Self-Assembled CurcuminâPoly(carboxybetaine methacrylate

Subscriber access provided by University of South Dakota

Biological and Environmental Phenomena at the Interface

Self-Assembled Curcumin-poly (Carboxybetaine Methacrylate) Conjugates: Potent Nano-Inhibitors against Amyloid #-Protein Fibrillogenesis and Cytotoxicity Guangfu Zhao, Xiaoyan Dong, and Yan Sun Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01921 • Publication Date (Web): 22 Aug 2018 Downloaded from http://pubs.acs.org on August 25, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

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

Page 1 of 46 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

Langmuir

1

Self-Assembled Curcumin-poly (Carboxybetaine Methacrylate)

2

Conjugates: Potent Nano-Inhibitors against Amyloid β-Protein

3

Fibrillogenesis and Cytotoxicity

4 5

Guangfu Zhao, Xiaoyan Dong, Yan Sun*

6 7

Department of Biochemical Engineering and Key Laboratory of Systems Bioengineering

8

of the Ministry of Education, School of Chemical Engineering and Technology, Tianjin

9

University, Tianjin 300072, China

10 11

KEYWORDS: Zwitterionic polymer; Nanoparticle; Hydration; Structural stabilization;

12

Amyloid β-protein

13

1

ACS Paragon Plus Environment

Langmuir 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

1

ABSTRACT:

2

Fibrillogenesis of amyloid β-protein (Aβ) is a pathological hallmark of Alzheimer’s

3

disease, so inhibition of Aβ aggregation is considered as an important strategy for the

4

precaution and treatment of AD. Curcumin (Cur) has been recognized as an effective

5

inhibitor of Aβ fibrillogenesis, but its potential application is limited by its poor

6

bioavailability. Herein, we proposed to conjugate Cur to a zwitterionic polymer, poly

7

(carboxybetaine methacrylate) (pCB), and synthesized three Cur@pCB conjugates of

8

different degrees of substitution (DS, 1.9 to 2.9). Cur@pCB conjugates self-assembled

9

into nanogels of 120-190 nm. The inhibition effects of Cur@pCB conjugates on the

10

fibrillation and cytotoxicity of Aβ42 was investigated by extensive biophysical and

11

biological analyses. Thioflavin T fluorescence assays and atomic force microscopic

12

observations revealed that the Cur@pCB conjugates were much more efficient than

13

molecular curcumin on inhibiting Aβ42 fibrillation, and cytotoxicity assays also indicated

14

the same tendency. Of the three conjugates, Cur1@pCB of the lowest DS (1.97) exhibited

15

the best performance; 5 µM Cur1@pCB functioned similarly with 25 µM free curcumin.

16

Moreover, 5 µM Cur1@pCB increased the cell viability by 43% but free curcumin at the

17

same concentration showed little effect. It is considered that the highly hydrated state of

18

the zwitterionic polymers resulted in the superiority of Cur@pCB over free curcumin.

19

Namely, the dense hydration layer on the conjugates strongly stabilized the bound Aβ on

20

curcumin anchored on the polymer, suppressing the conformational transition of the

21

protein to β-sheet-rich structures. This was demonstrated by circular dichroism

22

spectroscopy, in which Cur1@pCB was proven to be the strongest in the three conjugates.

23

The research has thus revealed a new function of zwitterionic polymer pCBMA and

24

provided new insights into the development of more potent nano-inhibitors for

25

suppressing Aβ fibrillogenesis and cytotoxicity.

26

2


Page 2 of 46

Page 3 of 46 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

Langmuir

1

INTRODUCTION

2

Alzheimer’s disease (AD) is a devastating neurodegenerative disorder,1,2 and

3

pathologically featured by the accumulation of extracellular amyloid plaques and

4

intracellular neurofibrillary tangles.3,4 The amyloid plaques are mainly composed of

5

various amyloid β-protein (Aβ) aggregates (oligomers, protofibrils and fibrils).5-7 Aβ40

6

and Aβ42 are the two major Aβ isoforms, which have different aggregation characteristics

7

and cytotoxicities.8-11 It is generally accepted that soluble oligomers are the most

8

neurotoxic species to brain cells associated with perturbations of synaptic function and

9

neural network activity that probably underlie the cognitive deficits in AD.10,12 Therefore,

10

inhibition of Aβ fibrillogenesis is considered as a promising therapeutic strategy for the

11

prevention and treatment of AD.

12

Till now, many inhibitors against Aβ aggregation and amyloid toxicity have been

13

reported, including peptides,13-16 small organic molecules,17-19 nanoparticles (NPs)20-22

14

and proteins.23-25 Among these inhibiting agents, small organic molecules such as

15

epigallocatechin gallate and curcumin were identified to show potent inhibitory

16

effects.26,27 Especially, curcumin has both obvious inhibiting capacity on Aβ

17

fibrillogenesis and disaggregation effect on mature fibrils.27,28 Nevertheless, there are

18

significant drawbacks that limit its application. For example, curcumin has extremely low

19

solubility in aqueous solution (0.27 µg/mL) and low stability in serum which results in its

20

rapid clearance from blood.29,30 To solve these problems that vitally limit its

21

bioavailability, several approaches including co-delivery with nanoparticles and

3


Langmuir 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 4 of 46

1

modification as prodrugs have been proposed.30,31 Among the approaches, conjugating

2

curcumin to polymers has attracted great attention, owing to their readily tailorable

3

chemical structure. In this work, we have proposed to conjugate curcumin to a

4

zwitterionic polymer, poly(carboxybetaine), to develop novel curcumin-polymer

5

conjugates.

6

Zwitterionic

polymers

such

as

poly(carboxybetaine)

(pCB)

and

poly

7

(sulfobetaine)32-34 are highly hydrophilic and excellent biocompatibility. Particularly,

8

pCB has unique functionalization capabilities for conjugating biomolecules or targeting

9

moieties.33 Thus, pCB is considered as good candidate for conjugation to improve the

10

solubility of curcumin and its efficacy. Considering the hydrophilicity of pCB and the

11

hydrophobicity of curcumin, the conjugation of curcumin to pCB could lead to

12

amphiphilic curcumin@pCB (Cur@pCB) conjugates. Moreover, due to the highly

13

hydration nature of pCB33 and hydrophobic nature of curcumin, the amphiphilic

14

Cur@pCB conjugates may present significantly higher inhibitory effects on Aβ

15

aggregation and cytotoxicity than molecular curcumin. This proposal has been validated

16

in this work by extensive biophysical and biological analysis. Based on the findings, a

17

mechanistic model was proposed to elucidate the effects of Cur@pCB conjugates on the

18

inhibitory effect on Aβ aggregation and toxicity.

19 20

EXPERIMENTAL SECTION

4


Page 5 of 46 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

Langmuir

1

Materials. 2-(Dimethyl amino) ethyl methacrylate (DMAEMA, 98%), pyrene

2

(99%),

3

4-dimethylaminopyridine (DMAP) were obtained from Sigma-Aldrich (St. Louis, MO,

4

USA). Curcumin (98%), phospho-tungstic acid, ethyl-2-Bromoisobutanoate (EBIB, 98%),

5

hexafluoro-2-propanol

6

3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium

7

purchased from JK Chemical (Beijing, China). 2,2'-Bipyridine (Bpy) and dimethyl

8

sulfoxide (DMSO) was purchased from Jiangtian Huagong (Tianjin, China). Aβ42 was

9

obtained from GL Biochem (Shanghai, China). The SH-SY5Y cell line was provided by

10

the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Dulbecco's

11

Modified Eagle Medium/Ham's F-12 (DMEM/F12) and fetal bovine serum (FBS) were

12

obtained from Gibco Invitrogen (Grand Island, NY, USA). Other chemicals were all the

13

highest purity available from local sources. Deionized water was used for all solution

14

preparations.

15

1,3-dicyclohexylcarbodiimide

(DCC),

(HFIP),

thioflavin

T

(ThT),

β-propiolactone, bromide

and

and (MTT)

was

Synthesis of Carboxybetaine Methacrylate. Carboxybetaine methacrylate (CBMA)

16

was synthesized as reported in literature32 and the reaction is shown in Scheme S1a. A

17

solution of 1.68 mL (10 mmol) DMAEMA dissolved in 25 mL anhydrous acetone was

18

allowed to cool in an ice bath under a nitrogen stream and magnetic stirring for 30 min

19

before 0.76 mL β-propiolactone (12 mmol) in 5 mL of anhydrous acetone was added

20

dropwise. The reaction was performed under 100 rpm at 15 °C for 5 h. The white rough

21

product was washed three times with anhydrous acetone and anhydrous ether in sequence. 5


Langmuir 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 6 of 46

1

After being dried under reduced pressure for 2 h, the recovered CBMA monomer was

2

stored at 4 °C before polymerization. Synthesis

3

of

Poly

(carboxybetaine

methacrylate).

