ex4/ProCP-2 nano-complex hydrogel group showed more suppressed initial release amount; 40% of loaded Ex-4 was detected in the release buffer until day 4. Then, continuous Ex-4 release was maintained for up to 3 weeks in the Ex-4/ProCP-2 nano-complex hydrogel. Between the Ex-4/ProCP-1 and Ex-4/ProCP-2 nano-complex hydrogel groups, the Ex-4 release behaviors were different based on the amounts of protamine group; the Ex-4/ProCP-2 nano-complex hydrogel showed more stable Ex-4 release behavior with more suppressed initial release than that of the Ex-4/ProCP-1 nano-complex hydrogel. These results depended on the different density of the positively charged protamine group on ProCP. The higher amount of protamine group in the ProCP-2 gave stronger positive charge than that of ProCP-1, which resulted in stronger ionic-complexes with negatively charged Ex-4. Consequently, strongly formed ionic-complexes provided the most effective release behaviors for the entire periods of hydrogel remaining and degradation.



Figure 3. In vitro exendin 4 (Ex-4) release study. (A) The hydrogel images were taken during the in vitro Ex-4 release study. (B) In vitro Ex-4 release behaviors of Ex-4/ContP nanoparticle and Ex-4/protamine-conjugated polymer (ProCP) nano-complex hydrogels. The released Ex-4 was detected from the phosphate-buffered saline (PBS) buffer. The hydrogels were transferred to freshly prepared PBS buffer at the pre-determined days. The amount of released Ex-4 was measured using enzyme-linked immunosorbent assay (ELISA) kit (n = 3).

In vivo retention studies of Ex-4 and Ex-4/ProCP ionic-nano-complex hydrogel depot by


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imaging of cy5.5-conjugated Ex-4 and AF-ProCP. In vivo clearance periods of Ex-4 were monitored when it was administered as an Ex-4 solution or an ionic-complex hydrogel form with ProCP. For tracking the loaded Ex-4 and ProCP, cy5.5 and aminofluorescein were conjugated to Ex-4 (cy5.5-Ex-4) and ProCP (AF-ProCP), respectively (Figure S6). Monitoring of cy5.5-Ex-4 and AF-ProCP was performed using real-time non-invasive near infrared (NIR) fluorescence after SC injection of cy5.5-Ex-4/AF-ProCP nano-complex hydrogel into the back of mice (Figure 4). Cy5.5-Ex-4 and AF-ProCP were observed using fluorescence imaging at the injection site and strongly showed luminescence under the SC tissue. The retention periods of AF-ProCP-1 and AF-ProCP-2 nano-particle hydrogels were monitored, and there were no notable differences in hydrogel biodegradation behaviors in vivo (Figure 4A). The AF-ProCP-1 and AF-ProCP-2 nano-particle hydrogels were gradually degraded with time and almost disappeared at the injection site after 3 weeks. Simultaneously, clearance of cy5.5-Ex-4 was monitored (Figure 4B). As a control, the cy5.5-Ex-4 solution without ProCP nano-particle hydrogel was injected by the same method of SC injection of the cy5.5-Ex-4/AF-ProCP nano-complex hydrogel. Timedependent behaviors of remaining cy5.5-Ex-4 were monitored with shorter interval than that of the hydrogel groups because of its fast clearance tendency. The cy5.5-Ex-4 solution without ProCP nano-complex system was dispersed under the SC tissue (Sub, in Figure 4B) in 15 min and showed strong kidney accumulation (Ki, in Figure 4B) after 30 min from the injection. The injected cy5.5-Ex-4 was entirely accumulated in kidney during 6 h, and complete clearance was monitored after 7 days from the injection. On the other hand, the cy5.5-Ex-4/AF-ProCP nano-complex hydrogel groups showed no distinct cy5.5-Ex-4 accumulation in kidney and any other specific organs. In the cy5.5-Ex-4 solution without ProCP nano-complex system, the concentration of cy5.5-Ex-4 in plasma rapidly increased and then decreased for a very short period after the injection. Hence, a high amount of cy5.5-



