Reactive Conjugated Polymers for the Modulation of Islet Amyloid

Research Article Cite This: ACS Appl. Mater. Interfaces 2019, 11, 22973−22978

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Reactive Conjugated Polymers for the Modulation of Islet Amyloid Polypeptide Assembly Han Sun,†,‡ Fengting Lv,†,‡ Libing Liu,*,†,‡ and Shu Wang*,†,‡ †

Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100910, P. R. China ‡ College of Chemistry, University of Chinese Academy of Sciences, Beijing 100049, P. R. China

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S Supporting Information *

ABSTRACT: Misfolding and abnormal assembly of proteins cause many intractable diseases. The modulation of the assembly process of these proteins could contribute to understanding and controlling amyloid protein aggregation. Previous works focused mainly on the inhibition of the assembly process. To broaden the interaction modality of modulators with proteins for developing new modulators, in this work, we designed and synthesized two reactive poly (pphenylene vinylene) polymers, respectively, functionalized with N-hydroxysuccinimide ester (PPV-NHS) and pentafluorophenol ester (PPV-PFP), which exhibited the prevention or co-assembly effect on the aggregation process of islet amyloid polypeptide (IAPP). Cell assays demonstrated that both of the two polymers could effectively eliminate the cytotoxicity of IAPP. Moreover, PPV-NHS also could irreversibly disrupt preformed IAPP fibrils. We envision that PPV-NHS and PPV-PFP might offer a new design method for the modulation of protein assembly. KEYWORDS: reactive conjugated polymers, islet amyloid polypeptide, assembly, disaggregation, detoxification

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chains, which have been used for detecting amyloid fibrils both in vivo and in vitro.14,15 Moreover, CPs showed the capability of inhibiting aberrant protein assembly.16 The complexes of CPs with amyloid proteins were also used as new functional materials.17,18 Although the interactions between amyloids have been established, the modulation of protein assembly and the role of side chains in this process have not been systematically studied. Herein, we investigated the modulation of islet amyloid polypeptide (IAPP) assembly by two reactive poly (p-phenylene vinylene) polymers (PPVs). IAPP is an important hormone for glucose homeostasis, but the aggregation of IAPP in diabetes contributes to the dysfunction and death of β-cells.19,20 Thus, rational design of molecules for inhibiting the assembly process and destroying the preformed aggregation to reduce the cytotoxicity of islet amyloid is significant.

INTRODUCTION Protein assembly plays an important role in living organisms. The assembly of tubulin maintains cell morphology and intracellular material transport. Besides, collagen is the main organic component of skin and bones. However, in some circumstances, proteins can assemble into undesirable aggregations called amyloidosis.1 These abnormal assemblies lose their original functions and cause damage to normal cells, which are considered as characteristic markers for many diseases, such as Alzheimer’s disease, Parkinson’s disease, and type-2 diabetes. Developing proper modulators to regulate the disease-associated protein assembly process to depress the cytotoxicity of amyloid aggregates is a promising way to treat these diseases.2 In recent years, inhibitors for slowing down the formation of protein aggregation were widely investigated, ranging from natural and synthetic small molecules,3,4 nanoparticles,5,6 graphene quantum dots,7 peptides,8 antibodies9,10 to artificial chaperones.11,12 These studies offer tools to further understand the aggregation mechanism of amyloid proteins and help in exploring the molecular etiology of associated diseases. In addition to inhibiting the assembly process, co-assembling with amyloid proteins into nontoxic aggregations has emerged as another modality for controlling protein assembly,13 which provides a new approach to develop modulators, although it is less investigated. Conjugated polymers (CPs) are characterized by a hydrophobic large π-conjugated backbone and modifiable side © 2019 American Chemical Society

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RESULTS AND DISCUSSION IAPP is a positively charged protein with 37 amino acids, in which one lysine residue and an N-terminal amino group can react with electrophiles. We designed and synthesized two negatively charged PPVs functionalized with N-hydroxysuccinimide ester (PPV-NHS) and pentafluorophenol ester (PPVReceived: March 24, 2019 Accepted: June 5, 2019 Published: June 5, 2019 22973

