Byproduct-Free Intact Modification of Insulin by Cholesterol End

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By-Product-Free Intact Modification of Insulin by Cholesterol End-Modified Poly(ethylene glycol) for In Vivo Protein Delivery Shoichiro Asayama, Kana Nagashima, Yoichi Negishi, and Hiroyoshi Kawakami Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00593 • Publication Date (Web): 11 Dec 2017 Downloaded from http://pubs.acs.org on December 19, 2017

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Bioconjugate Chemistry

1

By-Product-Free Intact Modification of Insulin by Cholesterol End-Modified Poly(ethylene glycol) for In Vivo Protein Delivery

Shoichiro Asayama,*,† Kana Nagashima,† Yoichi Negishi,‡ Hiroyoshi Kawakami†

†Department

of Applied Chemistry, Tokyo Metropolitan University, Hachioji, Tokyo 192-0397, Japan

‡School

of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192-0392, Japan

*Corresponding author. E-mail: [email protected]

ACS Paragon Plus Environment

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

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2

ABSTRACT Insulin is a key peptide hormone used for the treatment of both type I and type II diabetes. To maximize the effect of the treatment of these diseases, PEGylation methods for the insulin are widely developed.

Here, to make these PEGylation methods most simple, we report the

by-product-free intact modification of insulin by cholesterol end-modified poly(ethylene glycol) with urethane, propyl, and methoxy groups, that is, Chol-U-Pr-mPEG.

The noncovalent

PEGylation by the Chol-U-Pr-mPEG has been achieved by simple mixing of insulin with the Chol-U-Pr-mPEG in aqueous solution, followed by freeze-drying. Chol-U-Pr-mPEG/insulin

complex

has

proceeded

without

The formation of the by-products,

such

as

N-hydroxysuccinimide formed by the conventional covalent PEGylation using an active ester group. The by-product-free PEGylation has preserved insulin conformation as well as primary structure. The intact PEGylation has protected insulin from hydrolysis by protease.

The resulting insulin

modified by the Chol-U-Pr-mPEG has sustainably suppressed the level of blood glucose, as compared to naked insulin, in mice.

Consequently, the Chol-U-Pr-mPEG/insulin complex

formation offers the by-product-free intact PEGylation of insulin for in vivo protein delivery.


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3 INTRODUCTION Protein modification by poly(ethylene glycol) (PEG), that is, PEGylation, is the promising approach for biocompatibility to enhance half-life in blood.1-5

The resulting biocompatibility

applies proteins to biomedicine for various therapeutics to improve quality of life.6-9

To date,

covalent bonds are widely used for PEGylation such as site-selective,10-14 enzymatic,15-17 or releasable PEGylation.18,19

In spite of wide use, any covalent PEGylation changes the primary

structure of proteins and is difficult to reproduce native proteins with intact pharmaceutical activity. Conversely, few methods are reported for noncovalent PEGylation by hydrophobic interaction,20-22 coordination complex,23 polyion complex,24 lectin-carbohydrate interaction,25 or supramolecular host-guest interaction,26 as well as our mono-ion complex.27 For the above protein PEGylation, insulin is attractive target protein because insulin is a key peptide hormone used for the treatment of both type I and type II diabetes. diabetes are increasing steadily and prevalently.28-30

The patients with

To maximize the effect of the treatment of

these diseases, PEGylation methods for the insulin are widely developed.31-35

Here, to make

insulin PEGylation methods most simple, we report the by-product-free intact modification of insulin by cholesterol end-modified poly(ethylene glycol) with urethane, propyl, and methoxy groups, that is, Chol-U-Pr-mPEG (Figure 1).

The Chol-U-Pr-mPEG is expected to achieve the

PEGylation of insulin by hydrogen bond formation with urethane group as well as hydrophobic interaction with terminal cholesterol.



