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Synthesis and immunomodulatory activity of [60]fullerene–tuftsin conjugates
Biomaterials 32 (2011) 9940e9949
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Synthesis and immunomodulatory activity of [60]fullereneetuftsin conjugates Yingying Xua, Jiadan Zhua, Kun Xianga, Yuankai Lia, Ronghua Suna, Jie Mab, Hongfang Suna, *, Yuanfang Liua, c, ** a
Beijing National Laboratory for Molecular Sciences, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, PR China State Key Laboratory of Molecular Oncology, Cancer Institute, Chinese Academy of Medical Sciences, PUMC, Beijing 100021, PR China c Institute of Nanochemistry & Nanobiology, Shanghai University, Shanghai 200444, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 August 2011 Accepted 7 September 2011 Available online 19 September 2011
Immunomodulating peptide tuftsin (Thr-Lys-Pro-Arg) was covalently conjugated to fullerene C60 by two different ways to prepare NH2etuftsineC60 and C60etuftsineCOOH. The two new compounds were intensively characterized. The synthetic C60etuftsin conjugates were assayed for their stability against leucine aminopeptidase degradation. And the immunostimulating activities to murine peritoneal macrophages were investigated in vitro. Compared with the natural tuftsin, significant enhancement of phagocytosis, chemotaxis activities and major histocompatibility complex class II (MHC II) molecule expression were observed in macrophages stimulated by both of the conjugates. The two conjugates also exhibit complete resistance to enzymatic hydrolysis, and they are non-toxic to macrophages in the tested concentrations. On all accounts, these results suggest that the C60etuftsin conjugates can be used as potential candidates of immunomodulators and vaccine adjuvants. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Fullerene C60 Tuftsin Peritoneal macrophages Phagocytosis Chemotaxis MHC II
1. Introduction The nanomaterial fullerene C60 and its derivatives have exhibited many interesting activities in biomedical application, such as antitumor photodynamic therapy [1], antiviral activity against human immunodeficiency virus [2], DNA photocleavage [3], antioxidant activity [4] and enzyme inhibition [5]. Fullerene can penetrate through cellular membrane localizing within a cell, preferentially in the mitochondria [6]. And it can be used as a drug delivery vehicle, in particular for the anticancer drug delivery. For instance, C60 has been used to deliver anticancer drugs paclitaxel [7] and doxorubicin [8]. As a xenobiotic, when introduced to the organism, fullerenes will interact firstly with the immune system and may elicit a series of immune responses. Therefore, it is extraordinarily important to well understand the impact of fullerene and its derivatives on the immune systems, including both innate and adaptive immune response. Researchers have concerned about the immune activity of fullerenes since 1998, when Chen and his coworkers reported the
* Corresponding author. Tel.: þ86 10 62754127; fax: þ86 10 62755203. ** Corresponding author. Beijing National Laboratory for Molecular Sciences, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, PR China. Tel.: þ86 10 62757196; fax: þ86 10 62755203. E-mail addresses: [email protected] (H. Sun), [email protected] (Y. Liu). 0142-9612/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2011.09.022
production of anti-fullerene-specific antibody by immunization of mice with a C60 fullerene derivative conjugated to bovine thyroglobulin [9]. They found solubilized fullerene-bovine thyroglobulin conjugate can yield a population of fullerene-specific antibodies of the immunoglobulin G (IgG) isotype. Fullerene derivatives can regulate cytokine secretion [10e12], and some amino acidmodified C60 can enhance IgG production as an adjuvant [13]. Moreover, our previous studies have shown that polyhydroxylated C60, fullerol, can activate the innate immunity in mice and thereby inhibit the growth of tumor [14]. Tuftsin (Thr-Lys-Pro-Arg) is a naturally occurring tetrapeptide which possesses a wide range of biological activities, including stimulation of phagocytosis, motility and