Poly(carboxybetaine

4

methacrylate) (pCB) was prepared by atom transfer radical polymerization initiated by

5

Cu(I) as reported in literature.32,35 The reaction is presented in Scheme 1b and the

6

procedure is described as follows. CBMA (1.0045 g, 4 mmol), EBIB (3.665 µL, 0.05

7

mmol) and Bpy (31.2 mg, 0.29 mmol) were dissolved in 10 mL H2O/DMF (v/v = 2: 8) and

8

the solution was filled with N2 for 1 h. Thereafter, CuBr (14.3 mg, 0.094 mmol) and CuBr2

9

(2.20 mg, 0.005 mmol) were added into the reaction system. The mixtures were stirred and

10

filled with N2 for another 1 h at room temperature before it was rapidly sealed with a

11

piece of parafilm. The polymerization reaction continued with stirring at100 rpm and

12

25 °C for 24 h. Then, the polymer solution was dialyzed with a dialyzer with a membrane

13

of molecular weight cut-off of 3.5 kDa against deionized water for 5 d. Finally, the

14

polymer solution was lyophilized and kept under a dry environment (desiccator) before

15

use.

16

To synthesize pCB of higher molecular weight, another reaction was conducted with

17

different molar ratios of the reactants. That is, CBMA (1.145 g, 5 mmol), EBIB (1.83 µL,

18

0.025 mmol) and Bpy (31.2 mg, 0.29 mmol) were dissolved in 10 mL H2O/DMF (v/v = 2:

19

8) and the solution was filled with N2 for 1 h. Then, CuBr (14.3 mg, 0.094 mmol) and

20

CuBr2 (2.20 mg, 0.005 mmol) were added into the reaction system to initiate the

6


Page 7 of 46 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

Langmuir

1

polymerization. Other conditions and following operations were the same as those

2

described above.

3

Synthesis of Curi@pCB Conjugates. Cur@pCB conjugates were prepared

4

according to the method reported in the conjugation of curcumin to HA;36 the reaction is

5

illustrated in Scheme 1c. In Brief, pCB was dissolved in H2O by magnetic stirring for 1 h.

6

DCC and DMAP dissolved in DMSO was then dripped into the pCB solution to activate

7

the carboxyl groups of pCB. This activation reaction was continued for 1 h before the

8

resultant was dripped into a curcumin solution. In the conjugation reactions, the molar

9

ratio of DCC: DMAP: pCB: curcumin was kept at 1.5: 1.5: 1: n, in which n was set at 1, 2

10

and 3 to obtain three conjugates of different degrees of substitution (DS). The conjugation

11

reaction was carried out at 65 °C. The resulting Curi@pCB conjugates were purified by

12

dialysis against DMSO for 3 d and then against deionized water for another 3 d to remove

13

the non-reacted reactants and any by-products. Finally, the product, Curi@pCB (i=1, 2, 3),

14

was lyophilized and kept in a desiccator before use.

15

Characterization of Synthesized Chemicals. CBMA and pCB were characterized

16

by 1H-NMR (500 MHz Varian Inova, Varian Medical System, Inc., CA, USA). pCB and

17

Cur@pCB were analyzed by Fourier transform infrared spectroscopy (FT-IR) (Tensor 27,

18

Bruker Optics, Germany). Samples were prepared using the KBr pallet method as reported

19

in literature.32,33 The average molecular weight and molecular weight distribution of pCB

20

were measured by gel permeation chromatography (GPC). The GPC experiments were

21

carried out using a GPC column (TSK-gel G4000PWxl, Tosoh, Japan) with a 7


Langmuir 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

1

fractionation range of 40-300 kDa. The column was connected to Agilent 1100 Series

2

Quaternary Pump (G1314A, Agilent Technologies, CA, USA) and DAWN EOS

3

multi-angle laser light scattering instrument (Wyatt Technology, CA, USA) provided with

4

a data station. NaNO3 solution (0.1 µM) was used as the mobile phase at 0.6 mL/min.

5

One of the Cur@pCB conjugates, Cur1@pCB with a SD of 1.97, was scanned by UV-vis

6

spectroscopy (Lambda 35, Perking Elmer, USA).

7

Water Solubility of Curi@pCB. To evaluate the water solubility of Curi@pCB (i =

8

1, 2, 3), excess amount of the conjugate sample was added to water,36 and the mixture was

9

kept stirring for 5 min. It was then centrifuged at 14 000 rpm for 5 min in room temperature,

10

and the solution was separated and diluted with the same volume of DMSO to make a

11

solution in DMSO/water (v/v =1:1). The concentration was read from the calibration

12

curve of pure curcumin dissolved in DMSO/water (v/v=1:1) by spectrometric

13

measurement at 436 nm (Figure S1).

14

Dynamic Light Scattering (DLS). It is anticipated that Curi@pCB (i=1, 2, 3)

15

would form nanostructures spontaneously when it is dissolved in water because of its

16

amphiphilic characteristics. Hence, the size, polymer dispersity index (PDI) and zeta

17

potential of the nanogels at 1 mg/mL were measured using Zetasizer Nano (Malvern

18

Instruments, Worcestershire, UK). About 1 mL nanogel dispersion was added to the cell,

19

and the measurement was conducted at 25 °C with the backscatter angle detection at 173°

20

and refractive index of 1.46.

8


Page 8 of 46

Page 9 of 46 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

Langmuir

1

Determination of Critical Aggregation Concentration (CAC). The CAC of

2

Curi@pCB (i=1, 2, 3) was determined using pyrene as a fluorescence probe, which can

3

penetrate

4

aggregation-induced enhanced emission in aqueous solution.37 Briefly, Curi@pCB was

5

prepared in 100 mM phosphate buffer solution (PBS) (10 mM sodium chloride, pH7.4) at

6

different concentrations and mixed with 10 µL of 60 µM pyrene in acetone. Acetone was

7

volatilized under room temperature, and 1 mL Curi@pCB was added to each tube to form

8

nanogel solution at concentrations ranging from 1 × 10−4 to 0.5 mg/mL. The samples were

9

incubated at 50 °C for 1 h, and then left to cool down overnight at room temperature. The

10

fluorescence intensity of each sample was detected at wavelength of 378 and 381 nm using

11

fluorescence spectrophotometer (Perkin Elmer LS-55, MA, USA). The CAC value was

12

determined from the threshold concentration at the intensity ratio (I378/I381) vs the

13

logarithm of pyrene concentration plot, where a sharp increase in pyrene fluorescence

14

intensity can be observed in the presence of a CAC.

into

the

hydrophobic

core

of

polymer

nanogels

and

exhibits

15

Preparation of Aβ Monomer Solution. Aβ monomer was prepared as reported in

16

literature.36,38 First, the lyophilized Aβ42 protein was dissolved in HFIP to 1.0 mg/mL.

17

After the solution was kept in quiescence for at least 2 h, it was sonicated for 30 min in ice

18

bath to destroy the pre-existing Aβ aggregates. Thereafter, the solution was centrifuged at

19

16 000 g for 30 min at 4 °C to remove the existing Aβ aggregates. About top 75% of the

20

supernatant was collected and followed by the removal of HFIP under vacuum freeze

21

drying overnight. The freeze-dried protein was immediately stored in a refrigerator at 9


Langmuir 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

1

−20 °C. Before use, the lyophilized protein was dissolved in 20 mM NaOH, and after being

2

sonicated for 2 min in ice bath, the solution was centrifuged at 16 000 g for 20 min at 4 °C

3

to remove the aggregates. Then, the upper 75% of the supernatant was carefully collected

4

to produce Aβ monomers solution. Finally, the Aβ monomers were diluted with PBS to a

5

final concentration of 25 µM. This solution was used immediately for the following studies

6

of Aβ aggregation and cytotoxicity.