Page 4 of 25

4

Cholesterol End-modified Poly(ethylene glycol): Chol-U-Pr-mPEG H3C

Urethane (U) Methoxy (m)

CH3O

CH3

CH2CH2O

CH3

H3C

Propyl (Pr)

H3C

H CH2CH2CH2N C O n O

PEG

Cholesterol (Chol) By-Product-Free Intact Modification of Insulin COO-

Biocompatibility Hydrogen Bond Formation

N C

Hydrophobic Hydrogen-bondforming

Q Y L

L E N Y

G E R C G V F L F Y

Bovine Insulin Amino Acid S Sequence C V S

A-chain

+H3N G I V E Q C C

A

S

+H3N F V N Q H L C G

Y T

E

P K A COO-

V

S

B-chain

L A

Hydrophobic Interaction

S

H

L

Disulfide-bond-forming Acidic

Basic

Figure 1. Design concept of the cholesterol end-modified poly(ethylene glycol) (Chol-U-Pr-mPEG) for by-product-free intact modification of insulin.

RESULTS AND DISCUSSION Complex

formation

between

insulin

and

Chol-U-Pr-mPEG.

To

synthesize

Chol-U-Pr-mPEG, we added cholesterol chloroformate to aminopropyl mPEG in chloroform in the presence of trimethylamine (TEA) (Figure 2A).

The 1H NMR spectrum of the resulting polymer

showed the characteristic chemical shift derived from both a cholesterol group (1H, H-6 proton in the lowest magnetic field side) and a propyl PEG (3H, terminal methoxy protons) (Figure S1). well, the

13

As

C NMR spectrum of the resulting polymer showed the characteristic chemical shift


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5 derived from both a cholesterol group (approximately 10-60 ppm) and a propyl PEG (approximately 70 ppm) (Figure S2).

The yield is 79%.

Thus, we have succeeded in the

synthesis of the Chol-U-Pr-mPEG.

H3C

A

CH3 H3C CH3O

CH2CH2O

n

+

CH2CH2CH2NH2

Cholesterol chloroformate

O

Aminopropyl mPEG Cl

B Molar ratio

D Molar ratio

CH2CH2O

n

H3C

O

TEA CHCl3

CH3O

CH3 H3C

H CH2CH2CH2N C O O

Chol-U-Pr-mPEG

NHSmPEG

0 1.2 2.4 12 24 29

Chol-U-Pr-mPEG

NHSmPEG

0 1.2 2.4 12 24 29

CH3 H3C

C Molar ratio

E Molar ratio

CH3 H3C

Chol-U-Pr-mPEG

Chol-U-Pr-mPEG

NHSmPEG

0 1.2 2.4 12 24 2.9

Chol-U-Pr-mPEG

NHSmPEG

0 1.2 2.4 12 24 29

Figure 2. Complex formation between insulin and Chol-U-Pr-mPEG. A) Synthesis scheme of Chol-U-Pr-mPEG. B-E) Native-PAGE analysis of insulin incubated with Chol-U-Pr-mPEG under the following conditions: B) 37 °C for 6 h. C) 37 °C for 24 h. D) 4 °C for 6 h. E) 4 °C for 24 h. NHS-mPEG was used as a control of a reagent for covalent PEGylation. Molar ratio means the ratio of [R-mPEG] ([Chol-U-Pr-mPEG] or [NHS-mPEG]) to [insulin].



Page 6 of 25

6 To examine the complex formation between insulin and the resulting Chol-U-Pr-mPEG for non-covalent PEGylation, as shown in Figures 2B-2E, we performed native poly(acrylamide) gel electrophoresis (Native-PAGE) for the insulin incubated with the Chol-U-Pr-mPEG under various conditions.

Although the retardation band was so weak, a somewhat weak interaction between

insulin and Chol-U-Pr-mPEG should be applied for a strong point of native insulin release related to later results.

After the incubation of insulin with Chol-U-Pr-mPEG at 37 °C for 6 h, the

retardation of an insulin-containing band was observed at the highest [Chol-U-Pr-mPEG]/[insulin] ratio of 24 (Figure 2B). Chol-U-Pr-mPEG,

The band retardation suggests the complex formation of insulin with the

because

the

retardation

was

also

observed

in

case

of

monomethoxy-N-hydroxysuccinimide (NHS) -activated ester/carbonate-PEG (NHS-mPEG) as a control for conventinal covalent PEGylation (Figure S3).

The intensity of the retarded band

became stronger after the longer incubation at 37 °C for 24 h (Figure 2C).

Notably, the stronger

intensity of the retarded band was also observed when we incubated the mixture at 4 °C after the shorter incubation for 6h (Figure 2D).

It is worth noting that the retardation area broadened at the

molar ratio of 24 and appeared at the molar ratio of 12 after the longer incubation at 4 °C for 24 h (Figure 2E).