DOI: 10.1021/acsami.9b05247 ACS Appl. Mater. Interfaces 2019, 11, 22973−22978

Research Article

ACS Applied Materials & Interfaces

Scheme 1. (a) Illustration of IAPP Aggregation without and with Inhibitor PPV-NHS and Co-assembly Molecule PPV-PFP. (b) Synthetic Route of PPV-NHS and PPV-PFP

Figure 1. (a) Tricine−SDS−PAGE of IAPP (40 μM) after incubation with PPV-NHS (0, 40, 200, 400, and 800 μM) for 30 min at 37 °C. (b) Hydrodynamic diameters of PPV-NHS (10 μM) with and without IAPP (10 μM). (c) ζ-Potentials of PPV-NHS (10 μM) with and without IAPP (10 μM). (d) Tricine−SDS−PAGE of IAPP (40 μM) after incubation with PPV-PFP (0, 40, 200, 400, and 800 μM) for 30 min at 37 °C. (e) Hydrodynamic diameters of PPV-PFP (10 μM) in the absence and presence of IAPP (10 μM). (f) ζ-Potentials of PPV-PFP (10 μM) with and without IAPP (10 μM).

PFP), respectively. PPV-NHS and PPV-PFP bound to IAPP through electrostatic interactions and the active ester in polymer side chains reacted with amino groups of IAPP, forming a robust covalent bond, which could affect the aggregation of IAPP. The secondary structure conversion and

assembly of IAPP in the absence and presence of PPVs were investigated with circular dichroism (CD) spectroscopy and transmission electron microscopy (TEM). The results indicated that PPV-NHS and PPV-PFP showed different modulations of IAPP (Scheme 1a). PPV-NHS reacted with 22974


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band at around 216 nm, which indicated the formation of a βsheet structure (Figure 2a). In contrast, IAPP monomers

IAPP and subsequently prevented the conformational transition and polymerization of IAPP. While IAPP was treated with PPV-PFP, IAPP co-assembled with PPV-PFP into fibrils. Both of these two modalities exhibited decreased cytotoxicity of IAPP aggregations in living cells. The synthesis route of PPV-NHS and PPV-PFP is outlined in Scheme 1b. PPV-OH was synthesized according to the literature.21 PPV-OH reacted with succinic anhydride through nucleophilic substitution to afford PPV-COOH. Then, PPVNHS and PPV-PFP were obtained through the ester condensation reaction of PPV-COOH with N-hydroxysuccinimide and pentafluorophenol. The substitution degrees of NHS and PFP are 95 and 92% according to the NMR data, respectively. The weight-average molecular weights (Mw) of PPV-NHS and PPV-PFP are 31 980 and 32 030 with polydispersity indexes of 1.4 and 1.6, respectively, by gel permeation chromatography analysis. The absorption and fluorescence spectra of PPV-NHS and PPV-PFP are displayed in Figure S1. The critical micelle concentrations (cmc) of PPVNHS and PPV-PFP were measured by dynamic light scattering (DLS). The intensity of the scattered light increases due to the presence of micelles. Figure S2 is the plot of the intensity of the scattered light as a function of the PPV concentration. The intersection between the two lines at 1.25 μM concentration corresponds to the cmc of PPV-NHS. The cmc of PPV-PFP is 0.18 μM, which is much lower than that of PPV-NHS, indicating that PPV-PFP is more likely to assemble in solution. To investigate the interactions between PPVs and IAPP, tricine−sodium dodecyl sulfate (SDS)−polyacrylamide gel electrophoresis (PAGE) was performed to detect the extent of covalent reaction. IAPP was treated with PPVs at 37 °C for 30 min at ratios of 1:1, 1:5, 1:10, 1:20 (molar concentration of the repeating unit for polymers). As shown in Figure 1a,d, the band of IAPP became weaker in the presence of equivalent polymers, which indicated the conversion of the monomer IAPP to IAPP-PPVs conjugations. As the concentration of PPVs increased, the IAPP almost totally reacted with the polymer. The dynamic light scattering (DLS) analyses measured the hydrodynamic diameter changes of PPVs before and after reacting with IAPP. As expected, the size of PPVs became larger because of the formation of IAPP-PPV covalent conjugations (Figure 1b,e). ζ-Potential experiments were also performed to study the interactions between IAPP and PPVs (Figure 1c,f). Both PPV-NHS and PPV-PFP were negatively charged with ζ potential values of −20.2 ± 1.7 and −21.6 ± 0.4 mV, respectively. After treatment with IAPP, the surface charges of PPV-NHS and PPV-PFP shifted to −7.1 ± 0.1 and −11.4 ± 0.5 mV, respectively, suggesting the electrostatic interactions of PPVs with IAPP. According to the above results, we concluded that PPV-NHS and PPV-PFP could attach to IAPP by synergistic covalent interactions and electrostatic interactions. When the concentrations of PPVNHS and PPV-PFP were five times that of IAPP, IAPP reacted completely and converted into IAPP-PPV conjugates. Therefore, 5-fold excess PPVs would be used for the following study. We next investigated the effect of PPVs on IAPP aggregation. PPV-COOH, the precursor of reactive PPVs without active ester groups, was used as a negative control to study the function of covalent reactions in modulating IAPP assembly. Circular dichroism (CD) spectroscopy and Nile red assay were performed to measure the secondary structure conversion and aggregation of IAPP. After incubation of IAPP monomers for 12 h, the CD spectrum exhibited a negative