7

Thioflavin T Fluorescent Assay. Thioflavin T (ThT) is a benzothiazole dye that

8

exhibits enhanced fluorescence (with excitation and emission at 440 and 480 nm,

9

respectively) upon binding to amyloid fibrils and protofibrils,36 so ThT fluorescence assay

10

is a general and standard method to quantify the formation of amyloid fibrils. The ThT

11

fluorescence assay was conducted as described in literature.36,38 In the experiment, Aβ42

12

was mixed to a final concentration of 25 µM with various concentrations of curcumin, pCB

13

and Curi@pCB. The mixture was incubated by continuous orbital shaking at 150 rpm and

14

37 °C. The fluorescence was measured at 48 h using fluorescence spectrometer (Perkin

15

Elmer LS-55, MA, USA) at 25 °C with a slit width of 5 nm and excitation and emission

16

at 440 and 480 nm, respectively. Before each measurement, a sample (200 µL) was

17

withdrawn and mixed uniformly with 2 mL of ThT solution (25 µM ThT in 100 mM

18

phosphate buffer solution with 10 mM NaCl, pH7.4). The fluorescence intensity of the

19

sample without Aβ42 protein was subtracted as background from each read with Aβ42

20

protein. Each measurement was performed in triplicate; the average value is reported

21

with standard deviation described as an error bar in figures. 10


Page 10 of 46

Page 11 of 46 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

Langmuir

1

Atomic Force Microscopy. The morphology of Aβ42 aggregates was studied using

2

atomic force microscope (AFM). Aβ42 samples (10 µL) obtained by incubation without

3

and with inhibitors were dropped onto a piece of mica air-dried before observation by AFM

4

(CSPM5500, Benyuan, Beijing, China).

5

Circular Dichroism (CD) Spectroscopy. CD spectra of various Aβ42 samples were

6

measured using a J-810 spectrometer (JASCO, Tokyo, Japan) at 25 °C.39 A quartz cell with

7

1 mm path length was used for far-UV (190-260 nm) measurements with 1 nm bandwidth

8

at a scan speed of 100 nm/min. The CD spectra of solutions without inhibitors or Aβ42 were

9

subtracted as background. All spectra were the average of three consecutive scans for each

10

sample.

11

Cell Viability Assays. The MTT assay was employed to examine the cytotoxicity of

12

Aβ42. SH-SY5Y cells were cultured at 37 °C under 5% CO2 in DMEM/F12 supplemented

13

with 15% FBS, 2 mmol glutamic acid, 100 U/mL penicillin, and 100 µg/mL streptomycin

14

in a CO2 cell culture box (NAPCO 5410, Tualatin, Oregon, USA). A total of 5 × 103 cells

15

(80 µL) were seeded for 24 h in a 96-well polystyrene plate.38 Aβ42 stock solution (25 µM)

16

was incubated without or with different concentrations of curcumin or Curi@pCB (i = 1, 2,

17

3) at 37 °C and 150 rpm for 18 h. Then, the obtained samples (20 µL) were added to the

18

cells, and the cells were incubated for another 48 h, then 10 µL of 5 mg/mL MTT in PBS

19

was added into each well and incubated for another 4 h. The suspension was centrifuged at

20

1500 rpm for 10 min to remove the supernatant. Then, 100 µL DMSO was added and

21

followed by shaking at 150 rpm for 10 min to dissolve the cells. The cell viability was 11


Langmuir 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

1

calculated from the absorbance signals measured by a plate reader (Infinite M200, Tecan,

2

Austria) at a 570 nm. The wells containing medium only were subtracted as the

3

background from each reading. The cell viability data were normalized as a percentage of

4

the control group without Aβ42 and inhibitors.

5

All the cell viability data are representative of at least six independent experiments

6

carried out with different cell culture preparations and presented as mean ± standard

7

deviations. Analysis of variance was carried out for statistical comparisons using t-test,

8

and p < 0.05 or less was considered to be statistically significant.

9 10

RESULTS AND DISCUSSION

11

Characterization of CBMA, pCB and Cur@pCB

12

Scheme 1 shows the schematic for the synthesis of the monomer, carboxybetaine

13

methacrylate (CBMA), pCB and Curi@pCB conjugates by forming ester linkage between

14

pCB and curcumin. The structures of CBMA and pCB were confirmed by examination

15

with 1H nuclear magnetic resonance (1H-NMR) (see Figure S2 and data analysis in the

16

caption). Figure S3 shows the GPC of pCB, and the number-average molecular weight

17

(Mn) was determined to be 35,500 Da with a PDI of 1.51.

18

The initial pCB/curcumin ratio in the conjugation reaction was tuned to prepare

19

three Cur@pCB conjugates of different DS values of curcumin (Table 1). In this study, DS

20

was defined as the number of conjugated curcumin molecules per one hundred CBMA

21

repeating units in pCB. FT-IR spectroscopy of pCB and one of the Cur@pCB conjugates

12


Page 12 of 46

Page 13 of 46 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

Langmuir

1

(No.1 in Table 1) confirmed the successful synthesis of Cur@pCB conjugates (Figure S4).

2

Due to the structural symmetry of curcumin, the phenolic hydroxyl groups at the two ends

3

have the same reactivity. Thus, the reaction of curcumin and pCB might result in two

4

types of products, namely, mono-modified curcumin and di-modified curcumin.40,41 The

5

FT-IR spectrum of Cur1@pCB (Figure S4a) shows the signal of phenolic hydroxyl bond.

6

This indicates that one of the phenolic hydroxyl groups of curcumin remain in the

7

conjugate, though the presence of di-modification cannot be ruled out.

8

Curcumin has an intense absorption in the UV–visible spectral region in

9

DMSO/water (v/v = 1:1) with an absorption peak around 420 nm,37 but pCB does not

10

show any absorbance at the wavelength range (Figure S5). By contrast, the Cur@pCB

11

conjugate (No.1 in Table 1) shows an absorption peak at 436 nm. The red shift in the

12

peak position of Cur@pCB was caused by the ester linkage present in the Cur@pCB

13

conjugate, as often observed in polymer-curcumin conjugates.37 Then, curcumin

14

concentration in the Cur@pCB conjugates was determined with the calibration curve of

15

curcumin determined in DMSO/water (v/v = 1:1) by absorption at 436 nm (Figure

16

S1).36,37

17

Table 1 indicates that the solubility of curcumin in Curi@pCB (i=1, 2, 3) was

18

hundreds of times higher than that of molecular curcumin (0.27 µg/mL),37 and increased

19

with increasing the DS value. This was definitely due to the high hydrophilicity of pCB.

20

AFM observations confirmed the presence of nanogels (Figure S6), and DLS analysis

21

(Figure S7) revealed that the size of the Curi@pCB decreased with increasing the DS 13


Langmuir 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

1

value (Table 1). Similar trend was observed in AFM (Figure S6), but the particle sizes

2

observed in AFM were smaller than those from the DLS measurements, because AFM

3

images were obtained in a dry state.

4

Then, the self-assembly behavior of Cur@pCB conjugates in aqueous solution was

5

characterized by measuring the CAC using pyrene as a hydrophobic fluorescence probe.

6

As shown in Figure S8, the increase of fluorescence intensity ratio of I378/I381 was

7

observed with the increment of Curi@pCB (i = 1, 2, 3) concentration. From the plots, the

8

CAC values of the conjugates were estimated and are listed in Table 1. A distinct decrease

9

of CAC value with increasing DS was observed, because more conjugation of

10

hydrophobic curcumin led to the formation of more compact nanogel structure.42

11

Previously, curcumin was modified to hyaluronic acid (HA), but the CAC values of

12

HA-Cur were about 13.2 times larger than Cur@pCB conjugates at a similar DS

13

value.43,44 It is favorable for the Cur@pCB conjugates to possess low CAC values,

14

because it would make the Curi@pCB self-assemblies (nanogels) keep intact even

15

undergoing an extensive dilution in an in vivo administration.42 Due to the small size of

16

Aβ42 (4514 Da), the nanogel structure should have enough permeability for the protein to

17

diffuse into the hydrophobic core21 to interact with the conjugated curcumin.

18 19

Inhibitory Effects on Aβ Aggregation

20

First, we examined the inhibitory effects of curcumin and Curi@pCB on Aβ42

21

fibrillation at different molar ratios using ThT fluorescence assays. Aβ42 displayed faster

14


Page 14 of 46

Page 15 of 46 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

Langmuir

1

growth kinetics with little lag phase time and the steady state was reached in 24 h, as

2

reported previously.36 To make a fair comparison, we first used the maximum intensity of

3

ThT fluorescence obtained from pure Aβ42 incubation (a total amount of amyloid fibrils

4

being formed) to normalize other ThT profiles. As shown in Figure 1, addition of pCB

5

gave little influence on the ThT fluorescence intensity, indicating that pCB did not affect

6

Aβ42 aggregation. Then, curcumin or any of the Cur@pCB conjugates was incubated with

7

freshly prepared Aβ42 solution (25 µM) at three different curcumin to Aβ ratios. It is seen

8

that curcumin showed very weak inhibition effect on Aβ42 fibrillation at 5 µM, as

9

evidenced by only 15% decrease in the ThT intensity. The inhibition effect of curcumin

10

increased with its concentration, similarly to literature data;36 the ThT intensity decreased

11

about 40% in the presence of 25 µM curcumin. The figure also shows that the presence of

12

pCB did not affect the inhibition effect of curcumin on Aβ42 fibrillogenesis, further

13

confirming the lack of influence of free pCB on Aβ42 aggregation.