In general, the hydrogen bond formation is enhanced at lower temperature.

Hence,

outside the insulin, the hydrogen bond between the urethane group of the Chol-U-Pr-mPEG and the hydrogen-bond-forming amino acid of insulin is considered to become strong during the incubation at 4 °C.

From the corresponding molecular weight range (Figure S4), the enhancement of the

retardation of the insulin-containing band after the mixing with Chol-U-Pr-mPEG at lower


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7 temperature, as well as longer incubation, suggests the Chol-U-Pr-mPEG/insulin complex formation by hydrogen bond formation, as well as hydrophobic interaction. To examine further the the complex formation of insulin with the Chol-U-Pr-mPEG, we measured the particle size of the sample with the most retarded band corresponding to the molar ratio of 24, based on the results of above native-PAGE (Figure 2E).

Before the measurement of

the particle size of the sample, we examined whether the Chol-U-Pr-mPEG alone has a particle size by micelle formation (Table S1).

Although no particle size was observed up to 0.3 mg/mL,

average particle diameters appeared in approximately 10 nm range at 0.4 mg/mL or more, suggesting the micelle formation at higher concentration to mix insulin in the presence of the Chol-U-Pr-mPEG.

Furthermore, the micelle formation at higher concentration is supported by the

broaden signal of a cholesterol group in the 1H NMR spectrum of the Chol-U-Pr-mPEG in D2O (Figure S5).

On the other hand, insulin alone exhibited an average particle diameter of

approximately 360 nm, presumably due to aggregation.36

It should be noted that the particle

diameter of the insulin decreased to approximately 20 nm in the presence of the Chol-U-Pr-mPEG (Table S2).

The resulting decrease in the particle diameter is considered to be due to the

dispersion of the aggregated insulin by the Chol-U-Pr-mPEG with detergent properties.

In case of

the insulin reacted with NHS-mPEG as a control, the resulting diameter was approximately 1 nm. These results suggest the Chol-U-Pr-mPEG/insulin complex formation in spite of the mixing insulin with the Chol-U-Pr-mPEG forming micelle structure.



8 By-product-free intact formation of the Chol-U-Pr-mPEG/insulin complex.

To prove

the complex formation of insulin with the Chol-U-Pr-mPEG, based on the resulting particle diameter (Table S2), we carried out gel-filtration chromatography (GFC) of the sample that showed the most retarded band in Figure 2E.

As shown in Figure 3A, the GFC profile of insulin detected

by refractive index (RI) in the presence of Chol-U-Pr-mPEG was obtained earlier than that in the absence of the Chol-U-Pr-mPEG.

Especially, earlier elution from 8 to 9 min appeared.

The

earlier elution was also detected by the absorbance (ABS) at 280 nm attributed to aromatic amino C

6

7

8

9 10 11 Elution volume / mL

D

8


12

-6 -9

[θ]×10-5 / deg.cm2.dmol-1

-3

0

208 nm

222 nm

-2 -4 -6 -8

-10 204

214 224 234 Wavelength / nm

220 240 260 280 Wavelength / nm

300

6 [θ]×10-5 / deg.cm2.dmol-1

7

0

-12 200

12

B

3

3 0 -3 -6 -9

-12 200


RI


A

ABS at 280 nm

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

Page 8 of 25

0

208 nm

222 nm

-2 -4 -6 -8

-10 204

214 224 234 Wavelength / nm

220 240 260 280 Wavelength / nm

300

Figure 3. By-product-free intact formation of the Chol-U-Pr-mPEG/insulin complex. A, B) Gel-filtration chromatograms of insulin in the absence (dotted line) and presence of Chol-U-Pr-mPEG (solid line), as well as a control, NHS-mPEG (dashed line). Detection; A) refractive index (RI) and B) absorbance (ABS) at 280 nm. C, D) CD spectra of insulin in the absence (dashed line) and presence of C) Chol-U-Pr-mPEG (solid line), as well as a control, D) NHS-mPEG (solid line).

The insets show the expanded spectra.

The molecular ellipticities based on the molecular weight of insulin (7.0 µM) are represented. ACS Paragon Plus Environment

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9 acids of insulin, as shown in Figure 3B, proving the formation of the Chol-U-Pr-mPEG/insulin complex.