Figure 2. (a) CD spectra of IAPP (40 μM) in the absence and presence of PPV-NHS (200 μM), PPV-PFP (200 μM), and PPVCOOH (200 μM) after incubation for 12 h. (b) Fluorescence intensity of Nile red (10 μM) after incubation of IAPP (40 μM) with and without PPV-NHS (200 μM), PPV-PFP (200 μM), and PPVCOOH (200 μM) for 0 and 12 h. The excitation wavelength is 580 nm, and the emission wavelength is 640 nm. (c) TEM images of IAPP (40 μM) in the absence and presence of PPV-NHS (200 μM), PPVPFP (200 μM), PPV-COOH (200 μM) after incubation for 12 h. The scale bar is 200 nm.

treated with PPV-NHS showed two negative bands at around 208 and 222 nm, which are typically an α-helical conformation. While IAPP in the presence of PPV-PFP and PPV-COOH displayed β-sheet structures with the negative band at around 216 nm, PPV-NHS and PPV-PFP alone showed a very weak CD signal, which could not influence the signal of IAPP (Figure S3). These results suggested that PPV-NHS could inhibit the structure conversion of IAPP from α-helix to βsheet; nevertheless, PPV-PFP and PPV-COOH did not show the inhibition effect. For further investigating the influence of PPVs on IAPP assembly, Nile red was used to detect the formation of IAPP aggregations. As shown in Figure 2b, IAPP, IAPP treated with PPV-PFP, and IAPP treated with PPVCOOH showed obviously enhanced fluorescence intensity of Nile red after incubation for 12 h, which indicated the generation of IAPP aggregations. As expected, the fluorescence intensity of Nile red nearly did not change when IAPP was incubated with PPV-NHS, demonstrating that PPV-NHS could inhibit the assembly of IAPP, which was consistent with the CD results. TEM images were taken to directly visualize the morphology of IAPP aggregation. IAPP and IAPP treated with PPV-COOH assembled into similar fibrils after incubation (Figure 2c). IAPP treated with PPV-NHS did not show fibrils in TEM, further verifying the inhibition effect of PPV-NHS. IAPP treated with PPV-PFP formed fibrils that 22975