14

A dose-dependent effect of Cur@pCB conjugates Aβ42 aggregation was observed,

15

similar with free curcumin and many other chemical inhibitors, peptides and NPs.45-47 By

16

comparison to free curcumin, however, it is evident that the Cur@pCB conjugates offered

17

much higher inhibitory potencies than free curcumin at equivalent curcumin

18

concentrations (Figure 1). Moreover, it is obvious that the inhibitory effect increased

19

with decreasing DS at each equivalent curcumin concentration. Cur1@pCB offered a low

20

ThT fluorescence intensity (47% reduction) even at an equivalent curcumin concentration

21

as low as 5 µM and only 9% fluorescence intensity remained at 25 µM of equivalent 15


Langmuir 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

1

curcumin concentration. By contrast, free curcumin led to only 15% decrease of the

2

fluorescence intensity at 5 µM, and still 60% fluorescence intensity remained at 25 µM.

3

Namely, 5 µM Cur1@pCB showed better performance than 25 µM free curcumin. Thus,

4

the Cur@pCB conjugates displayed much stronger inhibitory effect on Aβ42 aggregation

5

than molecular curcumin at an equivalent curcumin concentration.

6

Previous study has reported curcumin-modified hyaluronic acid (HA), which formed

7

nanogels denoted as CHA, as inhibitors of Aβ aggregation.36 By comparison at equivalent

8

curcumin concentrations, it revealed that the inhibitory effect of Curi@pCB was much

9

stronger than the CHA nanogels on Aβ42 aggregation even at the optimum DS of CHA.36

10

For example, the ThT fluorescence reduced only 66% at the optimum DS (2.43) of CHA

11

with 25 µM curcumin equivalent, but only 9% ThT fluorescence was left with

12

Cur1@pCB (DS=1.97) with 25 µM curcumin equivalent.

13

AFM was used to observe the morphologies of Aβ42 aggregates obtained without and

14

with the inhibitors (Figure 2). After incubating Aβ42 (25 µM) at 37 °C for 48 h, serried and

15

entangled fibrils were observed (Figure 2a), consistent with literature results.48,49 The

16

morphological characteristics changed little in the sample with pCB (Figure 2b).

17

Co-incubating curcumin at 5 to 25µM with Aβ42 also led to the formation of fibrous

18

aggregates, though Aβ42 fibers became shorter and less tangled, the lengths and amount of

19

fibril decreased with increasing curcumin concentration (Figures 2d), consistent with

20

previous studies.27 In the presence of 25 µM curcumin + 0.97 mg/mL pCB, the

21

morphology did not change significantly as compared with the case 25 µM curcumin 16


Page 16 of 46

Page 17 of 46 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

Langmuir

1

(Figures 2c and 2d3). By contrast, the aggregate morphology changed significantly in the

2

presence of Curi@pCB (Figures 2e, 2f and 2g). For example, no mature fibrillar

3

amyloids but many amorphous aggregates were found in the presence of 25 µM

4

Curi@pCB (see Figure 2e3, 2f3 and 2g3). At lower equivalent curcumin concentrations

5

(5 and 10 µM) with Curi@pCB, some short fibrils were formed, and the amount of fibril

6

decreased with increasing the Curi@pCB concentrations (see e, f and g in Figure 2). The

7

AFM observations also confirmed the significantly increased inhibitory potency of

8

Curi@pCB as compared to molecular curcumin, and that Cur1@pCB was the best of the

9

three conjugates.

10 11

Protective Effect on Aβ42-Induced Cytotoxicity

12

To assess the cytotoxicities of Curi@pCB and Curi@pCB-mediated Aβ42 aggregates,

13

MTT assays were employed with the SH-SY5Y cell line. It was confirmed that pure pCB

14

had no obvious effect on the cell viability (Figure 3a), indicating that pCB was

15

biocompatible and non-toxic to the cells. However, curcumin displayed significant

16

cytotoxicity to the SH-SY5Y cells at 5 µM, at which the cell viability remained about

17

76% (Figure 3a). This was reasonable because previous researchers have reported the

18

antitumor effect of curcumin, and that curcumin caused the death of SH-SY5Y cells.27,36

19

By comparison, Cur@pCB conjugates showed much lower cytotoxicity than free

20

curcumin at each equivalent curcumin concentration, and the cell viability remained

21

about 91% with Cur1@pCB at 5 µM curcumin equivalent (Figure 3b). The decreased

17


Langmuir 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

1

cytotoxicity of Curi@pCB conjugates over curcumin might be due to the encapsulation

2

of curcumin into the inner core of the Cur@pCB nanogels, which protected the cells from

3

contacting curcumin.36

4

A comparison of suppression potencies of different inhibitors on the cytotoxicity of

5

Aβ42 aggregates is shown in Figure 3c, 3d and 3e. As controls, incubation with 5 µM

6

Aβ42 aggregates led to 49% death of SH-SY5Y cells, and pCB showed negligible effect

7

on the cell growth. Then, Aβ42 aggregates were obtained by co-incubating 25 µM Aβ42

8

with different inhibitors of different concentrations as indicated in the figure, and they

9

were added to SH-SY5Y cells by 5-fold dilution. It is seen that 5 and 10 µM curcumin

10

presented little reduction of the toxicity of Aβ42 aggregates (Figures 3c and 3d), and there

11

was only 37% viability increase (from 51% to 70%) when curcumin concentration was

12

increased to 25 µM (Figure 3e). In the presence of 25 µM curcumin + 0.97 mg/mL pCB,

13

the cell viability increased to 67% (Figure 3e), rather similar to the that with 25 µM

14

curcumin. By contrast, the inhibition effect of Cur@pCB conjugates on the amyloid

15

toxicity of Aβ42 aggregates were much higher than curcumin, as evidenced by the

16

significant cell viability increases at low concentrations (5 and 10 µM curcumin

17

equivalent) (Figure 3c and 3d). For example, the cell viability increased from 51% to

18

68% - 73% at 5 µM Curi@pCB (Figure 3c), similar to that with 25 µM curcumin (from

19

51% to 70%). Particularly, the same as those observed in the above ThT assays,

20

Cur1@pCB displayed the best protective effect on the amyloid toxicity of Aβ42

21

aggregation, which increased the cell viability to 73% at 5 µM, 77% at 10 µM, and 82% 18


Page 18 of 46

Page 19 of 46 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

Langmuir

1

at 25 µM. Moreover, it is of significance to see that 5 µM Cur1@pCB increased the cell

2

viability by 43% but free curcumin at the same concentration showed little effect.

3

With curcumin conjugated HA, Jiang et al. also found that CHA mitigated the

4

amyloid cytotoxicity more efficiently than free curcumin as evidenced at 25 µM

5

curcumin equivalent, but no data at curcumin concentrations lower than 25 µM was

6

reported.36 Recently, they reported more data at extensive curcumin concentrations with

7

CHA.50 It is seen that CHA of the optimum DS at 6.4 µM curcumin equivalent increased

8

the cell viability by 29% (from 49% to 63%). This increase was obviously lower than the

9

effect of Cur1@pCB at 5 µM (from 51% to 73%). Hence, cell viability data further

10

confirmed the superiority of Cur@pCB over CHA, in addition to the ThT results

11

discussed above.

12

It should be noted that due to the extremely low solubility of curcumin in aqueous

13

solution (0.27 µg/mL),29,30 1% DMSO (v/v) was added to prepare the experimental

14

solution

15

and to keep the same experimental condition, 1% DMSO was also contained in the cell

16

viability assays with Curi@pCB. To explore the effect of DMSO, a comparison

17

experiment was conducted with Curi@pCB in the absence of DMSO. As shown in Figure

18

S9, the cell viability was little affected by the presence of 1% DMSO.

in

the

above

cell

viability

19 20

Mechanistic Discussion

19


assays

with

curcumin,

Langmuir 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

1

Aβ fibrillation is a complex self-assembly process; hydrophobic interactions

2

including aromatic interactions (π−π stacking interactions) and electrostatic interactions

3

including hydrogen bonding among the side chains of proteins are believed to be the

4

driving forces, which govern the β-sheet assembly and provide amyloid stabilization.51

5

The inhibitory effect of curcumin has been well investigated and the inhibitory

6

mechanism of curcumin on Aβ aggregation has been explored. Some studies suggested

7

that curcumin interacts with Aβ via aromatic/hydrophobic interactions, which are able to

8

perturb the inter-molecular forces between protein molecules and thus suppress the

9

formation of Aβ oligomers and fibrils.47,52 However, other researches have demonstrated

10

that curcumin interacts with Aβ molecules and induces them to form “off-pathway”

11

aggregates with non- or less-toxicity.36,53

12

Herein, we conjugated curcumin to pCB, and Cur@pCB conjugates self-assembled

13

into nanogels. The conjugates not only drastically inhibited Aβ fibrillogenesis (Figure 1)

14

but also led to “off-pathway” aggregates as demonstrated by the aggregation morphology

15

changes at much lower equivalent curcumin concentrations than free curcumin (Figure 2).