Although the insulin peak remains to be the main peak of the GFC trace of insulin after

mixing the Chol-U-Pr-mPEG, from later results, the indicated weak interaction between insulin and the Chol-U-Pr-mPEG is considered to be applied for a strong point of native insulin release in vivo. Furthermore, in ultracentrifugation profiles,37 the sedimentation coefficient of above insulin sample in the absence (3.13 S) and presence (3.49 S) of the Chol-U-Pr-mPEG confirmed the formation of the Chol-U-Pr-mPEG/insulin complex (Figure S6).

Taking these results into account, we have

succeeded in the noncovalent PEGylation of insulin using Chol-U-Pr-mPEG. As a control for covalent PEGylation, the GFC profile of insulin detected by both RI (Figure 3A) and the ABS at 280 nm (Figure 3B) in the presence of NHS-mPEG appeared earlier and lager than that in the presence of Chol-U-Pr-mPEG, suggesting both higher degree of PEGylation and lager amount of PEGylated insulin.

The covalent PEGylation using NHS-mPEG is considered to

modify insulin with maximum three mPEG chains because the insulin has three primary amino groups, two N-terminals and one lysine (K) residue (Figure 1), to react with the NHS group. However, considerable large later elution detected by UV from 10 to 11 min appeared in the presence of NHS-mPEG (Figure 3B).

The later elution did not appear in the presence of

Chol-U-Pr-mPEG as well as insulin alone.

After the reaction of insulin with NHS-mPEG, NHS

separated from NHS-mPEG (Figure S3).

The separated NHS exhibited the ABS at 280 nm,

resulting in the later elution detected by UV.

Collectively, these results indicate that no

by-product such as NHS was obtained during the formation of the Chol-U-Pr-mPEG/insulin



Page 10 of 25

10 complex, that is, the noncovalent PEGylation of insulin using Chol-U-Pr-mPEG, as compared to the covalent PEGylation using NHS-mPEG. To examine whether the noncovalent PEGylation using Chol-U-Pr-mPEG affected the conformation of insulin, furthermore, we measured the circular dichroism (CD) of insulin in the absence and presence of the Chol-U-Pr-mPEG.

As shown in Figure 3C, the CD spectrum of the

Chol-U-Pr-mPEG/insulin complex was almost the same as that of native insulin.

The CD spectral

features are classic for α-helices with characteristic minima at 208 nm38 and 222 nm.39,40 Conversely, as shown in Figure 3D, the CD spectrum of the insulin reacted with NHS-mPEG, that is, a PEG-insulin conjugate, was especially disordered from 250 to 280 nm (near-ultraviolet) as compared to that of native insulin.

The disordered near-ultraviolet CD spectrum was observed in

case of a PEG-catalase conjugate27 and may be attributed to the induced interaction of aromatic side chains.41

Hence, although a recent crystal structure of a PEGylated protein demonstrates that the

protein structure was not affected by the PEG,42 the covalent PEGylation using NHS-mPEG is considered to affect the conformation of the insulin in aqueous solution as well as the primary structure from an amino group to an amide one.

From these results, the noncovalent PEGylation

using Chol-U-Pr-mPEG is considered to be intact for insulin conformation as well as primary structure. Properties for in vivo protein delivery by the Chol-U-Pr-mPEG/insulin complex.

In

spite of PEGylation, as shown in Figure 4A, the insulin in the Chol-U-Pr-mPEG/insulin complex, as well as a PEG-insulin conjugate, was recognized by a monoclonal anti-insulin antibody in the


Page 11 of 25

11 absence of a protease, trypsin, presumably due to no suppression for the antibody recognition by the PEGylation under these experimental conditions for enzyme-linked immunosorbent assay (ELISA). Conversely, after the incubation of the Chol-U-Pr-mPEG/insulin complex in the presence of trypsin, more insulin remained in spite of the trypsin digestion, as compared to the incubation of the PEG-insulin conjugate with almost the same level the considerable digestion of naked insulin. These results suggest that the noncovalent PEGylation using Chol-U-Pr-mPEG suppressed insulin degradation by trypsin digestion more effectively, as compared to the covalent PEGylation using