Ex was accumulated in the kidney because of the renal clearance system. The cy5.5-Ex4/AF-ProCP nano-complex hydrogels released a small amount of cy5.5-Ex-4 for a long period. There was no cy5.5-Ex-4 fluorescence in the renal region in the cy5.5-Ex-4/AFProCP nano-complex hydrogel groups, and hence, it could be inferred that the renal clearance system was functional enough to eliminate the low concentration of the released cy5.5-Ex-4. The loaded cy5.5-Ex-4 was identified at the injection site of in situ formed hydrogels for the same period with the hydrogel remaining; the fluorescence intensities of cy5.5-Ex-4 decreased with time and were still observed at day 21 at the hydrogel remaining sites. From these results, it was confirmed that cy5.5-Ex-4 was well trapped into the AF-ProCP nanoparticles via ionic-complex formation. These nano-complex forming system provided a significant advantage to control the release period of Ex-4 and the maintenance period of the nano-complex hydrogel depot. These results have demonstrated correlation with the results of in vitro Ex-4 release study. These two similar results could support the hypothesis that the duration of Ex-4 retention and release could continue until the hydrogel is entirely degraded. This prolonged cy5.5-Ex-4 release, and the entire degradation of AF-ProCP hydrogel at the same time may be beneficial as an anti-diabetes treatment system, because anti-diabetes treatment requires repeatable treatments.


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Figure 4. In vivo real-time non-invasive near infrared (NIR) fluorescence images of cy5.5exendin 4 (Ex-4) loaded aminofluorescein-protamine-conjugated polymer (AF-ProCP) nanocomplex hydrogels. (A) Images of retention and degradation behaviors of AF-ProCP nanoparticle hydrogels. (B) A solution state of cy5.5-Ex-4 and cy5.5-Ex-4/AF-ProCP nanocomplexes administered by subcutaneous (SC) injection into mice. The behaviors of cy5.5Ex-4 release and retention were monitored timely.

Pharmacokinetic (PK) study of sustained-released Ex-4 via one-time SC injection of the Ex-4/ProCP ionic-nano-complexes in Sprague-Dawley (SD) rats. The ability of controlled Ex-4 release of the Ex-4/ProCP nano-complex system was examined by PK study in SD rats. Ex-4 solution was administered with saline or nano-complex system at doses of 50 nmol. The



prepared Ex-4 and Ex-4/ProCP nano-complex solutions were administered at the back of rats by SC injection with 31-gauge needle. After the injection, bulk hydrogel depots were identified at the injection site of Ex-4/ProCP nano-complex solutions directly. Plasma concentration of released Ex-4 in the collected blood samples was examined using ELISA kit. The Ex-4 release behaviors and their PK parameters are shown in Figure 5 and Table 2. In detail, Ex-4 administered without ProCP nano-complex system was rapidly cleared from blood plasma. After injection of 50 nmol of Ex-4 with saline solution, serum concentration of Ex-4 sharply peaked and decreased during 12 h. The half-life was 1.2 h, and maximum concentration (Cmax) was 1,012.3 ng/mL at 1 h after the injection. On the other hand, significant extended half-lives and release periods were observed in the Ex-4/ProCP nanocomplex system. The initial burst release was considerably suppressed in both the Ex4/ProCP-1 and Ex-4/ProCP-2 nano-complex groups. The Ex-4/ProCP-1 nano-complexes showed 7-day sustained-release behaviors with half-life of 28.8 h, and its Cmax was 29.7 ng/mL at 24 h after the injection. The extended half-life was 24-fold higher than the Ex-4 solution group. In the Ex-4/ProCP-2 nano-complex group, half-life of 180.7 h was measured that showed 64.4-times increased value than the Ex-4 solution group. Ex-4/ProCP-2 nanocomplex showed 21 days of sustained-Ex-4 release with Cmax of 26.7 ng/mL at 48 h after the injection. In addition, the controlled Ex-4 release ability of the Ex-4/ProCP-2 nano-complex system drastically enhanced AUC levels; the Ex-4/ProCP-2 nano-complex group showed 2.0and 1.9-fold increased AUC levels compared to the Ex-4 solution and Ex-4/ProCP-1 nanocomplex groups. Additionally, a PK study with 100 nmol Ex-4 solution and Ex-4/ProCP-2 nano-complexes with 100 nmol Ex-4 groups was conducted (Figure S7 and Table S2). There was a slight dose-dependent increase in the AUC values. No significant differences in the other PK parameters, especially the Ex-4 release-periods according to the concentration of used Ex-4 were, however, observed.