16

More importantly, the Cur@pCB conjugates showed much stronger protective effect on

17

Aβ-induced cytotoxicity than molecular curcumin at equivalent curcumin concentrations

18

and Cur1@pCB of the lowest DS value exhibited the best performance among the three

19

Cur@pCB conjugates (Figure 3). In the protection of SH-SY5Y cells from Aβ-induced

20

toxicity, 5 µM Cur1@pCB functioned similarly with 25 µM free curcumin, which

21

showed little effect at 5 µM (Figure 3). This means that curcumin dosage can be reduced 20


Page 20 of 46

Page 21 of 46 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

Langmuir

1

to 1/5 when it is conjugated to pCB as Cur1@pCB. Hence, it is convincing that

2

Cur@pCB conjugates had an extra function on inhibiting Aβ aggregation and

3

cytotoxicity in addition to the effect of molecular curcumin itself.

4

For better understanding of the inhibitory effect of Curi@pCB conjugates on

5

suppressing Aβ fibrillogenesis and its corresponding cytotoxicity, the amino acid

6

sequences of Aβ42 are illustrated in Figure S10.38 It is seen that the protein has two

7

distinct hydrophobic parts, one in the central area (L17-S26) and the other on the C

8

terminal (I31-A42).54 It displays a random coil or α-helix structure before aggregating into

9

the toxic oligomers and fibrils with mainly β-sheet conformation at physiological

10

conditions.55

11

On the other hand, pCB has been widely recognized for its antifouling properties

12

when coating to surfaces, because it can electrostatically bind water molecules more

13

tightly than the hydrogen bonding displayed in other hydrophilic polymers (e.g. poly

14

(ethylene glycol), PEG).56 A recent research reported that each molecule of CBMA binds

15

9.3±0.5 molecules of water,57 implying that there is a dense hydration layer on pCB. Thus,

16

the Aβ molecules bound by the conjugated curcumin on Curi@pCB would be highly

17

influenced by the dense hydration layer, which embraces the bound Aβ molecules

18

(Figure 4). In this way, the hydration layer on pCB would greatly stabilize the

19

conformation of bound Aβ42 molecules, thus influencing Aβ42 aggregation pathway. This

20

is similar to the stabilization effects of zwitterionic materials on covalently conjugated

21

proteins.58,59 21


Langmuir 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

1

To verify the hypothesis about the effect of hydration layer of pCB on Aβ42

2

stabilization, we used circular dichroism (CD) spectroscopy to examine the secondary

3

structures of Aβ42 influenced by Curi@pCB. As shown in Figure 5, the CD spectra of

4

native Aβ42 displayed characteristic spectra of random coil structure but almost no

5

structural change was observed at 0 h in the presence of different inhibitors (Figure 5a1,

6

5b1, 5c1 and 5d1). After 48 h incubation, a positive peak at 198 nm and a negative valley

7

at 216 nm were observed, suggesting the structural transition from random coil to β-sheet

8

in the Aβ42 (Figure 5a2). By contrast, the conformational transition from random coil to

9

β-sheet was suppressed by curcumin, as manifested by the decrease of negative valley

10

intensity around 216 nm as compared to native Aβ42 (Figure 5a2). These results indicate

11

that curcumin affected Aβ42 aggregation by decreasing the content of β-sheet

12

conformation in the aggregates. The CD spectra of Aβ42 with the mixture of 25 µM

13

curcumin + 0.97 mg/mL pCB was rather similar with the curcumin experiment (Figure

14

5a1 and 5a2), indicating that free pCB did not influence the effect of curcumin. In the

15

presence of Curi@pCB, however, the conjugates completely suppressed the

16

conformational transition to β-sheet at higher concentrations (10 and 25 µM curcumin

17

equivalent) as evidenced by the lack of the negative valley intensity around 216 nm in the

18

CD spectra. Even at a dosage as low as 5 µM curcumin equivalent, the Cur@pCB

19

conjugates could significantly decrease the content of β-sheet structure, particularly with

20

Cur1@pCB (little negative valley intensity around 216 nm) (Figure 5b2, 5c2 and 5d2).

21

The results clearly show that the Cur@pCB conjugates were superior over molecular 22


Page 22 of 46

Page 23 of 46 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

Langmuir

1

curcumin or the mixture of curcumin and pCB on suppressing the conformational

2

transition of Aβ to β-sheet structure, demonstrating that both curcumin and its anchor,

3

pCB, influenced the conformational changes on the bound Aβ. In other words, it is the

4

dense hydration layer on pCB that suppressed the conformational transition of Aβ to

5

β-sheet structure, providing much higher inhibitory potency than molecular curcumin on

6

Aβ42 aggregation the amyloid toxicity.

7

Then, why Cur1@pCB showed the best performance of the three conjugates? This

8

can also be explained by the hydration of pCB. Due to the strong hydrophobicity of

9

curcumin29 and the super-hydrophilicity of pCB33, the conjugation of hydrophobic

10

curcumin to pCB would compromise the hydration of pCB, because a very small content

11

of hydrophobic moiety may cause a great damage to the hydration layer.60 So, the

12

Cur@pCB conjugates of lower DS value would possess higher hydration and present

13

higher effect on suppressing the conformational transition of Aβ to β-sheet structure. This

14

can be seen from the CD spectra affected by the Cur@pCB conjugates at 5 µM curcumin

15

equivalent discussed above. Of the three conjugates, Cur1@pCB almost completely

16

suppressed the secondary conformational transition to β-sheet structure as evidenced by

17

the little negative valley intensity around 216 nm. (Figure 5d2). So, it provided the

18

strongest inhibitory effect on Aβ fibrillation (Figures 1 and 2) and the amyloid toxicity

19

(Figure 3).

20

To provide more insight into the influence of Cur@pCB conjugates on the

21

aggregation of Aβ42, pCB of higher molecular weight (88,700 Da, Figure S11), was 23


Langmuir 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

1

synthetized at the second reaction condition described in the experimental section, and

2

pCB/curcumin ratio in the conjugation reaction was tuned to prepare four Cur@pCB

3

conjugates with different DS values of curcumin. The properties of the new group of

4

conjugates, Curj@pCB (j=Ⅰ, Ⅱ, Ⅱ and Ⅱ), are listed in Table S1. It is seen that in

5

comparison with Curi@pCB (i=1, 2, and 3), the size of Curj@pCB (j=Ⅰ, Ⅱ, Ⅱ and Ⅱ) is

6

larger than that of Curi@pCB (i=1, 2, and 3) at a similar DS. This might be attributed to

7

the difference in the molecule weights of pCB preparations in the conjugates. Then, the

8

inhibition effects of Curj@pCB (j=Ⅰ, Ⅱ, Ⅱ and Ⅱ) and Curi@pCB (i=1, 2, and 3) on

9

Aβ42 fibrillation were compared by ThT fluorescence assays. Figure S12 shows the

10

dependence of ThT fluorescence on DS for the two groups of conjugates at three different

11

curcumin concentrations. It is seen that the inhibition effects of both the conjugates

12

decreased with increasing DS at the three different concentrations, and it is interesting to

13

note that each group of the two lines for the two different conjugates at the same

14

curcumin concentration almost overlapped. This indicates that although the nanogel sizes

15

were different due to the difference in the molecular weights of pCB preparations, the

16

inhibition effects were little affected by the polymer size. This further confirmed DS to be

17

a key factor influencing the inhibition potency of the conjugates. That is, the increase in

18

DS compromised the hydration of pCB, which lead to the decrease of the inhibition effect

19

due to the weakened stabilization on Aβ42 conformation.