B

4.5

A

120

Blood glucose / mg.dL-1

4.0 3.5

ABS at 450 nm

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


3.0 2.5 2.0 1.5 1.0 0.5

0.0 Trypsin －－－－－－＋＋＋＋＋＋ Insulin －＋＋＋＋＋－＋＋＋＋＋ Molar 0 24 48 29 58 0 24 48 29 58 Ratio NHSNHSChol-U-Pr-mPEG mPEG Chol-U-Pr-mPEG mPEG

100

* **

80 60 40 20

0 Insulin － R-mPEG － Postinjection 1 h

－－

＋－

＋－

4h

1h

4h

＋＋

＋＋

＋＋

＋＋

1h

4h

1h

4h

Chol-U-Pr-mPEG NHS-mPEG

Figure 4. Properties for in vivo protein delivery by the Chol-U-PrmPEG/insulin complex. A) The protection from the protease digestion for insulin by the PEGylation using Chol-U-Pr-mPEG. The Chol-U-Pr-mPEG/insulin complex, as well as a control, PEG-insulin conjugate (NHS-mPEG), was incubated in the absence (-) and presence (+) of trypsin, followed by the detection of the resulting insulin by ELISA. Molar ratio means the ratio of [R-mPEG] ([Chol-U-Pr-mPEG] or [NHS-mPEG]) to [insulin]. Data are shown as mean and standard deviation (n = 3). B) The suppression of the blood glucose level in mice by the subcutaneous administration of the Chol-U-Pr-mPEG/insulin complex. The blood of the mice was collected after 1 h and 4 h post-injection. R-mPEG means Chol-U-Pr-mPEG or NHS-mPEG. Data are shown as mean and standard deviation (n = 3; student’s t-test, *p < 0.05, **p < 0.01).



Page 12 of 25

12 NHS-mPEG. Finally, as shown in Figure 4B, we examined whether the Chol-U-Pr-mPEG/insulin complex suppressed the blood glucose level in mice by subcutaneous administration.

After 1 h

post-injection, the Chol-U-Pr-mPEG/insulin complex lowered the level of blood glucose under the limit of detection, as well as naked insulin and the PEG-insulin conjugate.

After 4 h post-injection,

however, it is worth noting that the level of blood glucose in mice injected with the Chol-U-Pr-mPEG/insulin complex was significantly (p < 0.01) lower than that injected with naked insulin.

The insulin is considered to be gradually released from the Chol-U-Pr-mPEG/insulin

complex at physiological temperature (37 °C in vivo), in spite of the future detail investigation, because the Chol-U-Pr-mPEG/insulin complex formed at 37 °C (Figures 2B and 2C) was relative unstable, as compared to the injected complex formed at 4 °C (Figures 2D and 2E).

Although the

PEG-insulin conjugate also lowered the level of blood glucose, as compared to naked insulin, the significant difference (p < 0.05) was less than that by use of the Chol-U-Pr-mPEG/insulin complex. Consequently, the resulting insulin modified by the Chol-U-Pr-mPEG has sustainably suppressed the level of blood glucose, as compared to naked insulin, in mice. The resulting sustainable suppression of the blood glucose level is considered to be promising effect for future diabetes treatment.

CONCLUSIONS


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13 In summary, the noncovalent PEGylation by the Chol-U-Pr-mPEG has been achieved by simple mixing of insulin with the Chol-U-Pr-mPEG in aqueous solution without by-product.

The

resulting Chol-U-Pr-mPEG/insulin complex has preserved insulin conformation as well as primary structure, leading to intact PEGylation.

The by-product-free intact modification by

Chol-U-Pr-mPEG has been effective for the protection of insulin from hydrolysis by protease, succeeding in the sustainable suppression of the level of blood glucose in mice.

In this study, the

Chol-U-Pr-mPEG/insulin complex formation offers the by-product-free intact PEGylation of insulin for in vivo protein delivery with the possibility of various use of other therapeutic proteins.

EXPERIMENTAL PROCEDURES Materials.

Aminopropyl mPEG (molecular weight of 2000) and monomethoxy-

N-hydroxysuccinimide (NHS) -activated ester/carbonate-PEG (NHS-mPEG) (molecular weight of 2000) were purchased from NOF corporation (Tokyo, Japan).

Cholesterol chloroformate was

purchased from Tokyo Chemical Industry Co., LTD. (Tokyo, Japan). was purchased from Sigma-Aldrich Co. LLC. (St. Louis, MO).

