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Graphene Oxides Decorated with Carnosine as An Adjuvant to Modulate Innate Immune and Improve Adaptive Immunity in Vivo Chunchun Meng, Xiao Zhi, Chao Li, Chuanfeng Li, Zongyan Chen, Xusheng Qiu, Chan Ding, Lijun Ma, Hongmin Lu, Di Chen, Guangqing Liu, and Daxiang Cui ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.5b06750 • Publication Date (Web): 14 Jan 2016 Downloaded from http://pubs.acs.org on January 18, 2016
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Graphene Oxides Decorated with Carnosine as An Adjuvant to Modulate Innate Immune and Improve Adaptive Immunity in Vivo
Chunchun Meng1⊥, Xiao Zhi2⊥, Chao Li2, Chuanfeng Li1, Zongyan Chen1, Xusheng Qiu1, Chan Ding1,3, Lijun Ma4, Hongmin Lu5, Di Chen2, Guangqing Liu1,3*, Daxiang Cui2*
1
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 518 Ziyue Road,
Shanghai 200241, P. R. China 2
Institute of Nano Biomedicine and Engineering, Key Laboratory of Thin Film and Microfabrication
Technology of Ministry of Education, Department of Instrument Science and Engineering, School of Electronic Information and Electrical Engineering, National Center for Translational Medicine, Collaborative Innovational Center for System Biology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, P. R. China 3
Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and
Zoonoses, 48 Wenhui Road, Yangzhou 225009, P. R. China 4
Department of Oncology, Shanghai Tongren Hospital, Shanghai Jiao Tong University School of
Medicine, 1111 Xianxia Road, Shanghai 200336, P. R. China 5
Department of Oncology, Shanghai Renji Hospital, Shanghai Jiao Tong University School of Medicine,
160 Pujian Road, Shanghai 200127, P. R. China ⊥
equal contribution to this paper
Address correspondence to
[email protected] or
[email protected] ABSTRACT Current studies have revealed the immune effects of graphene oxide (GO) and have utilized them as vaccine carriers and adjuvants. However, GO easily induces strong oxidative stress and inflammatory reaction at the site of injection. It is very necessary to develop an alternative adjuvant based on graphene oxide derivatives for improving
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immune responses and decreasing side effects. Carnosine (Car) is an outstanding and safe antioxidant. Herein, the feasibility and efficiency of ultra-small graphene oxide decorated with carnosine as an alternative immune adjuvant were explored. OVA@GO-Car was prepared by simply mixing ovalbumin (OVA, a model antigen) with ultra-small GO covalently modified with carnosine (GO-Car). We investigated the immunological properties of the GO-Car adjuvant in model mice. Results show that OVA@GO-Car can promote robust and durable OVA specific antibody response, increase lymphocyte proliferation efficiency and enhance CD4+ T and CD8+ T cells activation. The presence of Car in GO also probably contribute to enhancing the antigen-specific adaptive immune response through modulating the expression of some cytokines, including IL-6, CXCL1, CCL2 and CSF3. In addition, the safety of GO-Car as an adjuvant was evaluated comprehensively. No symptoms such as allergic response, inflammatory redness swelling, raised surface temperatures, physiological anomalies of blood and remarkable weight changes were observed. Besides, after modification with carnosine, histological damages caused by GO-Car in lung, muscle, kidney and spleen became weaken significantly. This study sufficiently suggest that GO-Car as a safe adjuvant can effectively enhance humoral and innate immune responses against antigens in vivo. KEYWORDS: graphene oxide· carnosine· adjuvant· immunoenhancement
As anoxidized derivative of two-dimensional graphene, graphene oxide (GO) sheets have chemically functional groups, such as carboxylic acid, groups at their edges(according to the widely accepted Lerf–Klinowski model), and epoxy and hydroxyl groups on the basal planes [1]. Because of its large surface area, excellent adsorbing ability and physiological biocompatibility due to abundant hydrophilic groups [1], it has attracted substantial attention in biomedical applications as delivery carriers of drug [1, 2], nucleic acid [3, 4] and protein [5, 6].Surprisingly, several studies have revealed immune enhancement effects of GO in recent years [7-9]. However, most of the studies are constrained within experiments in vitro, and little is