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Figure 5. Pharmacokinetic study of exendin 4 (Ex-4) in Sprague-Dawley (SD) rats. Plasma concentration of Ex-4 in SD rats following subcutaneous injections of Ex-4 solution (50 nmol) (■), Ex-4/protamine-conjugated polymer 1 (ProCP-1) nano-complexes (50 nmol) (▲), Ex4/ProCP-2 nano-complexes (50 nmol) (♦). Error bars represent standard deviation (n = 4).

Table 2. Pharmacokinetic parameters of Figure 5. Ex-4 Sol. (50nmol/rat)

Ex-4/ProCP-1 (50nmol/rat)

Ex-4/ProCP-2 (50nmol/rat)

AUC

1572.1

1676.0

3172.8

T 1/2 (hour)

1.2

28.8

108.7

Tmax (hour)

1.0

24.0

48.0

Cmax (ng/mL)

1012.3

29.7

26.7



Biological efficacy of the Ex-4/ProCP ionic-nano-complex system in diabetes (db/db) mice. Biological efficacy of the sustained-released Ex-4 via one-time injection of the Ex4/ProCP nano-complex system was examined in diabetes (db/db) mice. Pharmacodynamic parameters such as blood glucose levels, body weight, and food uptake were monitored for 14 days from the injection (Figure 6). Before the injections of the Ex-4 and Ex-4/ProCP nanocomplex solutions, each parameter was monitored. The mice were fasted for a day and fed right after the injection. Every Ex-4 and Ex-4/ProCP nano-complex solution was freshly prepared before the injection with 50 nmol of Ex-4. The prepared Ex-4 and Ex-4/ProCP nano-complex solutions were injected at the back of mice subcutaneously. After injection, bulk hydrogel depots were identified at the injection site of Ex-4/ProCP nano-complex solutions directly. The blood glucose levels of each experimental group are shown in Figure 6A and Figure S8. The three experimental groups showed sharp fluctuations of blood glucose levels at 1 h after the injection. The most drastic fluctuation was monitored in the Ex-4 solution group (from 380 ± 53.73 mg/dL on fasting day to 73.66 ± 6.01 mg/dL). However, the Ex-4 solution group showed rapid return pattern to high glucose levels (238.30 ± 18.56 mg/dL) at 3 h after the injection. In contrast, Ex-4/ProCP nano-complex groups showed extended periods of reducing effect on blood glucose levels. In the Ex-4/ProCP-1 nanocomplex group, reduced blood glucose levels were observed until day 1 (under 200 mg/dL); 313.93 ± 47.73 mg/dL was detected at the day 2. On the other hand, the Ex-4/ProCP-2 nanocomplex group showed a significantly prolonged glucose-lowering effect; the lowered blood glucose levels (under 200 mg/dL) were maintained for 4 days, and the blood glucose levels were closed to the original levels for 14 days. As for changes of body weight (Figure 6B), the Ex-4 solution group showed immediate weight-gain after food supply, then the increased body-weight exceeded the previous body weight in 2 days. In the Ex-4/ProCP-1 nanocomplex group, the effect of reducing body weight was monitored for a day, and it took 4