chemotaxis of phagocytes, e.g. neutrophils, monocytes and macrophages [15]. Tuftsin exerts its biological activities through binding with specific receptors existing on the surface of phagocytic cells [16]. It is demonstrated that tuftsin has a marked therapeutic potential as antibiotics and anticancer agent based on its immunostimulating activities [17]. However, the degradation of the peptide in serum, especially caused by leucine aminopeptidase, greatly limits its clinical use [15]. Therefore, it is crucial to develop potent and metabolically stable analogs of tuftsin. In this work, the rational of conjugating tuftsin with fullerene C60 is based on the following considerations. Firstly, the fullerene conjugation is expected to protect tuftsin from the enzyme degradation and consequently increase its serum stability. And
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thereby the formation of the C60etuftsin complex might prolong the blood circulation time of tuftsin. Secondly, on observing that fullerene derivatives preferentially enter the mononuclear phagocyte system due to their nanoparticulate nature [18], we therefore speculate that C60 might enhance the targeting of tuftsin to phagocytes and thus improve the bioavailability of tuftsin as well. Herein, we synthesized two conjugates of C60 and tuftsin, NH2etuftsineC60 and C60etuftsineCOOH, by covalently coupling C60 to the carboxyl or amino terminal of the tetrapeptide tuftsin, respectively. The C60etuftsineCOOH conjugate was prepared by covalently linking tuftsin with a pre-functionalized fullerene via a solid-phase peptide synthesis protocol, whereas the NH2etuftsineC60 conjugate was synthesized in a solution phase. Then the primary murine peritoneal macrophages were employed to evaluate the immunostimulating activities of the synthesized conjugates to phagocytosis, chemotaxis and MHC II molecule expression. 2. Materials and methods 2.1. Materials All solvents were dried before use. HOBT (1-hydroxybenzo-triazole), HBTU (OBenzotriazole-N,N,N0 ,N0 -tetramethyl-uronium-hexafluoro-phosphate), DIEA (diisopropylethylamine), EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) DBU
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(1,8-diazabicyclo[5,4,0]undecen-7-ene), TIS (triisopropylsilane) and TFA (trifluoroacetic acid) were purchased from SigmaeAldrich (USA). Wang resin, FmocArg(Pbf)eOH, Fmoc-ProeOH, Fmoc-Lys(Boc)eOH, and Fmoc-Thr(tBu)eOH were obtained from Chinapeptide Co. (Shanghai, China). The completely protected tuftsin, BocNH-Thr(tBu)-Lys(Boc)-Pro-Arg(Pbf)eOH, was customized by the same company. Electrospray ionization mass spectrometry (ESI-MS) analysis was performed on a Bruker APEX IV (USA) instrument.
2.2. Synthesis and characterization of NH2etuftsineC60 conjugate To conjugate C60 to the carboxyl terminal of tuftsin, we prepared compound 1 (Scheme 1) following the previously reported method in Ref [19]. Then EDC (14.3 mg, 0.075 mmol) was added slowly to a solution of compound 1 (40 mg, 0.05 mmol), BocNH-Thr(tBu)-Lys(Boc)-Pro-Arg(Pbf)eCOOH (75 mg, 0.075 mmol), HBTU (29 mg, 0.075 mmol), and DIEA (24 mL, 0.15 mmol) in DCM/DMF (5:1, 4.8 mL, DCM, dichloromethane; DMF, dimethyl sulfoxide). The mixture was stirred at room temperature for 2 h and the product was purified by silica gel chromatography (toluene/ethanol 3:1). The product was deprotected with TFA/TIS/H2O (95:2:3) for 1 h. The resulting NH2etuftsineC60 conjugate was purified by Sephadex G-15 with 1% acetic acid and then lyophilized. The conjugate was characterized by reverse phase high performance liquid chromatography (RP-HPLC) and ESI-MS. RP-HPLC equipped with YMC C4 analytic column was employed for purity analysis. A linear gradient of 30e100% B (A: 0.05% TFA in water; B: 0.05% TFA in acetonitrile) in 30 min at a flow rate of 1 mL/min was used for elution. The elution time was 14.5 min and the product purity was about 95%. The final yield was 52 mg (80%). ESI-MS: m/z, calculated 1288, found 1289.4[M þ H]þ. The particle size in aqueous solution determined with dynamic light scattering (DLS, Zetasizer, Malven Nano ZS90) was 161 19.2 nm.
Scheme 1. Synthesis of NH2etuftsineC60 and C60etuftsineCOOH conjugates.