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known about comprehensive evaluation of immunological effects of GO in vivo. In particular, when GO was used for an immunopotentiator, the side effects of GO, oxidative stress and inflammatory reaction [10, 11], have always not been taken care of seriously [12, 13]. The oxidative stress in cells is caused by the generation of reactive oxygen species (ROS). Normally, cells require adequate levels of antioxidant defenses in order to avoid the harmful effect of an excessive production of ROS and to prevent damage to themselves. And antioxidant could inhibit the oxidative stress. Carnosine, the dipeptide β-alanyl-l-histidine, was discovered in 1900 by Gulewitsch and Amiradzbi [14]. This dipeptideis present at rather high concentrations in skeletal muscle and in the olfactory bulb of mammals [15, 16]. Carnosine possesses numerous biological roles including pH-buffering capacity in muscle tissue at physiological pH [17], regulation of enzyme activity [18], protection of proteins from glycation [19], antioxidant activity [20, 21] and antineoplastic effect [22]. L-Histidine, and more specifically its imidazole moiety, appears to be the prime bioactive component. Our previous investigation demonstrated that GO modified by biocompatible molecules possesses good immunological biocompatibility and immunoenhancement effects in vitro, and is likely to be an available candidate of immunoadjuvant in the future [23]. Herein, we report the preparation of graphene oxide (GO) sheets with lateral dimensions less than 50 nm. And we imparted distinctive immune enhancement to the GO by covalently grafting L-carnosines onto the chemically activated edge rich in carboxyl groups. RESULTS AND DISCUSSION
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Figure 1. (A) Photographs of aqueous dispersions of GO before and after modification with Car, (B) UV-vis spectra of carnosine, GO and GO-Car, (C) Infrared spectra of GO and GO-Car powers, (D) High-resolution transmission electron microscopy (HR-TEM) image of monolayer GO sheets, (E) Histogram of diameters from 200 GO platelets. The mean width is 18.2±3.2 nm.
Characterization of GO and GO-Carnosine (GO-Car). GO used in these studies was synthesized using modified Hummers method and our previous reports [24-27]. As shown in Figure 1A, the aqueous dispersion of GO sheets was brown, and it became black after functional modification with carnosine. The UV-vis spectra of carnosine solution (Figure 1B, black line) shows a strong absorption peak at 220 nm [28]. Before modification, the UV-vis spectra of GO aqueous dispersion (Figure 1B, blue line) shows a strong absorption peak at 230 nm (π-π* transitions of C=C) and a weak shoulder peak at 300nm (n-π* transitions of C=O) [29, 30]. After modification with carnosine (Figure 1B, red line), the strong absorption peak blue-shifted to 215 nm, the 15 nm blue shift owing to the change of the GO electronic ground state induced by carnosine conjugation, and a new absorption peak showed up at 240 nm (imidazole ring of carnosine). But, the shoulder peak at 300 nm did not change
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significantly. Fourier-transform infrared spectroscopy (FT-IR) spectra were recorded using a pressed disc of powder combined with KBr. As shown in Figure 1C, the intensities of the FT-IR peaks of GO corresponding to the oxygen functionalities, such as the stretching and deformation vibration peak of O-H at 3440 cm-1 and 1410 cm-1 respectively, the C=O carbonyl stretching vibration peak at 1720 cm-1,the vibration peaks of C=C and the adsorbed water molecules at 1632 cm-1, the in-plane OH bending vibration peak of carboxylic group at 1385 cm-1,the stretching vibration peak of C-OH at 1226 cm-1, and the C-O stretching vibration peak at 1052 cm-1 [31-34]. After modification of GO with carnosine, the hydroxyl vibration peaks at 1410 cm-1 and 1226 cm-1 decreased dramatically, intimating the reduction of hydroxyl of GO by carnosine. In addition, some new peaks shown up, such as the CHN deformation vibration (Amide II vibration) peak at 1560 cm-1 and C-N stretching vibration peak (Amide III vibration) at 1265 cm-1 [35]. Figures 1D shows a high-resolution transmission electron microscopy (HR-TEM) image of monolayer GO sheets. The mean width of GO sheets is 18.2±3.2 nm (Figure 1E).