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days to exceed the initial body weight. In contrast with the Ex-4 solution and Ex-4/ProCP-1 nano-complex groups, a more effective loss of body weight was monitored in the Ex4/ProCP-2 nano-complex group. The body weights of the Ex-4/ProCP-2 nano-complex group were maintained under the initial body weight for 6 days, and the average body weights during the experimental period were the least among the three experimental groups. In addition, reduced food-uptake behaviors, well-known anti-diabetic effects of Ex-4, were monitored. Whereas there were short-term reduction effects on food uptake in the Ex-4 solution and Ex-4/ProCP-1 nano-complex groups, the Ex-4/ProCP-2 nano-complex showed 13 days reduced food uptake levels compared to the other groups (Figure 6C). Controls comprised daily Ex-4 injection, daily saline injection, and untreated groups were additionally evaluated (Figure S9 and Figure S10). Once-daily injections significantly reduced blood glucose levels, body weight, and food uptake compared with other controls. Blood glucose levels in the oncedaily injection group were between 250 mg/dL and 400 mg/dL. In the twice-daily injection group, blood glucose levels were lower than those in the once-daily injection group (between 200 mg/dL and 250 mg/dL). From the comparison of the blood glucose levels in the Ex-4/ProCP nano-complex systems groups and daily injection groups, it was construed that the Ex-4/ProCP nano-complex system may be advantageous for long-term and continuous antidiabetic effects, even with a lower dose of Ex-4, and offers a convenient treatment method. These prolonged anti-diabetes activities

via one-time injection of the Ex-4/ProCP-2 nano-complex system show its great potential as a long-term effective Ex-4 delivery system.



Figure 6. Pharmacodynamic studies of released exendin 4 (Ex-4) via one-time subcutaneous (SC) injection of the Ex-4/protamine-conjugated polymer (ProCP) nano-complex system in diabetes (db/db) mice. (A) Blood glucose level, (B) body weight, (C) and food uptake were monitored for 14 days from the injection (n = 4).

▪ CONCLUSION Treatment of diabetes mellitus aims not only to keep blood glucose levels as normal as possible, but also to prevent from well-known diabetic complications such as ketonuria, inflammation, and micro- and macro-vascular complications resulting in heart disease, stroke, vision loss, and chronic kidney disease.27 In addition, as diabetes is a long-term metabolic disorder, patients with diabetes have to look after their health very carefully for the rest of their life. Development of long-term effective anti-diabetes system could reduce the daunting task of frequent medication for maintaining normal blood glucose level of diabetes patients. A new injectable and sustained-Ex-4 releasing nano-complex system was suggested in this study as an alternative anti-diabetes treatment, reducing treatment and the resulting patientinconvenience. As described in Figure 7, a solution state of Ex-4/ProCP nano-complex can be administered by injection, and in situ formation of bulk hydrogel depot occurs at the injection site because of the body temperature. The loaded Ex-4 is released after dissociation of the Ex4/ProCP nano-complex from the bulk hydrogel. Therefore, the Ex-4 release period can be


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prolonged as much as the hydrogel remaining period. These prolonged periods of the Ex-4 release and equivalent period of hydrogel sustenance may be beneficial for the treatment of diabetes patients, who need a lifelong treatment regime that is reproducible and effective.

Figure 7. Schematic illustration of injectable and sustained-exendin 4 (Ex-4) releasing nanocomplex system. Loaded Ex-4 and protamine-conjugated polymer (ProCP) nano-particles form an ionic- and nano-complexes by their opposite electric charges. Ex-4 could be trapped in the ProCP nano-complexes. After injection of the Ex-4/ProCP nano-complex solution (10 wt% of polymer in 90% phosphate-buffered saline (PBS) solution) into the body, the Ex4/ProCP nano-complexes were localized via forming a hydrogel based on the body temperature. The Ex-4/ProCP nano-complex is sustained-released from the bulk hydrogel. Then, the entrapped Ex-4 is released via dissociation of Ex-4/ProCP nano-complex and degradation of ProCP. The Ex-4 release and hydrogel remaining show almost similar periods; therefore, the Ex-4/ProCP nano-complex system had benefits as a long-term active and repeatable anti-diabetes treatment.