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then added to the preformed IAPP aggregations, and the morphology changes of IAPP fibrils were observed under TEM. As shown in Figure 4, the number of fibrils decreased

were much longer than those of IAPP, which indicated that PPV-PFP may co-assemble with IAPP to form new aggregations. PPV-NHS and PPV-PFP alone could not assemble into fibrils after incubation (Figure S4). PPVCOOH without a reactive group has a little influence on IAPP assembly, suggesting that the covalent interactions played predominant roles in the binding of PPVs with IAPP. With the above results together, we can conclude that PPVNHS and PPV-PFP displayed different modulation modalities on IAPP assembly. The NHS side chains of PPV-NHS reacted with IAPP, stabilizing the conformation of IAPP in α-helix conformation; the assembly of IAPP was disturbed because of the steric hindrance caused by PPV-NHS. However, when IAPP was incubated with PPV-PFP, IAPP exhibited a β-sheet structure and they co-assembled into new fibrils. This might be because that except for reacting with IAPP, excess planar pentafluorophenyl could stack with other through π−π interactions, and the unique hydrophobicity and lipophilicity of the fluorine atoms offered strong interactions in polymers, as well as polymers and proteins.22 These factors are in favor of further assembling into hierarchical structures after formation of the polymer−IAPP conjugates, leading to co-aggregation of PPV-PFP with IAPP together into new fibrils. To examine the rescue effect of PPVs from IAPP-induced toxicity, INS-1 cells were used as model cells. The biocompatibilities of PPV-NHS, PPV-PFP, and PPV-COOH were first evaluated. As shown in Figure 3a, PPVs incubated

Figure 4. TEM of preformed IAPP fibrils treated with PPV-NHS for 0, 2, 4, and 72 h. The scan bar is 200 nm.

with the extension of time. After treatment with PPV-NHS for 2 h, the fibrils decreased obviously, and just a few short fibrils could be observed after incubation for 4 h. The fibrils did not reform as long as 3 d incubation. These data suggested that PPV-NHS was capable of disaggregating mature IAPP fibrils, which could be further used as effective covalent agents of disassembly in pathological tissues.

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CONCLUSIONS In summary, two conjugated polymers PPV-NHS and PPVPFP were employed to modulate IAPP assembly. PPV-NHS and PPV-PFP exhibited inhibition and promotion effects, respectively, on the assembly process. Both of the two modalities could rescue INS-1 cells from IAPP-induced cytotoxicity. The co-assembly strategy breaks through the traditional design ideas, offering new views for controlling protein aggregations. Moreover, PPV-NHS also displayed the capability to disrupt preformed IAPP fibrils, which provides possibilities to eliminate amyloidosis through covalent breakers.

Figure 3. (a) Cell viability of INS-1 cells after incubation with PPVNHS, PPV-PFP, and PPV-COOH. (b) Inhibition effect of PPV-NHS, PPV-PFP, and PPV-COOH on the cytotoxicity of IAPP. [IAPP] = 5 μM, [PPV-NHS] = 25 μM, [PPV-PFP] = 25 μM, and [PPV-COOH] = 25 μM.

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with INS-1 cells at the concentrations ranging from 1.56 to 100 μM showed good cell viability. When INS-1 cells were treated with IAPP preincubated for 12 h, the cell viability of INS-1 was about 65%. As expected, PPV-COOH showed a little influence on cytotoxicity because of the weak interactions with IAPP. In contrast, for IAPP in the presence of PPV-NHS or PPV-PFP, the cell viability increased to about 90% (Figure 3b), which indicated that both the inhibition and co-assembly effect could attenuate IAPP-induced cytotoxicity. Disassembling protein aggregation was still a big challenge because the strong intermolecular forces among proteins were difficult to break, and the disassembled proteins may reaggregate again as the time extends.5,23 Since it has been confirmed that the excellent inhibition effect of PPV-NHS was mainly based on the irreversible covalent interactions between PPV-NHS and IAPP, we wondered whether PPV-NHS could disrupt preformed IAPP aggregations. To test this, IAPP was first incubated for 24 h to obtain mature fibrils. PPV-NHS was