Figure 2. Tapping mode AFM images of GO (A) and GO-Car (B) sheets obtains at RH of 60%.
Figure 2 depicts the typical atomic force microscopy (AFM) images of the GO (Figure 2A) and GO-Car (Figure 2B) sheets we obtained. The thickness, measured from the height profile of the AFM image, Figure 2B, is about ～1.2 nm, which is consistent with the data reported in the literature, indicating that the formation of the
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single layered GO [36].The thickness measured of monolayer GO-Car is about ～1.6 nm. The thickness difference, about 0.4 nm, should correlate with the average size of a carnosine layer of noncovalent adsorption on the GO basal planes.
Figure 3. (A) The C1s XPS spectra of GO, (B) The C1s XPS spectra of GO-Car, (C) Elemental analyses of GO and GO-Car by XPS, (D) 532 nm excited Raman spectra of GO and GO-Car.
Figure 3(A, B) shows the C1s XPS spectra of GO before and after the functional modification with carnosine. Before modification (Figure 3A), The C1s component of GO has been deconvolved four different peaks centered at 284.8 eV (C-C in aromatic rings), 286.7 eV (C-O), 287.6 eV (C=O) and 288.6 eV (O-C=O). After the modification with carnosine (Figure 3B), two new peaks of C–N (amine) and N-C=O (amide in imidazole ring of carnosine) showed up at 285.8 eV and 289.2 eV respectively, revealing that carnosines were modified on the GO sheets [37, 38]. Theoretically, the C/O atomic ratio will increase as a result of the de-oxygenation of the functional groups of GO. Indeed, there is a significant change of the C/O atomic ratios from 2.21 of GO to 2.88 of GO-Car (Figure 3C), which suggests that carnosine is able to deoxidize GO partly. In Figure 3D, two prominent peaks of GO appear at around 1358 and 1595 cm-1,
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which are attributed to D and G band, respectively [39, 40]. After modification with carnosine, the D and G peaks still exist but with a higher frequency D peak (1377 cm-1) than that in GO (1358 cm-1) and decreased D/G intensity ratio (0.78) compared to that of GO (0.86). This phenomenon can be attributed to increased disorder caused by conjugated carnosines on the edge of GO [41, 42]. Besides D and G band in the Raman spectra of GO-Car, we also observed vibrations related imidazole ring, such as tortuosity vibration of ring (789 cm-1), deformation vibration of ring (898 cm-1), C-H stretching vibration of ring (995 cm-1, 1100 cm-1) and N-C-N stretching and N-H deformation vibration of ring (1188 cm-1) [43]. The above analysis indicated that carnosine molecules had been conjugated on the edge and adsorbed on the basal of GO sheets successfully. We also found that -OH groups of GO sheets had been reduced partly by carnosines in the modification process. In addition, the as-prepared GO easily aggregated in phosphate buffer solution (PBS, pH=7.4). But, the aggreation of GO-Car in PBS was greatly reduced. However, they all have a good stability in water, PBS with OVA (1 mg·mL-1, pH=7.4), fetal bovine serum (Supporting Information Figure S1).
Figure 4. The specific anti-OVA antibody titers in mice serum vaccinated with 50 µg OVA based materials per mouse or PBS. The mice were immunized three times at ten days intervals (days 0, 10, 20) and sacrificed on 2 weeks (A), 6 weeks (B), following the last immunization. The OVA antibody intensity in mice serum was measured using ELISA.
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(*P>0.05, **P