▪ MATERIALS AND METHOD Materials. Exendin 4 (Ex-4) was purchased from Aldrich. Hexachlorocyclotriphosphazene was acquired from Aldrich and purified by sublimation at 55 C under vacuum (about 0.1 mmHg). Poly(dichlorophosphazene) was prepared as described in the previous method.28 αAmino-ω-methoxy-poly(ethylene glycol) (AMPEG) with molecular weights of 550 was prepared by a published method.29 The ethyl esters of isoleucine (IleOEt) was prepared according to the literature.30 Glyglycine ally ester trifluoroacetic acid salt (GlyGlyOAll·TFA) was prepared by our previous study.31 Tetrahydrofuran (THF) was dried by reflux over sodium metal and distilled. Trimethylamine (TEA) was distilled with BaO. Both distillation processes were performed under dry nitrogen. All other reagents were purchased from commercial suppliers and used as received. The Animal Care Ethnic Committee (ACEC) of Korea of Science and Technology (KIST) approved all animal experiments. Synthesis of protamine conjugated poly(organophosphazenes). All reactions were carried over an atmosphere of nitrogen by using standard Schlenk-line techniques. a. [NP(IleOEt)1.14 (GlyLacOEt)0.03(AMPEG550)0.83]n (ContP). As a control polymer, a polymer lacking protamine group was synthesized. Poly(dichlorophosphazene) was prepared as described previously.32 First, L-isoleucine ethyl ester hydrochloride (IleOEt) (7.70 g, 39.35 mmol) suspended in anhydrous THF (100 ml) containing triethylamine (20.0 ml) was added slowly to a flask of poly(dichlorophosphazene) (4.0 g, 34.52 mmol) dissolved in dry THF (100ml). The reaction was performed for 24 h at 55 C and then for 24 h at room temperature. To this mixture, prepared ethyl-2-(O-glycyl) lactate ammonium oxalate (depsipeptide, GlyLacOEt·AO) (0.23 g, 1.04 mmol) and prepared AMPEG (31.51 g, 57.30 mmol) were gradually added. The final reaction was performed for 24 h at 55 C. After the reaction, the


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mixture was filtered. The final product was obtained by concentration and precipitation using n-hexane. Then, purification of the final product was performed by dialysis in methanol for 3 days and then in distilled water at 4 °C for 3 days. The dialyzed solution was freeze-dried to obtain pure ContP. Yield: 83%, 1H NMR (300 MHz, CDCl3, δ), d (ppm): 0.8–1.0 (s, 6H, CH3), 1.6–1.9 (b, 1H, CH), 2.8–3.1 (b, 2H, CH2), 3.4 (s, 3H, CH3), 3.5–3.9 (b, 62H, CH2), 3.9–4.1 (b, 4H, CH2), 5.0–5.1 (b, 1H, CH). b.

[NP(IleOEt)1.16

(GlyGlyOH)0.17

(AMPEG)0.67]n

(Pre-ProCP-1).

Poly(dichlorophosphazene) (4 g, 34.52 mmol) dissolved in THF was reacted stepwise with IleOEt (7.83 g, 40.04 mmol), GlyGlyOAll (1.98 g, 6.90 mmol), and AMPEG (15.53 g, 20.71 mmol). Then, carboxylic acid-terminated polymer was synthesized by the allyl ester cleavage reaction of polymer using 0.2 equivalent of tetarkis(triphenylphosphine)-palladium(0) and 20 equivalent of morpholine. The final reaction was performed for 12 h at room temperature. After the reaction, the mixture was filtered. The final product was obtained by concentration and precipitation using n-hexane. Then, purification of the final product was performed by dialysis in methanol for 3 days and then in distilled water at 4 °C for 3 days. The dialyzed solution was freeze-dried to obtain pure Pre-ProCP-1. Yield: 90%. 1H NMR (CDCl3),  (ppm): 0.8-1.0 (s, 6H), 1.1-1.3 (b, 3H), 1.3-1.6 (b, 2H), 1.6-1.9 (b, 1H), 3.2 (s, 2H), 3.3 (s, 3H) 3.4-3.8 (b, 44H), 3.9 (s, 2H), 4.0-4.1 (b, 1H), 4.1-4.3 (b, 2H). c. [NP(IleOEt)1.16(GlyGlyOH)0.15 (Protamine)0.02(AMPEG)0.67]n (ProCP-1). Pre-ProCP-1 (5.00 g, 22.44 mmol) was dissolved in 50 ml of distilled THF. After cooling at 0 C, TEA (0.51 ml, 3.71 mmol) and IBCF (0.24 ml, 1.85 mmol) were added to the reaction flask. The mixture was transferred to another reaction flask containing protamine (18.90 g, 3.71 mmol) in 0.01 M HEPES (pH 7.5) with TEA (1.03 ml, 7.41 mmol). Then, the reaction mixture was stirred at 4 C for 18 h and at room temperature for 6 h. After reaction, the solution was concentrated, and it was purified by dialysis in methanol for 3 days and then in distilled water