20

In the above, we have discussed that the inhibitory effects of Cur@pCB conjugates

21

were much stronger than curcumin-conjugated HA (CHA) in both ThT fluorescence and 24


Page 24 of 46

Page 25 of 46 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

Langmuir

1

cell viability assays.50 Researchers have suggested that hydrations of pCB and HA are

2

attributed to their repeating units, indicating that each CBMA molecule binds 9.3±0.5

3

molecules of water,57 and that a disaccharide unit of HA binds about 15 water

4

molecules.61 From the molecular weight of CBMA (Mn=229.3) and the disaccharide unit

5

(Mn=776) of HA, it can be estimated that pCB could bind over two times more water

6

than HA on the same mass basis. The larger value of bound water on pCB than HA

7

indicates the presence of higher hydration layer on pCB than HA. It would be the reason

8

for the stronger inhibitory effect of Cur@pCB conjugates than CHA.

9 10

CONCLUSIONS

11

Despite proven efficacy and relative safety of curcumin, its clinical applications are

12

severely limited because of its poor solubility under physiological conditions. In order to

13

improve the solubility of curcumin, we have conjugated the hydrophobic agent to pCB of

14

very strong hydrophilicity. It proved that the Cur@pCB conjugates were superior over

15

molecular curcumin on inhibiting Aβ aggregation and cytotoxicity, as evidenced by the

16

similar inhibitory potency of Cur@pCB conjugates with molecular curcumin at 1/5

17

equivalent curcumin concentration. Such a superior inhibitory capacity of Curi@pCB

18

was most likely attributed to the high hydration nature of pCB that anchors the inhibitor.

19

CD spectroscopy revealed that the conjugates strongly interfered with the conformational

20

transition of bound Aβ on the conjugated curcumin, leading to the suppression of the

21

conformational transitions from α-helix to β-sheet-rich structures. The finding suggests

25


Langmuir 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

1

that the property of a polymer for inhibitor conjugation can be a key factor influencing

2

the inhibitory potency of the conjugate. Hence, further work should direct towards two

3

aspects, one towards the use of pCB for fabrication of more effective amyloid inhibition

4

systems, and the other towards the design or discovery of polymers of even higher

5

hydration for development of more potent polymer-based Aβ inhibitors.

6 7

ASSOCIATED CONTENT

8

Supporting Information.

9

Properties of Curj@pCB conjugates (Table S1). Calibration curve of curcumin in

10

DMSO/Water (v/v = 1:1) (Figure S1). 1H NMR spectra of CBMA and pCB (Figure S2).

11

Gel permeation chromatography (GPC) profile of pCB for Curi@pCB (Figure S3). FT-IR

12

spectra of Curi@pCB conjugates (No.1 In Table 1) and pCB (Figure S4). UV–vis spectra

13

of pCB, curcumin and Cur@pCB conjugate in DMSO/Water (v/v = 1:1) (Figure S5).

14

AFM images of Cur@pCB conjugates (Figure S6). Size distributions of Cur@pCB

15

conjugates (Figure S7). Critical aggregation concentration plots of Cur@pCB conjugates

16

(Figure S8). Effect of 1% DMSO on the viability of SH-SY5Y cells (Figure S9). Amino

17

acid sequence of Aβ42 (Figure S10). GPC profile of pCB for Curj@pCB (Figure S11).

18

The dependence of ThT fluorescence on DS for the two groups of conjugates at 3 different

19

concentrations (Figure S12). The Supporting Information is available free of charge on

20

the ACS Publications website at http://pubs.acs.org.

21

26


Page 26 of 46

Page 27 of 46 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

Langmuir

1

AUTHOR INFORMATION

2

Corresponding Author:

3

*Tel: +86 22 27403389; Fax: +86 22 27403389; E-mail address: [email protected] (Y.

4

Sun).

5

ORCID

6

Xiaoyan Dong: 0000-0002-8040-5897

7

Yan Sun: 0000-0001-5256-9571

8

Author Contributions

9

Y.S. designed the research; G.F.Z. performed the experiments and analyzed the data;

10

G.F.Z., X.Y.D. and Y.S. wrote or contributed to the writing of the manuscript.

11

Notes

12

The authors declare no competing financial interest.

13 14

ACKNOWLEDGEMENTS

15

This work was supported by the National Natural Science Foundation of China (Grant Nos.

16

91634119 and 21621004).

17

27


Langmuir 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

1

REFERRENCES

2

(1) Park, J.; Lee, B. K.; Jeong, G. S.; Hyun, J. K.; Lee, C. J.; Lee, S. H.,

3

Three-dimensional brain-on-a-chip with an interstitial level of flow and its application as

4

an in vitro model of Alzheimer's disease. Lab Chip 2014, 15, 141-150.

5

(2) Vitiello, G.; Mariono, D. S.; D'Ursi, A. M.; D'Errico, G. Omega-3 fatty acids regulate

6

the interaction of the Alzheimer's Aβ(25-35) peptide with lipid membranes. Langmuir

7

2013, 29, 14239-14245.

8

(3) Mattson, M. P. Pathways towards and away from Alzheimer’s disease. Nature 2004,

9

430, 631-639.

10

(4) Williams, T. L.; Day, I. J.; Serpell, L. C., The effect of Alzheimer's Aβ aggregation

11

state on the permeation of biomimetic lipid vesicles. Langmuir 2010, 26, 17260-17268.

12

(5) Cohen, S. I.; Rajah, L.; Yoon, C. H.; Buell, A. K.; White, D. A.; Sperling, R. A.;

13

Vendruscolo, M.; Terentjev, E. M.; Dobson, C. M.; Weitz, D. A. Spatial propagation of

14

protein polymerization. Phys. Rev. Lett. 2014, 112, 098101.

15

(6) Lesné, S.; Ming, T. K.; Kotilinek, L.; Kayed, R.; Glabe, C. G.; Yang, A.; Gallagher,

16

M.; Ashe, K. H. A specific amyloid-β protein assembly in the brain impairs memory.

17

Nature 440, 352-357. Nature 2006, 440, 352-357.

18

(7) Jiang, D. L.; Rauda, I.; Han, S. B.; Chen, S.; Zhou, F. M. Aggregation pathways of

19

the amyloid β(1-42) peptide depend on its colloidal stability and ordered β-sheet stacking.

20

Langmuir 2012, 28, 12711-12721.

21

(8) Jarrett, J. T.; Berger, E. P.; Lansbury, P. T. The carboxy terminus of the β amyloid 28


Page 28 of 46

Page 29 of 46 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

Langmuir

1

protein is critical for the seeding of amyloid formation: implications for the pathogenesis

2

of Alzheimer's disease. Biochemistry 1993, 32, 4693-4697.

3

(9) Liao, Y. H.; Chang, Y. J.; Yoshiike, Y.; Chang, Y. C.; Chen, Y. R. Negatively Charged

4

Gold Nanoparticles Inhibit Alzheimer's Amyloid‐β Fibrillization, Induce Fibril

5

Dissociation, and Mitigate Neurotoxicity. Small 2012, 8, 3631–3639.

6

(10) Jin, M.; Shepardson, N.; Yang, T.; Chen, G.; Walsh, D.; Selkoe, D. J. Soluble

7

amyloid beta-protein dimers isolated from Alzheimer cortex directly induce Tau

8

hyperphosphorylation and neuritic degeneration. Proc. Natl. Acad. Sci. U. S. A. 2011,

9

108, 5819-5824.

10

(11) Wang, Q.; Zhao, J.; Yu, X.; Zhao, C.; Li, L.; Zheng, J. Alzheimer Abeta(1-42)

11

monomer adsorbed on the self-assembled monolayers. Langmuir 2010, 26, 12722-12732.

12

(12) Ren, B. P.; Jiang, B. B.; Hu, R. D.; Zhang, M. Z.; Chen, H.; Ma, J.; Sun, Y.; Jia, L.

13

Y.; Zheng, J. HP-β-cyclodextrin as an inhibitor of amyloid-βaggregation and toxicity.

14

Phys. Chem. Chem. Phys. 2016, 18, 20476-20485.

15

(13) Xiong, N.; Dong, X. Y.; Zheng, J.; Liu, F. F.; Sun, Y. Design of LVFFARK and

16

LVFFARK-Functionalized Nanoparticles for Inhibiting Amyloid β-Protein Fibrillation

17

and Cytotoxicity. ACS Appl. Mater. Interfaces 2015, 7, 5650-5662.

18

(14) Xiong, N.; Zhao, Y. J.; Dong, X. Y.; Zheng, J.; Sun, Y. Design of a Molecular Hybrid

19

of Dual Peptide Inhibitors Coupled on AuNPs for Enhanced Inhibition of Amyloid

20

β‐Protein Aggregation and Cytotoxicity. Small 2017, 13, 1601666.

21

(15) Xiao, B.; Si, X.; Han, M. K.; Viennois, E.; Zhang, M.; Merlin, D., Co-delivery of 29


Langmuir 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

1

camptothecin and curcumin by cationic polymeric nanoparticles for synergistic colon

2

cancer combination chemotherapy. J Mater. Chem. B 2015, 3, 7724-7733.