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2.3. Synthesis and characterization of C60etuftsineCOOH conjugate
2.8. Chemotaxis assay
C60etuftsineCOOH conjugate was prepared with 50 mg Wang resin (0.6 mmol/ g). After swelling the resin in DCM for 15 min, Fmoc-Arg(Pbf)eOH in a 3-fold excess was coupled for 8 h using N,N-diisopropylcarbodiimide and HOBT in 1 mL DMF, as activating agents. For all subsequent couplings, a 3-fold excess of amino acid was used, and the reaction was activated with HBTU/HOBT/DIEA in 1:1:1:3 ratio in 2 mL DMF. Fmoc deprotection was performed using 20% perperidine in DMF solution. After the deprotection of the last residue (Thr), compound 2 [19] (Scheme 1) was coupled in a 1 mL mixture of DCM/DMF (1:1). The reaction was carried out for 8 h at room temperature. Fmoc deprotection was repeated twice with 2 mL of 5% of DBU for 2 min. The final product was removed from the resin using TFA/TIS/H2O (95:2:3) mixture. The crude fraction was washed with cold diethyl ether and lyophilized to remove excess TFA. Final purification was performed on Sephadex G-15 with 1% acetic acid. RP-HPLC equipped with YMC C4 analytic column was used for the purity analysis (eluant: A: 0.05% TFA in water and B: 0.05% TFA in acetonitrile, 30e100% B in 30 min at 1 mL/min). The elution time was 17.6 min and the product purity was about 98%. The final yield was 48 mg (63%). ESI-MS: m/z, calculated 1417, found 1418.4 [M þ H]þ, 710.2 [M þ 2H]2þ, 473.8 [M þ 3H]3þ. The particle size in aqueous solution determined with DLS was 132 27.2 nm.
Chemotaxis assay was performed as described by Sun et al. [23]. Briefly, peritoneal macrophages (5 106/mL) were suspended in a binding medium (DMEM lacking serum and containing 0.1% BSA and 25 mM HEPES). The upper chamber of the system was filled with the suspension of cells; the lower chamber was filled with the conjugates or tuftsin at tuftsin-equivalent concentration of 2, 10, 20 mmol/L. An adhesive polycarbonate filter with 8 mm pores (Corning, NY) was used to separate the two chambers. The binding medium was set as the negative control and was used for the dilution of the test substances. The cells were incubated at 37 C in a humidified atmosphere (5% CO2 and 95% air) for 90 min. The filter membrane was then rinsed, fixed and stained. The number of migrating cells was counted at 400 in three separate fields by the light microscope. Chemotaxis index is defined as the ratio of the migrating cell number in a chemoattractant gradient to the migrating cell number in a medium control.
2.4. Stability analysis with leucine aminopeptidase Enzymatic hydrolysis of tuftsin as well as the synthesized conjugates by leucine aminopeptidase (EC 3.4.11.1, SigmaeAldrich Co., USA) was carried out at 37 C in 0.2 M phosphate buffer of pH 7.4. An aliquot of 50 mL hydrolyzed peptide solution was taken at the time 0, 5, 15, 30, 60, 90, 120, 180 and 240 min, respectively. The samples were intermittently added to 450 mL ethanol to denature the enzyme. The mixture was thoroughly stirred and then centrifuged at 8000 g for 10 min. The subsequent supernatants were filtrated with a 0.45 mm Nylon membrane filter and analyzed by HPLC (eluant: A: 0.05% TFA in water and B: 0.05% TFA in acetonitrile, 30% B at 1 mL/ min). The peak area of the peptide at time 0 is taken as the 100% control. 2.5. Detection of the endotoxin The Limulus Amebocyte Lysate test (LAL test, Xiamen, China) was performed to detect the endotoxin content of the water solutions of tuftsin, NH2etuftsineC60, and C60etuftsineCOOH within the tested concentrations. Lipopolysaccharide (LPS) was used as the standard, and the content was expressed as endotoxin unit (EU) per mL. 2.6. Macrophages Specific pathogen-free, male BALB/c mice (6e8 weeks) were provided by the Experimental Animal Center, Academy of Military Medical Sciences (Beijing, China). The mice were housed under the normal laboratory conditions with free access to standard rodent chow and water. Peritoneal macrophages were collected as described by Ichinose M. et al. [20]. Briefly, the peritoneal exudate cells were harvested by washing the peritoneum with cold phosphate-buffered saline (PBS) and pooled together. The collected cells were washed three times with cold PBS, then resuspended in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 mg/mL streptomycin and 100 unit/mL penicillin G. The cell viability determined by the trypan blue exclusion method is always greater than 98%. The animal experiments were conducted following the approval of the Institutional Animal Care and Use Committee of Peking University.