Insulin from bovine pancreas

All other chemicals of a special

grade were used without further purification. Synthesis of Chol-U-Pr-mPEG.

A typical procedure is as follows (Figure 2A):

Cholesterol chloroformate (59.20 mg: 0.1318 mmol) was added to aminopropyl mPEG (120 mg: 0.06 mmol) in 10 mL of chloroform in the presence of 8.36 µL (0.06 mmol) of triethylamine (TEA) and incubated at 40 °C for 24 h.

After the incubation, the resulting mixture was dried in vacuo.



Page 14 of 25

14 Then, ultrapure water was added to the dried sample, followed by filtration.

The resulting sample

was dialyzed against distilled water using a Spectra/Por 7 membrane (103 molecular weight cut-off) and then freeze-dried. 1

H NMR spectroscopy.

atom % deuterium; Acros, NJ).

The polymer (5 mg) was dissolved in 600 µL of CDCl3 (99.8 1

H NMR spectra (500 MHz) were obtained on a Bruker AV500

spectrometer (Billerica, MA). Particle size measurement.

The size of the samples was measured by a dynamic light

scattering spectrophotometer (ELS-Z2, Otsuka Electronics Co., Ltd., Tokyo, Japan). the micelles from Chol-U-Pr-mPEG was measured in 60 µL of H2O.

The size of

The size of the complexes

between Chol-U-Pr-mPEG (200 µg, 83 nmol) and insulin (20 µg, 3.5 nmol), which were incubated at 4 °C for 24 h in 102 µL of H2O and then freeze-dried, were measured in 60 µL of PBS(−).

As a

control, for the synthesis of a insulin–PEG conjugate, NHS-mPEG (200 µg, 100 nmol) and insulin (20 µg, 3.5 nmol) were incubated at 4 °C for 24 h in 102 µL of borate buffer (pH 8.5) and then freeze-dried, followed by the measurement in 60 µL of PBS(−). Native poly(acrylamide) gel electrophoresis (Native-PAGE).

Various amounts of

Chol-U-Pr- mPEG (5–100 µg, 2.1–41 nmol) and insulin (10 µg, 1.7 nmol) were mixed in 20 µL of H2O.

As a control, for the synthesis of a insulin–PEG conjugate, NHS-mPEG (10 µg or 100 µg, 5

nmol or 50 nmol) and insulin (10 µg, 1.7 nmol) were mixed in 20 µL of borate buffer (pH 8.5). The resulting mixture, as well as free insulin (no PEG), was incubated at 4 °C or 37 °C for 6 h or 24 h, followed by freeze-drying.

Then, 18 µL of PBS(−) was added to the dried sample, mixed with


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15 loading buffer (2 µL), and loaded onto 15% polyacrylamide gel.

The prepared gel was run using

buffer (pH 8.3) comprising 50 mM Tris and 38 mM glycine.

Electrophoresis was performed at

room temperature for 1 h, and the current was kept at 20 mA.

Coomassie brilliant blue was used

to observe the insulin-containing bands.

After washing with 7% acetic acid solution, the

insulin-containing bands were visualized. Gel-filtration chromatography (GFC).

(50 µg, 8.7 nmol) were mixed in 250 µL of H2O.

Chol-U-Pr-mPEG (500 µg, 207 nmol) and insulin As a control, for the synthesis of a insulin–PEG

conjugate, NHS-mPEG (500 µg, 250 nmol) and insulin (50 µg, 8.7 nmol) were mixed in 250 µL of borate buffer (pH 8.5). freeze-drying.

The resulting mixture was incubated at 4 °C for 24 h, followed by

Then, 250 µL of PBS(−) was added to the dried sample.

GFC was carried out

using a Jasco PU-980 pumping system (Tokyo, Japan) at a flow rate of 1.0 mL/min with a Shodex OHpak SB-804 HQ column (Showa Denko K.K., Tokyo, Japan). PBS(−) was used as the mobile phase. column.

The aqueous solution containing

The resulting samples in PBS(−) were injected into the

The eluate was detected with a RI detector (RI-1530; Jasco) and a UV detector

(UV-2077; Jasco). Circular dichroism (CD) measurements.

Chol-U-Pr-mPEG (400 µg, 166 nmol) and

insulin (40 µg, 7.0 nmol) were mixed in 204 µL of H2O.