3

(16) Jha, A.; Kumar, G. M.; Gopi, H. N.; Paknikar, K. M. Inhibition of β-Amyloid

4

aggregation Through a Designed β-hairpin Peptide. Langmuir 2017, 34, 1591−1600.

5

(17) Pallauf, K.; Rimbach, G., Autophagy, polyphenols and healthy ageing. Ageing Res.

6

Rev. 2013, 12, 237-252.

7

(18) Ren, B. P.; Liu, Y. L.; Zhang, Y. X.; Zhang, M. Z.; Sun, Y.; Liang, G. Z.; Xu, J. X.;

8

Zheng, J. Tanshinones inhibit hIAPP aggregation, disaggregate preformed hIAPP fibrils,

9

and protect cultured cells. J. Mater. Chem. B 2018, 6, 56-67.

10

(19) Thapa, A.; Vernon, B. C.; De la Peña, K.; Soliz, G.; Moreno, H. A.; López, G. P.; Chi,

11

E. Y. Membrane-mediated neuroprotection by curcumin from amyloid-β-peptide-induced

12

toxicity. Langmuir 2013, 29, 11713-11723.

13

(20) Cabaleirolago, C.; Szczepankiewicz, O.; Linse, S. The effect of nanoparticles on

14

amyloid aggregation depends on the protein stability and intrinsic aggregation rate.

15

Langmuir 2012, 28, 1852-1857.

16

(21) Zhang, M.; Mao, X.; Yu, Y.; Wang, C. X.; Yang, Y. L.; Wang, C. Nanomaterials for

17

Reducing Amyloid Cytotoxicity. Adv. Mater. 2013, 25, 3780-3801.

18

(22) Ramesh, N. K.; Sudhakar, S.; Mani, E. Modeling of the inhibitory effect of

19

nanoparticles on amyloid β fibrillation. Langmuir 2018, 34, 4004−4012.

20

(23) Mangrolia, P.; Yang, D. T.; Murphy, R. M. Transthyretin variants with improved

21

inhibition of β-amyloid aggregation. Protein Eng. Des. Sel. 2016, 29, 209-218. 30


Page 30 of 46

Page 31 of 46 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

Langmuir

1

(24) Kerr, M. L.; Gasperini, R.; Gibbs, M. E.; Hou, X.; Shepherd, C. E.; Strickland, D.

2

K.; Foa, L.; Lawen, A.; Small, D. H. Inhibition of Aβ aggregation and neurotoxicity by

3

the 39‐kDa receptor‐associated protein. J. Neurochem. 2010, 112, 1199-1209.

4

(25) Li, X.; Xie, B. L.; Dong, X. Y.; Sun, Y. Bifunctionality of Iminodiacetic

5

Acid-Modified Lysozyme on Inhibiting Zn2+-Mediated Amyloid β-Protein Aggregation.

6

Langmuir 2018, 34, 5106−5115.

7

(26) Zhang, J. N.; Zhou, X. B.; Yu, Q. Q.; Yang, L. C.; Sun, D. D.; Zhou, Y. H.; Jie, L.

8

Epigallocatechin-3-gallate (EGCG)-stabilized selenium nanoparticles coated with Tet-1

9

peptide to reduce amyloid-β aggregation and cytotoxicity. ACS Appl. Mater. Interfaces

10

2014, 6, 8475-8487.

11

(27) Potter, P. E. Curcumin: a natural substance with potential efficacy in Alzheimer’s

12

disease. J. Pharmacol. Exp. Ther. 2013, 5, 23-31.

13

(28) Halder, U. C.; Pal, S.; Maity, S.; Sardar, S.; Parvej, H.; Das, N.; Chakraborty, J.

14

Curcumin inhibits the Al(III) and Zn(II) induced amyloid fibrillation of β-lactoglobulin in

15

vitro. Rsc Advances 2016, 6, 111299–111307.

16

(29) El, K. E.; Abiad, M.; Kassaify, Z. G.; Patra, D. Green synthesis of curcumin

17

conjugated nanosilver for the applications in nucleic acid sensing and anti-bacterial

18

activity. Colloids Surf. B: Biointerfaces 2015, 127, 274-280.

19

(30) Dhumal, D. M.; Kothari, P. R.; Kalhapure, R. S.; Akamanchi, K. G.

20

Self-microemulsifying drug delivery system of curcumin with enhanced solubility and

21

bioavailability using a new semi-synthetic bicephalous heterolipid: in vitro and in vivo 31


Langmuir 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

1

evaluation. Rsc Advances 2015, 5, 90295-90306.

2

(31) Dey, S.; Kunnatheeri, S., Conjugating Curcumin to Water Soluble Polymer

3

Stabilized Gold Nanoparticles via pH Responsive Succinate Linker. J. Mater. Chem. B

4

2015, 3, 824-833.

5

(32) Zhang, Z.; Timothy Chao; Chen, S. F.; Jiang, S. Y. Superlow Fouling Sulfobetaine

6

and Carboxybetaine Polymers on Glass Slides. Langmuir 2006, 22, 10072-10077.

7

(33) Zhang, Z.; Chen, S. F.; Jiang, S. Y. Dual-functional biomimetic materials: nonfouling

8

poly(carboxybetaine) with active functional groups for protein immobilization.

9

Biomacromolecules 2006, 7, 3311-3315.

10

(34) Lin, W. F.; Ma, G. L.; Ji, F. Q.; Zhang, J.; Wang, L. G.; Sun, H. T.; Chen, S. F.

11

Biocompatible long-circulating star carboxybetaine polymers. J. Mater. Chem. B 2014, 3,

12

440-448.

13

(35) Eugenia K.; Vladimir A. I.; Svetlana A. S. Electrostatic Layer-by-Layer

14

Self-Assembly of Poly(carboxybetaine)s: Role of Zwitterions in Film Growth.

15

Macromolecules 2007, 40, 3663-3668.

16

(36) Jiang, Z. Q.; Dong, X. Y.; Liu, H.; Wang, Y. J; Zhang, L.; Sun, Y. Multifunctionality

17

of self-assembled nanogels of curcumin-hyaluronic acid conjugates on inhibiting amyloid

18

β-protein fibrillation and cytotoxicity. React. Funct. Polym. 2016, 104, 22-29.

19

(37) Bisht, S.; Feldmann, G.; Soni, S.; Ravi, R.; Karikar, C.; Maitra, A.; Maitra, A.

20

Polymeric nanoparticle-encapsulated curcumin. J. Nanobiotechnol. 2007, 5, 1-18.

21

(38) Xie, B. L.; Li, X.; Dong, X. Y.; Sun, Y. Insight into the inhibition effect of acidulated 32


Page 32 of 46

Page 33 of 46 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

Langmuir

1

serum albumin on amyloid β-protein fibrillogenesis and cytotoxicity. Langmuir 2014, 30,

2

9789-9796.

3

(39) Guo, J. J.; Sun, W. Q.; Li, L.; Liu, F. F.; Lu, W. Y. Brazilin inhibits fibrillogenesis of

4

human islet amyloid polypeptide, disassembles mature fibrils, and alleviates cytotoxicity.

5

Rsc Advances 2017, 7, 43491-43501.

6

(40) Gao, X. L.; Zhao, Y. J.; WANG, Z. Synthesis and performance study of

7

folate-conjugated curcumin prodrug dendrimer derivative. Journal of International

8

Pharmaceutical Research 2013, 40, 359-364.

9

(41) Shi, W.; Sukanta D.; Samar R.; Afshan H.; Hussnain T.; Saadyah A.; William L'A.;

10

Abdeslem, E. I.; Probal B.; Krishnaswami R. Synthesis of Monofunctional Curcumin

11

Derivatives, Clicked Curcumin Dimer, and a PAMAM Dendrimer Curcumin Conjugate

12

for Therapeutic Applications. Org. Lett. 2007, 9, 5461-5464.

13

(42) Zheng, W.; Tu, C. L. Synthesis of Amphiphilic Chitosan Derivatives and Their

14

Self-aggregation Phenomenon. Chem. J. Chinese U. 2008, 29, 419-424.

15

(43) Manju, S.; Sreenivasan, K. Conjugation of curcumin onto hyaluronic acid enhances

16

its aqueous solubility and stability. J. Colloid Interface Sci. 2011, 359, 318-325.