2.9. Flow cytometry Fluorescence-activated cell sorting (FACS) was employed to investigate the effect of the conjugates and tuftsin on the expression of MHC II on the surface of peritoneal macrophages. Macrophages were plated into a 24-well plate at the concentration of 8 105 cells per well with a medium of DMEM containing 10% fetal bovine serum, 100 mg/mL streptomycin and 100 unit/mL penicillin G. After 3 h incubation and following purification as described above, the cells were cultured overnight. Tuftsin, NH2etuftsineC60 and C60etuftsineCOOH at a tuftsin-equivalent concentration of 20 mmol/L was added respectively to the medium and the cells were cultured for 24 h. Macrophages treated with 100 ng/mL LPS and without LPS were set as the positive control and negative control, respectively. After stimulation, cells were harvested, and washed twice with FACS buffer. The surface Fc receptor was blocked by incubation with 200 mL of 5% normal rabbit serum (Dingguo, China) for 30 min on ice. After another twice-washing with cold FACS buffer, aliquots of 2 105 cells in 100 mL of cold FACS buffer were added into Falcon tubes. Ten mL of FITC-labeled mouse antibodies specific for MHC II and the mouse IgG1 isotypes (eBioscience, USA) were added to each tube, respectively. After 30 min on ice in the dark, cells were washed three times with FACS buffer and resuspended in 350 mL cold FACS buffer. Flow cytometry (FACS Calibur, BD Bioscience, USA) was employed to analyze the surface markers of each sample. 2.10. Toxicity assay The peritoneal macrophages were plated into a 96-well plate at a density of 1 105 per well and cultured overnight at 37 C in a humidified atmosphere (5% CO2 and 95% air). The cell viability was assayed by 2-(2-methoxy-4-nitrophenyl)-3-(4nitrophenyl)-5-(2,4-disulf-opheyl)-2H-tetrazolium monosodium salt (WST-8), using Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan). The conjugates and tuftsin were introduced to the cells at a tuftsin-equivalent concentration of 2, 10, 20 mmol/L, respectively. The cells cultured in the blank medium were taken as the control. After 24 h incubation, 10 mL CCK-8 solution was added to each well, followed by incubation for another 3 h. The absorbance value at 450 nm was measured with a plate reader (Varioskan Flash, Thermo Electron, Finland). 2.11. Statistical analysis At least three parallel experiments were conducted for each sample. The data were processed by Origin 8.0. Results were expressed as mean standard deviations (SD). Means of each treated group were statistically compared using the two-tailed Student’s t-test. p < 0.05 was considered statistically significant.
2.7. Phagocytosis assay Phagocytosis assay was carried out as described by Terashima M. et al. [21] with a few modifications. The collected macrophages were plated into a 48-well plate at a concentration of 3 105 cells per well. After incubation for 2 h at 37 C in a CO2 incubator, the cells were washed twice with PBS to eliminate the non-adherent cells, and cultured in DMEM. Fluorescent latex beads were incubated with 1% bovine serum albumin (BSA) for 30 min at 37 C followed by ultrasonication. The macrophages were washed twice with HEPES balanced bath solution (pH 7.5, 10 mM HEPES, 140 mM NaCl, 5 mM KCl, 2 mM CaCl2 and 1 mM MgCl2) and preincubated for 15 min at room temperature. After adding the fluorescein-labeled latex beads (beads to cells ratio 30:1) and the indicated concentration of conjugates or tuftsin, the cells were further incubated for 15 min at 37 C. The beads that were not phagocytosed were removed partly by discarding the culture medium and washing the cells with cold PBS for three times. Each macrophage preparation was fixed with 10% glutaraldehyde. The cells were observed under a fluorescent microscope (Leica DMI4000B, Germany), which permits examination with both visible and UV fluorescence light. The number of phagocytosed cells was counted at 400 in three separate fields. The cell surface-adhered particles and the phagocytosed particles can be distinguished by finely adjusting the focal depth in the microscopic observation [22]. The internalized particles usually locate in the same layer of macrophages, whereas surfaceadhered particles do not.
