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Protein Enables Conformation Transition of a Hydrogel Based on Pentapeptide and Boosts Immune Response in vivo Yune Zhao, Zhen Wang, Chenyang Mei, Zhengxuan Jiang, Yifan Feng, Rongrong Gao, Qinmei Wang, and Jinhai Huang Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00044 • Publication Date (Web): 10 Apr 2018 Downloaded from http://pubs.acs.org on April 12, 2018
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Bioconjugate Chemistry
Protein Enables Conformation Transition of a Hydrogel Based on Pentapeptide and Boosts Immune Response in vivo Yune Zhaoab*, Zhen Wangc*, Chenyang Meib*, Zhengxuan Jiangd, Yifan Fenge, Rongrong Gaoab, Qinmei Wangab#, Jinhai Huangab# a
School of Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China 325027.
Key Laboratory of Vision Science, Ministry of Health Peoples Republic of China, Wenzhou, Zhejiang, China 325027.
b
Education Ministry Key Laboratory of Laboratory Medicine, Zhejiang Provincial People's Hospital, People's Hospital of Hangzhou medical College, Hangzhou, China 310014.
c
Department of Ophthalmology, The Second Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China 230601. d
e
Department of Ophthalmology, Zhongshan Hospital, Fudan University, Shanghai, China 200032.
Supporting Information Place holder tein structures, it is very difficult to fold into helical conformation as single peptide chain.29-32 Several strategies including constrained peptides33-35 and foldamers36, 37 are developed for inducing α-helix formation of short peptide, which are already applied in drug delivery, anticancer therapy. However, the synthetic procedure and degradation problems hinder further application of these α-helical materials. Recently, mixing macromolecule that originated from nature with peptide can dramatically change the properties of peptidic materials. For example, Ulijn group used cooperative self-assembly of peptides and protein to tune the morphological and mechanical properties of hydrogels formed by aromatic peptide amphiphiles.38 Incorporating clay into small molecule hydrogel can dramatically improve the mechanical properties of resulted hydrogel.39 Combination of surfactants and small molecule gelator can selfassemble orthogonally.40 Stupp group have shown that macroscopic membranes can be generated by mixing peptide amphiphiles and polysaccharide, which further induced differentiation of stem cells.41 However, few of the work have shown the macromolecules can alter the conformation transition of resulted hydrogel.
ABSTRACT: We report a supramolecular hydrogel based on
di-histidine containing pentapeptide serve as a novel vaccine delivery system. Protein encapsulated into the hydrogel not only enhances the mechanical property up to 15 times, but also changes the conformation of resulted nanostructure from β-sheet to α-helix. The resulted hybrid hydrogel enhances antigen uptake and moderately promotes dendritic cell (DC) maturation in vitro. More importantly, the pentapeptide hydrogel promotes antigen-specific antibody production in vivo and splenocytes proliferation ex vivo.
Hydrogel, one kind of soft materials which contains more than 99% of water and less than 1% of gelator,1, 2 has been applied in various fields including drug delivery,3-7 wound healing,8, 9 regenerative medicine,10-12 cell culture,10, 13-15 drug screening,16 controlling cell fate,17-20 analyte detection21, 22 etc.23 Among various candidates for constructing hydrogel, peptide or amino acid derivatives attracted more and more attention in recent years, due to their unique advantages, such as easy preparation and scale up, low toxicity and self-immunity, easy functionality, good degradation etc.24-27 Among these reported studies, the conformation of hydrogels is usually a βsheet structure. Although the advances of these hydrogels have achieved, it is remains difficult to design αhelical hydrogel based on short peptide,28 a major obstacle for the further development of peptidic biomaterials.