As a control, for the synthesis of a

insulin–PEG conjugate, NHS-mPEG (400 µg, 200 nmol) and insulin (40 µg, 7.0 nmol) were mixed in 204 µL of borate buffer (pH 8.5). The resulting mixture was incubated at 4 °C for 24 h, followed by freeze-drying. The final concentration of insulin was adjusted to 40 µg/mL (7.0 µM).


The CD


Page 16 of 25

16 spectrum from 200 nm to 300 nm of the resulting sample was measured with a Jasco J-820 spectropolarimeter (Tokyo, Japan). Measurement of the insulin degradation.

Chol-U-Pr-mPEG (30 µg or 60 µg, 12 nmol

or 25 nmol), as well as NHS-mPEG (30 µg or 60 µg, 15 nmol or 30 nmol), and insulin (3 µg, 0.5 nmol) were used for the preparation of the complex, as well as the conjugate, as above, and were incubated at 4 °C for 24 h, followed by freeze-drying.

The resulting complex or conjugate was

dissolved in 1.5 mL of PBS(−) in the absence and presence of 10 µg/mL trypsin and then incubated at 37 °C for 4 h.

Then, the insulin in the resulting sample was detected by Mercodia ultrasensitive

insulin ELISA kit (Uppsala, Sweden), according to the manufacture’s protocol. In Vivo Insulin Delivery by Subcutaneous Administration.

Chol-U-Pr-mPEG (347 µg,

144 nmol), as well as NHS-mPEG (347 µg, 174 nmol), and insulin (34.7 µg, 6.1 nmol) were used for the preparation of the complex, as well as the conjugate, as above, and were incubated at 4 °C for 24 h, followed by freeze-drying. of PBS(−).

The resulting complex or conjugate was dissolved in 1.0 mL

ICR mice (5 weeks old, male) were anesthetized with pentobarbital and were

subcutaneously administrated with 200 µL of the resulting PBS(−) solution.

The blood of the mice

was collected after 1 h and 4 h post-injection, and the blood glucose level was determined with glucose meter by Oriental Yeast Co., Ltd. (Tokyo Japan). Animals.

The use of animals and relevant experimental procedures were approved by the

Tokyo University of Pharmacy and Life Science Committee on the Care and Use of Laboratory Animals.


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17

ASSOCIATED CONTENT Supporting Information

The Supporting Information is available free of charge on the ACS Publications website. 1

H NMR spectrum of Chol-U-Pr-mPEG, modification of insulin with NHS-mPEG as a covalent

PEGylation, and particle size of Chol-U-Pr-mPEG and Chol-U-Pr-mPEG/Insulin complex. This material is available free of charge via the Internet at http://pubs.acs.

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Phone: +81 42 677 1111 (ext.) 4976.

Fax: +81 42 677 2821.

ORCID

Shoichiro Asayama: 0000-0002-5109-6052 Notes

The authors declare no competing financial interest.

ACKNOWLEDGEMENTS

This research was partially supported by a Grant-in-Aid for Scientific Research (B) from Japan Society for the Promotion of Science (JSPS KAKENHI grant number 16H03183).


We are


Page 18 of 25

18 grateful to Dr. Fumio Arisaka, an honorary professor of Tokyo Institute of Technology, for ultracentrifugation analysis.

ABBREVIATIONS

PEG, poly(ethylene glycol); Chol-U-Pr-mPEG, cholesterol (Chol) end-modified PEG with urethane (U), propyl (Pr), and methoxy (m) groups; NHS, N-hydroxysuccinimide; NHS-mPEG, monomethoxy-NHS-activated ester/carbonate-PEG; TEA, triethylamine; Native-PAGE, native poly(acrylamide) gel electrophoresis; GFC, Gel-filtration chromatography; CD, circular dichroism; ELISA, enzyme-linked immunosorbent assay

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25

Table of Contents Graphic

H3C

Chol-U-Pr-mPEG : Cholesterol-Urethane-PropylMethoxy-Poly(ethylene glycol) CH3O

CH2CH2O

n

CH3 H3C

CH3 H3C

H CH2CH2CH2N C O O

Insulin

Most Simple Noncovalent PEGylation

7

8



By-product by conventional covalent PEGylation 12

Byproduct-Free Intact Modification of Insulin by Cholesterol End

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