3. Results 3.1. Synthesis of C60etuftsin conjugates The synthesis of the conjugates of C60etuftsin is achieved according to Scheme 1. Namely, C60 is linked respectively to the carboxyl and amino terminals of tuftsin to obtain two conjugates, NH2etuftsineC60 and C60etuftsineCOOH. The NH2etuftsineC60 conjugate is synthesized in a solution phase, in which an equimolar combination of EDC and HBTU is employed as a coupling agent. The completely protected tuftsin [BocNH-Thr(tBu)-Lys(Boc)-ProArg(Pbf)eOH] is linked to the amino group of the C60 derivative, compound 1 (Scheme 1a). The resultant conjugate is highly hydrophobic and easily separated from the excess reactants by the silica gel chromatography. After deprotection process the NH2etuftsineC60 conjugate becomes highly hydrophilic. Then the crude product was purified by Sephadex G-15, and the red brown
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fraction was collected. Finally, the purity was tested by analytical YMC C4 HPLC, which shows a purity of 95% (Fig. 1a). On the other hand, the C60etuftsineCOOH conjugate was synthesized by a solidphase peptide synthesis protocol following previous work (Scheme 1b) [19,24]. The crude product was also highly hydrophilic and purified by Sephadex G-15 similarly. The resultant compound was analyzed by the RP-HPLC, which shows a purity of 98% (Fig. 1b). Both conjugates were characterized by ESI-MS, and Fig. 2 shows the correct spectra in accordance with their chemical structures. The DLS measurement shows the average size of the two conjugates are both in the range of 100e200 nm. The result demonstrates that the synthetic conjugates form homogenous aggregates in aqueous solution. Additionally, we assessed the endotoxin level of the aqueous solutions of the conjugates, as well as tuftsin with Limulus Amebocyte Lysate test (LAL test), because bacterial endotoxin LPS is an effective macrophages stimulator that might influence the following biological evaluation tests. The results show that at the highest concentration used in the following experiments (20 mM), the LPS content of the three samples are all lower than the detection limit. Therefore, these samples are considered free of endotoxin.
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carboxypeptidase B, pronase and subtilisin [25]. The LAP locating on the plasma membrane of human neutrophils, could remove the N-terminal threonine to yield a tripeptide Lys-Pro-Arg, which is an inhibitor of tuftsin activity [26]. Herein, the stability of the synthesized C60etuftsin conjugates as well as tuftsin per se against LAP degradation was investigated. The conjugates or tuftsin were incubated with LAP at 37 C, and the aliquots were taken at various time intervals. The amount of intact peptides was evaluated by the analytical HPLC. For tuftsin, there are two peaks in the HPLC chromatogram at 120 min. The right one at 1.86 min is the hydrolysate from tuftsin, which is probably the tripeptide Lys-ProArg [26]. While the C60etuftsin conjugates could not be recovered from the supernatant because they coagulated with the enzyme during the denaturing process by ethanol. Then we tried to quantify the degradation of the conjugates by the product generated during the hydrolysis. And it turns out that both of the conjugates show no such hydrolysate within 2 h incubation (Fig. 3a). Therefore, the results demonstrate that C60etuftsin conjugates have an excellent stability against LAP hydrolysis. Moreover, Fig. 3b indicates that the hydrolysis rate of tuftsin is quite fast with a half-life of about 16 min. After 1 h, the enzymatic hydrolysis reaches the equilibrium. 3.3. Stimulation of phagocytosis by C60etuftsin
3.2. Stability against leucine aminopeptidase (LAP) degradation One major complication for the clinical use of tuftsin is the instability against a battery of enzymes in serum. It has been reported that the biological activity of tuftsin is reduced by LAP,
Since the main target cells of tuftsin are phagocytes [16], we chose the murine peritoneal macrophages to investigate the immunomodulatory activity of the two synthesized conjugates. At first we assayed the phagocytosis activity of macrophages after
Fig. 1. RP-HPLC chromatogram of the purified conjugates. (a) NH2etuftsineC60, (b) C60etuftsineCOOH. The inset shows the UVeVis spectrum of the corresponding conjugate.