EXPERIMENTAL SECTION

Materials. All organic solvents were purchased from Beijing Chemical Works and used without further purification. Chemicals were purchased from Sigma-Aldrich Chemical Company and used as received. IAPP was purchased from ChinaPeptides Co., Ltd. (Suzhou, China). Chemicals for tricine−SDS−PAGE were purchased from Solarbio. Rat insulinoma INS-1 cells were obtained from the cell culture center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). Modified RPMI 1640 (RP 1640) was purchased from HyClone/ThermoFisher (Beijing, China). Fetal bovine serum was purchased from Sijiqing Biological Engineering Materials (Hangzhou, China). Penicillin and streptomycin were purchased from Life Technologies (Carlsbad). 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and nitrocellulose membranes were purchased from Xinjingke Biotechnology Co., Ltd (Beijing, China). Deionized water (18.2 MΩ·cm) was acquired from a Milli-Q system (Millipore, Bedford, MA). Measurements. The 1H NMR spectra were recorded on Bruker Avance 400 MHz spectrometers. The fluorescence intensity of Nile red was measured on a Varioskan Flash (Thermo Scientific 22976


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ACS Applied Materials & Interfaces Company) at a wavelength of 640 nm, and the excitation wavelength was 580 nm. The absorbance for MTT analysis was recorded on a microplate reader (BIO-TEK Synergy HT) at a wavelength of 570 nm. Cell counting was measured on an automated cell counter (Countess, Invitrogen). CD spectra were recorded on a Hitachi JascoJ815 circular dichroism spectrophotometer. TEM images were observed on a Hitachi S-7700 transmission electron microscope. Synthesis of PPV-COOH. Succinic anhydride (20 mg, 0.2 mmol) was added to the solution of PPV-OH (15 mg, 0.01 mmol) in 4 mL of anhydrous dichloromethane and stirred at 0 °C for 1 h and then at room temperature for 24 h under argon. The resultant reaction mixture was diluted by dichloromethane and washed with a brine solution three times. The collected organic layer was dried with anhydrous Na2SO4, concentrated, and precipitated into ethyl ether to afford a red oil liquid (7.5 mg, 44%). 1H NMR (300 MHz, CDCl3, δ): 7.43 (br, 2H), 7.17 (br, 2H), 4.23−4.12 (br, 5H), 3.93 (br, 4H), 3.75−3.50 (br, 36H), 3.34 (s, 3H), 2.62 (s, 4H). Synthesis of PPV-NHS. To the solution of PPV-COOH (20 mg, 0.013 mmol) in 4 mL of anhydrous dichloromethane were added Nhydroxysuccinimide (NHS) (4.5 mg, 0.039 mmol) and 1-ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride (EDCI) (6.32 mg, 0.033 mmol) under 0 °C. The resulting solution was further stirred at room temperature for 24 h. Then, the mixture was diluted by dichloromethane and washed with water three times. The collected organic layer was washed with brine solution, dried with anhydrous Na2SO4, and concentrated to give the desired red oil liquid (18 mg, 80%). 1H NMR (300 MHz, CDCl3, δ): 7.42 (br, 2H), 7.15 (br, 2H), 4.25−4.12 (br, 5H), 3.91 (br, 4H), 3.73−3.49 (br, 36H), 3.34 (s, 3H), 2.93 (t, 2H), 2.85 (s, 4H), 2.75 (t, 2H). Synthesis of PPV-PFP. To the solution of PPV-COOH (20 mg, 0.013 mmol) in 4 mL of anhydrous dichloromethane were added pentafluorophenol (PFP) (7.0 mg, 0.039 mmol) and 1-ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride (EDCI) (6.32 mg, 0.033 mmol) under 0 °C. A catalytic amount of 4-dimethylaminopyridine was added. The resulting solution was further stirred at room temperature for 24 h. Then, the mixture was diluted by dichloromethane and washed with water three times. The collected organic layer was washed with brine solution, dried with anhydrous Na2SO4, and concentrated to give the desired orange oil liquid (16 mg, 67%). 1 H NMR (300 MHz, CDCl3, δ): 7.42 (br, 2H), 7.15 (br, 2H), 4.28− 4.11 (br, 5H), 3.92 (br, 4H), 3.74−3.49 (br, 32H), 3.33 (s, 3H), 2.98 (t, 1.8H), 2.78 (t, 1.9H), 2.59 (m, 0.4H) Preparation of Peptides. IAPP was prepared according to the literature.24 Briefly, the lyophilized peptides were dissolved in 1,1,1,3,3,3-hexafluoroisopropanol (HFIP). After bath-sonication for 10 min, the samples were incubated for 2 h with shaking at 4 °C. Then, the resultant samples were divided into aliquots. IAPP was stored at −20 °C after HFIP was evaporated. Tricine−SDS−PAGE. IAPP (40 μM) was incubated with PPVNHS and PPV-PFP at molar concentration ratios of 1:1, 1:5, 1:10, and 1:20 in 10 mM phosphate-buffered saline at 37 °C for 30 min. The resultant samples were denatured in 2X Tris−tricine−SDS− PAGE loading buffer at 90 °C for 10 min. After cooling to room temperature, the samples were separated in a vertical tricine−SDSPAGE system with 16.5% separating, 10% interlayer, and 4% stacking layers and stained with Coomassie brilliant blue. Circular Dichroism (CD) Spectroscopy Measurement. HFIPtreated IAPP films (140 μg) were dissolved in 99.9% pure DMSO (50 μL). The above IAPP solution was diluted by phosphate buffer (PB) (10 mM, 950 μL) to a final concentration of 40 μM. PPV-NHS, PPVPFP, and PPV-COOH were incubated with IAPP at molar ratios of 5:1 for 30 min at 37 °C. The mixtures were dialyzed at 37 °C for 12 h to remove DMSO in a solution of 10 mM NaOH (10% of the final volume), 10 mM PB (90% of the final volume), and 200 mM NaCl. The samples (150 μL) were added to a 0.1 cm quartz cell for far-UV (190−260 nm) measurements. The bandwidth was 2 nm. The scanning speed was 100 nm/min with a response time of 4 s. Each spectrum was an average of three scans. Transmission Electron Microscopy (TEM) Measurement. The samples (5 μL) were dropped on the carbon-coated copper