Inspired by the mentioned works, we decide to use the naturally derivative protein to change the conformation of hydrogel based on pentapeptide and improve the bioactivity of the protein. Our studies showed that the molecule of 1 (Fig. 1A) in PBS solution can self-assemble to form stable hydrogel. The addition of protein of OVA to the hydrogel of 1 (G1) can alter conformation transition from β-sheet to α-helix and enhance the mechanical property of the resulted hybrid hydrogel (G2). The cooperative interaction of 1 and protein can boost antibodies production in vivo. This work provides an alternative
The α-helix conformation is the major secondary structural motif of protein, and plays crucial role in mediating bimolecular interaction including protein-protein and protein-DNA interactions. Although the peptide sequence of α-helix conformation is stable in whole pro-
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approach to develop supramolecular structures consisting of α-helix structure and functionality. In order to explore the influence of protein on the conformation transition of self-assembly short peptide, we rationally designed hydrogelator 1 (G1) for the following reasons: i) dipeptide FF is a well-established building block for promoting self-assembly both in aqueous solution and biological milieu.42 ii) Nap, the most employed capping group in peptidic materials, provide aromaticaromatic interaction to enhance the self-assembly ability.43 iii) Histidine, an amino acid that is often involved in metal ion coordination in proteins, is hypothesized to decrease the solubility of peptide in weak alkali condition.44 We chose ovalbumin (OVA) as a model protein since this protein is the well explored protein with known structure and function.45
Figure 1. A) Molecular structure of Nap-HHFF (hydrogelator 1, G1) and the proposed illustration of self-assembly (SA) of G1. Optical image represents the formation of hydrogel by G1 in PBS buffer. B) Rheology property and C) TEM image of hydrogel formed by G1 at concentration of 0.5 wt% in PBS buffer. Scale is 500 nm. After obtained 1 by Solid phase peptide synthesis (SPPS) and characterized by NMR (Fig. S1) and LC-MS (Fig. S2), we first test the self-assembly ability of 1 in PBS buffer. As shown in Figure 1A, 1 forms transparent hydrogel at the concentration of 0.5 wt% (denoted as G1) in PBS solution. The critical gelation concentration of 1 is 0.25 wt%, indicating 1 is a good gelator for selfassembly. We next use rheometer to determine the viscoelastic properties of the obtained hydrogel. We use dynamic strain sweep to determine the proper condition for the dynamic frequency sweep of the hydrogel. The result (Fig. S3) shows that the storage modulus (G’) and loss modulus (G’’) of the hydrogel at the concentration of 0.5 wt% exhibited a weak dependence on the strain from 0.1% to 10% (with G’ dominating G’’). We then use the dynamic frequency sweep to study the mechanical property with the strain of 0.5%. As shown in Fig. 1B, the G’ and G’’ slightly changes with the increase of frequency from 0.1 to 100 Hz, the value of G’ is larger than G’’ among the tested frequency, indicating the external force has negligible effect on the viscoelastic property of the hydrogel. Moreover, rheology experiments also indicate the hydrogel formed by 1 is a thixotropic hydrogel (Fig. S4). Hydrogel usually consists of 3D interacted network of nanostructures, which can be visualized by
transmission electron microscopy (TEM). Negative straining TEM shows the hydrogel of G1 consists of uniform flexible nanofibers with several micrometer in length and with the average width of 8 ± 2 nm. The results of rheology and TEM indicated that the hydrogel formed by precursor 1 is a thixotropic hydrogel with nanofibrous networks, which are the prerequisite properties for protein delivery.46, 47 We next test the influence of protein OVA on the gelation property of 1. Encapsulation of OVA into G1 cannot prevent the hydrogel formation of 1, as shown by the optical image of the hydrogel (Fig. S5). Dynamic strain sweep (Fig. 2A) indicates that the hydrogel (denoted as G2) formed by 1 and OVA is sensitive to breaking force. G’ and G’’ of G2 exhibit almost no change among the value from 0.1 to 1% of the strain, however, they decrease quickly when the value of strain increased from 1 to 10%. The cross point of G’ and G’’ is 2% of the strain, indicating higher strain could lead the disruption of the hydrogel. We then set the strain amplitude at 0.5%, the linear response regime of strain amplitude, to study the effect of external force on the hydrogel. As shown in Fig. 2B, both of G’ and G’’ of G2 increased with the increase of frequency from 0.1 to 100 Hz, the value of G’ is about three times larger than the value of G’’, indicating the hydrogel of G2 is tolerant to external force among the tested frequency. The value of G’ of G1 is 20 Pa, while this value is about 300 Pa for G2, which is 15 times larger than G1, indicating encapsulation of OVA into G1 can increase the mechanical property of the hydrogel of G1. These results are consistent with most reported work that macromolecule could influence the self-assembly property of small molecules.48
Figure 2. A) Strain sweep (Arrow indicates the cross point for phase transition of hydrogel) B) Frequency sweep of the hydrogel formed by 1 and OVA. C) TEM image of hydrogel (G2) contains OVA. Scale is 100 nm. D) CD spectrum of G1 and G2. To get insight into how OVA influence the selfassembly property of 1, we first use TEM to explore the internal networks in the hydrogel of G2. As shown in Fig. 2C, the hydrogel of G2 not only consists of flexible
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Bioconjugate Chemistry nanofibers with several micrometre length and 7± 2 nm in width, but also contains spherical structure. We can also observe more aggregated site of the nanofibers in the TEM image, suggesting OVA could lead the nanofibers to form more clustering structures. This result also is consistent with the viscosity of hydrogel, more entangled nanofibers could lead to higher mechanical property of the resulted hydrogel. To understand the arrangement of molecule in the hydrogel, we use circular dichroism (CD) to study the secondary structure of both G1 and G2. OVA itself exhibits a negative peak around 225 nm (Fig. S6), meanwhile, the hydrogel of G1 exhibits positive peak at 190 nm and a negative peak around 208 nm (Figure 2D), indicating the conformation of G1 is βsheet structure. While G2 exhibit two negative peaks of 220 nm and 232 nm, suggesting the major conformation is α-helix like structure, the red shift of the peak may due to the n-π* interaction.49 Our results may provide an alternative strategy for inducing α-helix formation of short peptide.