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Fig. 2. Electrospray mass spectra of the conjugates. (a) NH2etuftsineC60 (H2O), (b) C60etuftsineCOOH (H2O).
stimulation with the conjugates. It was reported that the natural tuftsin showed a bell-shaped activity profile in phagocytosis stimulation of macrophages [27], and the highest phagocytosis rate was observed at the concentration of 10 mmol/L. The same activity profile of tuftsin is observed in our experiment, but the two conjugates show different phagocytosis stimulation activity and concentration profiles (Fig. 4). NH2etuftsineC60 exhibits the similar activity profile as the natural tuftsin. Besides, NH2etuftsineC60 shows more potent stimulation ability than that of tuftsin at the same concentration. For C60etuftsineCOOH, the stimulation activity increases with increasing concentration, and at the same concentration the conjugate shows more potent activity than that of tuftsin too. 3.4. Chemoattractant effect of C60etuftsin Chemotaxis is the directed cell motility along a chemical gradient, and it is an important mechanism by which the inflammatory cells accumulate at the local sites. We conducted chemotaxis assay to evaluate the chemotactic activity of macrophages toward the C60etuftsin conjugates. As shown in Fig. 5, both NH2etuftsineC60 and C60etuftsineCOOH trigger a significant chemoattractant effect in the tested concentration 2e20 mmol/L. The activity increases with increasing concentration in both cases of the two conjugates. It was reported that tuftsin exerts chemotactic effect on granulocytes and monocytes in the concentration range of
103e102 mmol/L [28]. In our experiment, we also observe a significant chemotactic activity of tuftsin, especially at the concentration of 10 mmol/L. Whereas, the chemotactic index decrease slightly at 20 mmol/L. Comparing with tuftsin, the two conjugates exhibit significant enhanced chemoattractant activity, especially for NH2etuftsineC60. And the chemotactic index increases with increasing concentration for both the two conjugates, which is different from that of tuftsin.
3.5. Stimulation of MHC II molecule expression by C60etuftsin We also measured the MHC II molecule expression on the surface of macrophages. Macrophages are essential in the activation of the adaptive immune system via phagocytosing, processing and presenting of antigens. Since both NH2etuftsineC60 and C60etuftsineCOOH conjugates can up-regulate the phagocytic and chemotactic activities of macrophages, we speculate that they may also influence the presentation of antigens. MHC II is mainly expressed on the antigen-presenting cell surface, such as macrophages, dendritic cells and B cells. The main function of MHC II is to bind and present fragments of exogenous antigen to T cells. Our results in Fig. 6 show that both NH2etuftsineC60 and C60etuftsineCOOH can significantly up-regulate MHC II expression on the surface of murine peritoneal macrophages, whereas tuftsin per se does not influence the expression of MHC II.
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Fig. 3. Stability of tuftsin and C60etuftsin conjugates against enzymatic degradation by leucine aminopeptidase. (a) Representative HPLC chromatogram of aliquots at indicated time point (0 and 120 min). (b) Percentage of the intact peptide in aliquots taken at different time points.
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Fig. 4. Analysis of the phagocytosis of fluorescent latex beads by peritoneal macrophages in vitro. Data are presented as mean SD (n ¼ 3). *p < 0.05, compared with the control group.
3.6. Toxicity of the C60etuftsin conjugates To assess the biocompatibility of the synthesized conjugates, macrophages were exposed to tuftsin or conjugates at the concentration range of 2e20 mmol/L. As Fig. 7 shows, the cell viabilities of all the tested materials have no significant difference to the control, which indicates that they are non-toxic to macrophages. 4. Discussion Tuftsin has been discovered for several decades, however, practical use of tuftsin in cancer therapy and microbial infections still needs to develop more stable and potent tuftsin analogs. In years past researchers have investigated various approaches to improve the stability of tuftsin and preserve its biological activity as well. These approaches include substitution of the N-terminal threonyl residue, substitution of L-amino acids by D-amino acids and elongation of the tetrapeptide chain by the addition of further
Fig. 5. Chemotactic effect of peritoneal macrophages toward tuftsin or conjugates. Data are presented as mean SD (n ¼ 3). *p < 0.05, compared with control group.