Page 26 of 35

for 3 days at 4 C. The final dialyzed solution was freeze-dried to obtain pure ProCP-1. Yield: 87%. 1H NMR (D2O),  (ppm): 0.6-1.0 (br, 12H), 1.0-1.3 (br, 3H), 1.4-2.0 (br, 93H), 2.1-2.3 (s, 3H), 2.9-3.1 (s, 42H), 3.1-3.3 (s, 3H) 3.4-3.8 (b, 44H), 3.3-4.1 (br, 60H), 4.1-4.2 (br, 21H), 4.2-4.6 (br, 9H). d.

[NP(IleOEt)1.21(GlyGlyOH)0.23

(AMPEG)0.56]n

(Pre-ProCP-2).

Poly(dichlorophosphazene) (4.00 g, 34.52 mmol), IleOEt (8.17 g, 41.76 mmol), GlyGlyOAll (2.96

g,

10.35

mmol),

AMPEG

(18.12

g,

24.16

mmol),

0.2

equivalent

of

tetarkis(triphenylphosphine)- palladium(0), and 20 equivalent of morpholine were used. After reaction, the solution was concentrated, and it was purified by dialysis in methanol for 3 days and then in distilled water for 3 days at 4 C. The final dialyzed solution was freeze-dried to obtain pure Pre-ProCP-2. Yield: 75 %. 1H NMR (CDCl3),  (ppm): 0.8-1.0 (s, 6H), 1.1-1.3 (b, 3H), 1.3-1.6 (b, 2H), 1.6-1.9 (b, 1H), 3.2 (s, 2H), 3.3 (s, 3H) 3.4-3.8 (b, 44H), 3.9 (s, 2H), 4.0-4.1 (b, 1H), 4.1-4.3 (b, 2H). e. [NP(IleOEt)1.21(GlyGlyOH)0.18 (Protamine)0.05(AMPEG)0.56]n (ProCP-2). Pre-ProCP-2 (5.00 g, 28.68 mmol) was dissolved in 50 ml of distilled THF. After cooling at 0 C, TEA (0.56 ml, 4.00 mmol) and IBCF (0.26 ml, 2.00 mmol) were added to the reaction flask. The mixture was transferred to a reaction flask containing protamine (20.41 g, 4.00 mmol) in 0.01 M HEPES (pH 7.5) with TEA (1.12 ml, 8.00 mmol). After reaction, the solution was concentrated, and it was purified by dialysis in methanol for 3 days and then in distilled water for 3 days at 4 C. The final dialyzed solution was freeze-dried to obtain pure ProCP-2. Yield: 90%. 1H NMR (D2O),  (ppm): 0.6-1.0 (br, 12H), 1.0-1.3 (br, 3H), 1.4-2.0 (br, 93H), 2.1-2.3 (s, 3H), 2.9-3.1 (s, 42H), 3.1-3.3 (s, 3H) 3.4-3.8 (b, 44H), 3.3-4.1 (br, 60H), 4.1-4.2 (br, 21H), 4.2-4.6 (br, 9H). Characterization of poly(organophosphazenes). The synthesized polymers were