grid and adsorbed for 20 min. Then, redundant samples were removed with a filter paper, and remanent samples were stained with uranyl acetate (5 μL of 3%) for 2 min. The samples were then observed using TEM. Cell Assays. Rat insulinoma INS-1 cells were cultured in RP 1640 medium containing 10% fetal bovine serum, 1% penicillin/ streptomycin, and 2% INS-1 stock solution [500 mM N-(2hydroxyethyl)piperazine-N′-ethanesulfonic acid, 100 mM L-glutamine, 100 mM sodium pyruvate, and 2.5 mM β-mercaptoethanol] under 5% CO2 at 37 °C. The cells were seeded at a density of 5000 cells per well. IAPP with and without PPV-NHS, PPV-PFP, and PPVCOOH were incubated for 12 h at 37 °C prior to the addition to cells. The samples were diluted with RP 1640 medium and incubated with cells for 48 h. The mixture in each well was replaced with MTT (1 mg/mL in medium, 100 μL/well) and incubated for 4 h, Then, the supernatant was replaced with 100 μL of DMSO per well, and the plates were shaken for 10 min. Absorbance values of formazan were read with a microplate reader at 570 nm. Wells with neither PPVs nor IAPP were served as a control group. Disaggregation of Preformed IAPP Fibrils. IAPP monomers (40 μM) were incubated in 10 mM NaOH (10% of the final volume), 10 mM PB (90% of the final volume), and 200 mM NaCl under agitation at 37 °C for 24 h to grow mature IAPP fibrils. Five-fold excess PPV-NHS was added to aged IAPP fibrils and incubated for 0, 2, 4, and 72 h. The morphology changes of IAPP were detected by TEM.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.9b05247.

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Materials; measurements; synthesis and characterization of polymers; preparation of IAPP; tricine−SDS−PAGE; CD; TEM; cell assays; and disaggregation assays (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (L.L.). *E-mail: [email protected] (S.W.). ORCID

Han Sun: 0000-0001-5519-6846 Fengting Lv: 0000-0003-3748-3096 Libing Liu: 0000-0003-4827-6009 Shu Wang: 0000-0001-8781-2535 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to the National Natural Science Foundation of China (Nos. 21533012 and 21661132006) and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16020804).

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REFERENCES

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Reactive Conjugated Polymers for the Modulation of Islet Amyloid

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