cells via endocytosis. After 4 h incubation, free OVAFITC is still co-localized with lysosome (Fig.S8). On the other hand, some of green fluorescence in G2 group does not co-localize with red signals, indicating OVA can escape from late endosome/lysosome into cytosol. Furthermore, we also investigate the effect of G1 on DC maturation using flow cytometry. The result shows that G1 have no effect on expression of CD40 and CD80 (Fig. S9), but moderately enhance the production of IL-6 and IFN-γ (Fig. 3c and D), indicating that G1 slightly induce DC maturation. Above all, the ability of G1 on promoting antigen escapes from lysosome into cytosol and DC maturation may play a role in inducing adaptive immunity.
Improving the bioavailability and activity of protein is the key issue in the current protein drug design.50-52 Since the protein of OVA is a model protein for exploring the immunotherapy, several groups also reported that small molecule hydrogel could enhance the antibody production of HIV-DNA,53 proteins54 as well as peptidic antigens.55 To explore the application of hydrogel, we also test its bioactivity in vitro. We first study the cytotoxicity of G1 on Raw 264.7 cells and L929 cells. As shown in Fig. S7, both two cells show a near 100% cell viability even when the concentration is up to 320 µg/ml after the treatment of G1 for 48 h, indicating G1 has good biocompatibility, which may be used as a vaccine delivery system. As we known, the antigen uptake by antigen presentation cells (APC) such as dendritic cells or macrophages, is the primary step of vaccine-induced adaptive immune response .56 Next, we investigate the effect of G2 on cellular uptake of protein (OVA-FITC) by flow cytometry and confocal laser scanning microscopy (CLSM). As shown in Figure 3B, comparing with free OVA, G2 dramatically enhances OVA uptake by bone marrow dendritic cells (BMDCs) about 2.1-fold, indicating its strong ability of promoting antigen uptake. CLSM also confirms that (Fig. 3A and S8) G2 group shows stronger green fluorescence intensity than that of free OVA-FITC group at 2 h time point. The enhancement of antigen uptake by the hydrogel may be caused by the hybrid nanostructure formed by 1 and protein. As we known, the nanostructures can be easily uptake by cells, especially DCs.57 After uptake by APCs, antigen is then digested in APC, and presented to activate different T cells through different major histocompatibility complex (MHC) molecules, which is a crucial step for APCprimed T cell activation.56 In this process, the location of antigen in cellular compartment of APCs is very important for the downstream immune responses.58 Therefore, we investigate the effect of G2 on antigen location in APCs. As shown in Fig. 3A, most of the green fluorescent dots (OVA-FITC) co-localize with the red dots (Lyso-Tracker) at 2 h time point in both G2 and free OVA-FITC groups, suggesting the uptake of OVA by DC
Figure 3. A) CLSM images of BMDC cells which were treated with OVA-FITC encapsulated in Nap-HHFF (G2) at 37 °C for 2 h or 4 h, and then strained with Lyso-Tracker. Magnification: 63×. B) BMDCs incubate with G2 or free OVA-FITC (2 µg/ml) at 37 °C for 0.5 h, and then analysed using flow cytometry. The percentage of antigen uptake was determined by OVA-FITC positive cells. C) BMDCs incubate with Medium (Med), G1 (80 µg/ml) and LPS (500 ng/ml) for 24 h. The cytokines (IL-6 and IFN-γ) in the supernatant were detected using ELISA kit. Bars shown are mean ± SEM, and differences among groups are determined using T-test (B) or one-way ANOVA analysis (C and D). The asterisk indicates that difference between OVA (Med) group and other group. *: p