17

(44) Tian, C. H.; Asghar, S.; Xu, Y. R.; Chen, Z. P.; Zhang, M.; Huang, L.; Ye, J. X.; Ping,

18

Q. N.; Xiao, Y. Y., The effect of the molecular weight of hyaluronic acid on the

19

physicochemical characterization of hyaluronic acid-curcumin conjugates and in vitro

20

evaluation in glioma cells. Colloids Surf. B: Biointerfaces. 2018, 165, 45-55.

21

(45)

Adessi, C.; Frossard, M. J.; Boissard, C.; Fraga, S.; Bieler, S.; Ruckle, T.; 33


Langmuir 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

1

Vilbois, F.; Robinson, S. M.; Mutter, M.; Banks, W. A. Pharmacological profiles of

2

peptide drug candidates for the treatment of Alzheimer's disease. J. Biol. Chem. 2003,

3

278, 13905-13911.

4

(46) Landreth, G.; Cramer, P.; Wesson, D.; Cirrito, J.; Brunden, K.; Wilson, D. 44

5

ApoE-directed therapeutics for the treatment of Alzheimer's disease. Neurobiol. Aging

6

2012, 33, S19-S19.

7

(47) Fuson, K. S.; Boggs, L. N.; May, P. C. 431 Zinc promotes Aβ aggregation but

8

attenuates Aβ neurotoxicity. Neurobiol. Aging 1996, 17, S108.

9

(48) Ouberai, M.; Dumy, P.; Chierici, S.; Garcia, J., Synthesis and Biological Evaluation

10

of Clicked Curcumin and Clicked KLVFFA Conjugates as Inhibitors of β-Amyloid Fibril

11

Formation. Bioconjugate Chem. 2009, 20, 2123-2132.

12

(49) Ungureanu, A. A.; Benilova, I.; Bael, M. J. V.; Haesendonck, C. V.; Bartic, C. In

13

AFM investigation of the aggregation behavior of Alzheimer's disease Aβ peptides,

14

Nanotechnology, 2012, pp 1-2.

15

(50) Jiang, Z. Q.; Dong, X. Y.; Yan, X.; Liu, Y.; Zhang, L.; Sun, Y. Nanogels of dual

16

inhibitor-modified hyaluronic acid function as a potent inhibitor of amyloid β-protein

17

aggregation and cytotoxicity. Sci. Rep. 2018, 8, 3505.

18

(51) Amaro, M.; Birch, D. J.; Rolinski, O. J. Beta-amyloid oligomerisation monitored by

19

intrinsic tyrosine fluorescence. Phys. Chem. Chem. Phys. 2011, 13, 6434-6441.

20

(52) Jr, S. W.; Dahlgren, K. N.; Krafft, G. A.; Ladu, M. J., In vitro characterization of

21

conditions for amyloid-beta peptide oligomerization and fibrillogenesis. J. Biol. Chem. 34


Page 34 of 46

Page 35 of 46 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

Langmuir

1

2003, 278, 11612-11622.

2

(53) Crespo, R.; Villaralvarez, E. M.; Taboada, P.; Rocha, F. A.; Damas, A. M.; Martins,

3

P. M. Insoluble Off-Pathway Aggregates as Crowding Agents During Amyloid Fibril

4

Formation. J. Phys. Chem. B 2017, 121, 2288-2298.

5

(54) Weber, D. K.; Gehman, J. D.; Separovic, F.; Sani, M. A. Copper Modulation of

6

Amyloid Beta 42 Interactions with Model Membranes. Aust. J. Chem. 2012, 65, 472-479.

7

(55) Pellarin, R.; Caflisch, A., Interpreting the Aggregation Kinetics of Amyloid Peptides.

8

J. Mol. Biol. 2006, 360, 882-892.

9

(56) Cao, Z. Q.; Jiang, S. Y. Super-hydrophilic zwitterionic poly(carboxybetaine) and

10

amphiphilic non-ionic poly(ethylene glycol) for stealth nanoparticles. Nano Today 2012,

11

7, 404-413.

12

(57) Lin, W. F.; Ma, G. L.; Wu, J.; Chen, S. F. Different in vitro and in vivo behaviors

13

between Poly(carboxybetaine methacrylate) and poly(sulfobetaine methacrylate).

14

Colloids Surf. B: Biointerfaces. 2016, 146, 888-894.

15

(58) Liu, E. J.; Sinclair, A.; Keefe, A. J.; Nannenga, B. L.; Coyle, B. L.; Baneyx, F.;

16

Jiang, S. Y. EKylation: Addition of an Alternating-Charge Peptide Stabilizes Proteins.

17

Biomacromolecules 2015, 16, 3357-3361.

18

(59) Keefe, A. J.; Jiang, S. Y. Poly(zwitterionic)protein conjugates offer increased

19

stability without sacrificing binding affinity or bioactivity. Nat. Chem. 2011, 4, 59-63.

20

(60) Hallenga, K.; Grigera, J. R.; Berendsen, H. J. C., Influence of hydrophobic solutes

21

on the dynamic behavior of water. J. Phys. Chem. 1980, 84, 2381-2390. 35


Langmuir 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

1

(61) Dahiya, P.; Kamal, R., Hyaluronic Acid: A Boon in Periodontal Therapy. N. Am. J.

2

Med. Sci. 2013, 5, 309-315.

3

36


Page 36 of 46

Page 37 of 46 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

Langmuir

1

Table 1. Properties of Curi@pCB conjugates. Curi@pCB

DS

Solubility a

Size b

Zeta potential b

PDI b

CAC c

(µg/mL)

(nm)

(mV)

(-)

(mol/L)

1

1.97

75.1

190.1 ± 2.9

4.51 ± 0.54

0.539 ± 0.025

6.83×10-8

2

2.56

125.3

141.8 ± 3.2

4.64 ± 0.75

0.473 ± 0.031

5.96×10-8

3

2.92

142.3

122.4 ± 2.7

5.12 ± 0.43

0.464 ± 0.038

3.41×10-8

2

a

Solubility of conjugated curcumin in water.

3

b

Determined with DLS in 100 mM phosphate buffer solution (PBS) (10 mM sodium chloride, pH7.4).

4 5

c

Concentration of Curi@pCB conjugate.

6

37


Langmuir 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

1

Scheme 1. Syntheses of (a) CBMA, (b) pCB and (c) Cur@pCB.

2

38


Page 38 of 46

Page 39 of 46 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

Langmuir

1 2

Figure 1. ThT fluorescence intensity of 25 µM Aβ42 incubated for 48 h without and with

3

different concentrations of curcumin and Cur@pCB conjugates. The ThT fluorescence

4

intensity of Aβ42 only system was set as 100%. In the control experiments with pCB, pCB

5

concentration was 0.97 mg/mL, the same with the content of pCB in Cur1@pCB at 25

6

µM of curcumin.

7

39


Langmuir 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

1

Figure 2. AFM images of Aβ42 aggregates obtained by incubating 25 µM Aβ42 for 48 h

2

without and with different inhibitors. (a) Aβ42 only, (b) 0.97 mg/mL pCB, (c) 25 µM 40


Page 40 of 46

Page 41 of 46 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

Langmuir

1

curcumin + 0.97 mg/mL pCB, (d1) 5 µM curcumin, (d2) 10 µM curcumin, (d3) 25 µM

2

curcumin, (e1) 5 µM Cur3@pCB, (e2) 10 µM Cur3@pCB, (e3) 25 µM Cur3@pCB, (f1)

3

5 µM Cur2@pCB, (f2) 10µmol L-1 Cur2@pCB, (f3) 25 µM Cur2@pCB, (g1) 5 µM

4

Cur1@pCB, (g2) 10 µM Cur1@pCB and (g3) 25 µM Cur1@pCB.

41


Langmuir 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

1 2

Figure 3. Viability of SH-SY5Y cells (5×103/well) incubated with 5 µM Aβ42 aggregates

3

that were obtained by incubating 25 µM Aβ42 aggregates without and with different

4

agents at the concentrations indicated in the figure. The control group was incubated with

5

5 µM Aβ42. pCB concentration in the control group of pCB was 0.97 mg/mL. Curcumin

6

and Curi@pCB concentrations given in (a) and (b) indicate curcumin or curcumin

42


Page 42 of 46

Page 43 of 46 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

Langmuir

1

equivalent concentrations in the incubation of SH-SY5Y cells, while curcumin and

2

Curi@pCB concentrations expressed in (c) to (e) indicate curcumin or curcumin

3

equivalent concentrations in the incubation of 25 µM Aβ42 aggregates with the different

4

inhibitors, which becomes 1/5 of them in the later incubation with SH-SY5Y cells due to

5

a 5-fold dilution. *** p

Self-Assembled CurcuminâPoly(carboxybetaine methacrylate

Recommend Documents

Self-Assembled CurcuminâPoly(carboxybetaine methacrylate