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characterized by 1H nuclear magnetic resonance (NMR) (300 MHz, CDCl3, δ). Molecular weights (MW) of polymers were measured by gel permeation chromatography (GPC) system (Waters 1515) with a refractive index detector (Waters 2410) and two stryragel columns (Waters styragel HR 4E and HR 5E) connected in line at a flow rate of 0.8 ml/min at 35 °C. THF containing 0.1 wt% of tetrabutylammonium bromide was used as a mobile phase. Polystyrenes (Mw: 1270; 3760; 12,900; 28,400; 64,200; 183,000; 658,000; 1,050,000; 2,510,000; 3,790,000) were used as standards to calibrate the column. Characterization of thermosensitive sol-gel transition of polymer solutions. The ConP and ProCPs were dissolved in phosphate-buffered saline (PBS) by stirring at 4 °C (15 wt% polymers). The viscosity measurements of the aqueous polymer solutions were performed on a Brookfield RVDV-III+ viscometer between 5 and 70 °C under 0.1−1 shear rate. The measurements were carried out with a set spindle speed of 0.2 rpm and 1 °C raise in temperature for 3 minutes. Preparation of Ex-4/ProCP ionic-nano-complexes. All Ex-4/ProCP ionic-nano-complexes were freshly prepared prior to use. The ContP and ProCPs were dissolved in PBS by stirring at 4 °C (15 wt% polymers), then the Ex-4 was loaded prepared polymer solutions. The mixture was incubated at room temperature for 20 min for ionic-nano-complex formation. Characterization of Ex-4/ProCP ionic-nano-complexes. The ProCP nano-particles and Ex4/ProCP ionic-nano-complexes were confirmed by transmission electron microscopy after negatively stained using a droplet of 2 wt% aqueous uranyl acetate (TEM, CM 200 electron microscope, Philips). Hydrodynamic diameter of ProCP and Ex-4/ProCP ionic-nanocomplexes were measured by dynamic light scattering (DLS) method and zeta potentials were investigated simultaneously (Zetasizer Nano ZS, Malvern instruments Ltd., Malvern, UK). For preparation of the Ex-4/ProCP ionic-nano-complex solution, 50 nmol Ex-4 and 200 µl of 15 wt% of ProCP nano-particle solution were used, then thirtyfold diluted before the



measurement. The complex formation ability of protamine conjugate with Ex-4 was confirmed by Native-polyacrylamide gel electrophoresis (PAGE) using a Bio-Rad MiniProtean 3 cell electrophoresis system. Gels were prepared discontinuously with stacking and running gel of 5% and 10% polyacrylamide, respectively. After loading of the complexes onto the polyarylamide gel, electrophoresis was carried out in a constant voltage mode at 100 V using a Bio-Rad power supply in a Tris/glycine buffer at pH 8.3. The gels were stained with Coomassie Brilliant Blue R-250 staining solution (Rio-Rad, USA) for the observation of Ex-4 bands. The complex formation was confirmed a degree of gel retardation. In vitro release study of Ex-4. The polymers were dissolved in PBS by stirring at 4 °C. After dissolving, Ex-4 was added to the polymer nano-particle solutions with stirring at 4 °C for 1 hour in order to make ionic interactions between Ex-4 and ProCP nano-particles. After that, 0.2 ml of Ex-4/ProCP ionic-nano-complex solutions were loaded into millicell (12 mm of diameter, Millipore, USA) and incubated at 37 °C to transform to hydrogels (the final concentration of Ex-4: 50 nmol/well). These prepared hydrogels were soaked in 4 ml of PBS with 0.02 wt% of sodium azide and then incubated in a water bath (KMC-12055W1, Vision, Korea) at 37 °C under mild shaking motion (50 rpm). The PBS was renewed periodically with fresh buffer. The released amount of Ex-4 were quantified by Exendin 4 enzyme-linked immunosorbent assay (ELISA) kit (Phoenix Pharmaceuticals, Inc., Burlingame, CA). Each of established standard curves was used to the quantification of the release experiments. Study of local retention behaviors of Ex-4 and ProCP nano-particle hydrogel in nude mice. For cy-5.5 labeled Ex-4, 1 mg of Cy5.5-NHS (GE Healthcare, UK) was added to the solutions including 100 mg of the Ex-4 for 1 h at room temperature. Using Centricon (Millipore, USA), labeled Ex-4 solution was washed 5 times with 2 ml of 0.15 M NaCl. For aminofluorescenin (AF) labled ProCP, the AF was conjugated to carboxylic-acid residues of the protamine-conjugate polymers using EDC/NHS at room temperature for 6 h. A mixing


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solution of AF-protamine conjugate and Cy5.5-Ex-4 was incubated for 30 min at room temperature for complex formation. Then, the Cy5.5-Ex-4/AF-ProCP ionic-nano-complex solution was injected subcutaneously into nude mice (male, 6-8 weeks old, Orient Co., Korea). The mice were received Ex-4 at a dose of 12.5 nmol/mouse with 100 µl of Cy5.5-Ex4/AF-ProCP ionic-nano-complex solution. Fluorescent optical images were obtained using a Kodak Image Station 4000 MM (Digital Imaging Systems, USA). Pharmacokinetic study of Ex-4 in SD rats. Pharmacokinetic study of Ex-4 from the Ex4/ProCP ionic-nano-complex system was examined in male Sprague-Dawley rats (6-weeksold (300±15g)) (Orientbio, Korea). The rats were randomly assigned into five treatment groups (n = 4 mouse per group). 50 or 100 nmol Ex-4 in PBS solution was administered as controls into back of rats. Before administration of Ex-4/ProCP ionic-nano-complex solutions, 50 or 100 nmol of Ex-4 was loaded into freshly prepared ProCP ionic-nano-particle solutions and incubated for 30 min at room temperature for complex formation. Then, 200 µl of solution of Ex-4/ProCP ionic-nano-complex solution was injected subcutaneously in to the back of rats using 31-gauge needle. Blood samples were obtained from tail vein at every predetermined time, and the plasma concentration of released Ex-4 was detected analyzed by enzyme-linked immunosorbent assay (ELISA) (Phoenix Pharmaceuticals, Inc., Burlingame, CA). Pharmacokinetic parameters such as area under the curve (ACU) and half-life (t½) were calculated by WinNonlin program (Pharsight Corporation, USA). Study of biological efficacy in diabetes (db/db) mice. The biological efficacies of released Ex-4 from the Ex-4/ProCP-1 and Ex-4/ProCP-2 ionic-nano-complex systems were examined by measurement of blood glucose level, food uptake, and body weight in diabetes (db/db) mice (6-weeks-old, Inc. CHARES RIVER Lab., USA). The mice were randomly assigned into three treatment groups (n = 4 mouse per group): one-time injection of Ex-4 solution, a



Page 30 of 35

single injection of Ex-4/ProCP-1 ionic-nano-complex solution, and a single injection of Ex4/ProCP-2 ionic-nano-complex solution. Each administration was injected by subcutaneously using syringes with 31-gauge needles. The mice of all three groups were received Ex-4 at a dose of 50 nmol/mouse with saline solution or 100 µl of Ex-4/ProCP ionic-nano-complex solutions. The blood glucose level, food uptake, and body weight were monitored during 14 days from post injection. As controls, daily Ex-4 injection, daily saline injection, and untreated groups were evaluated (5 nmol of Ex-4 was injected for every injections, n=5).

▪ ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Figures S1-S10 and Tables S1-S2 (PDF)

▪ AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Notes The authors declare no competing financial interest.

▪ ACKNOWLEDGMENTS The Korea Institute of Science and Technology (2E27930) and the Korea National Research

Foundation

(2014M3A9B6034220,

2018M3A9H1024872) supported this work.


2017M3A9F5027614,

and

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