amino acid residues [29]. With these approaches the metabolic stability of tuftsin can be enhanced in some extent, whereas the biological activity usually decreases or even be abrogated at all. In this work, we have established an effective approach by conjugating tuftsin with the nanocarrier C60. The stability assay have demonstrated that both the NH2etuftsineC60 and C60etuftsineCOOH conjugates are much stable than the natural tuftsin. Furthermore, the subsequent biological evaluation tests all suggest that the immunostimulating activities of tuftsin are well preserved and even significantly enhanced. Therefore, we are confident that the approach reported in this paper is rather promising for the further application of tuftsin in the clinic. Herein, to achieve high synthetic yield, different synthesis protocols were employed respectively for the preparation of NH2etuftsineC60 and C60etuftsineCOOH conjugates. It has been demonstrated that solid-phase synthesis is an useful and effective method for the construction of fullereneepeptide conjugates [24,30]. In the synthesis of C60etuftsineCOOH, a fulleropyrrolidinoglutamic acid (compound 2, Scheme 1) was used for the conjugation of C60 to tuftsin. The result illustrates that this fullerene amino acid is a good building block for the solid-phase synthesis of fullereneepeptide conjugate. On the other hand, solution phase synthesis is also a convenient route for the construction of fullereneepeptide conjugate. Using the activation agent EDC/HBTU, we obtained the NH2etuftsineC60 with a quite high yield. Moreover, we found that Sephadex G-15 size exclusion chromatography is an effective method for the purification of the C60etuftsin conjugates. At the beginning, we tried to use a semi-preparative C4 RP-HPLC column, whereas almost all the product retained on the column and could not be eluted due to the interaction between C60 and the stationary phase of RP-HPLC. Then the Sephadex G-15 chromatography was used instead. And the results turn out that a high yield and purity have been achieved. The stability of the synthetic conjugates is a main concern in our present work, since it is an obstacle for the potential clinical use of tuftsin. Fortunately the above degradation test with leucine aminopeptidase (LAP) has shown that both of the conjugates are fully resistant to the LAP enzymatic hydrolysis. The inhibition effect of fullerene derivatives to various enzymes has been investigated [5,31,32], however the interaction between fullerene and LAP has not been reported. We speculate that there might be steric hindrance effect of C60 toward LAP, hence results in the inhibition of C60etuftsin conjugates degradation. Anyhow, further study on this issue is needed to clarify the inhibition mechanism. It is noticed that, there are somewhat differences lying between the two conjugates. In the phagocytosis assay, NH2etuftsineC60 exhibits a highest phagocytosis activity at 10 mmol/L concentration, while for C60etuftsineCOOH, the phagocytosis rate increases with increasing concentration. In the chemotaxis assay, the chemoattractant activity of the both conjugates increases along with increasing concentration. And at all the three tested concentrations, NH2etuftsineC60 exhibits more potent chemoattractant effect than C60etuftsineCOOH. Additionally, in the MHC II expression assay, the stimulation activity of NH2etuftsineC60 is also more potent than that of C60etuftsineCOOH. The different activities of NH2etuftsineC60 and C60etuftsineCOOH are probably determined by their chemical structures, since the C60 conjugation may influence the interaction of tuftsin and its receptor on the surface of macrophages. Based on the previous structureefunction studies of tuftsin, elongation on the amino terminal may decrease the activity, since the integrity of the free N-terminus of the Thr1 residue is found crucial for the manifestation of biological activity [17]. In the synthesis of NH2etuftsineC60, C60 is conjugated to the carboxyl terminal of tuftsin, presumably the activity of tuftsin is not affected. Therefore, in most of the cases the biological activity of
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Fig. 6. (a). Flow cytometry analysis of MHC II molecule expression of peritoneal macrophages after 24 h stimulation by tuftsin or conjugates at tuftsin-equivalent concentration of 10 mmol/L. The shadows and solid lines represent isotype control and FITC-labeled mouse antibodies specific for MHC II stained cells respectively. The percentage of positive cells is shown in the middle of each histogram. (b). Comparison on the percentage of positive cells for MHC II molecule expression on peritoneal macrophages induced by tuftsin and conjugates. Data are presented as mean SD (n ¼ 3). *p < 0.01 compared with negative controls.
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phagocytosis and chemotaxis of murine peritoneal macrophages. Moreover, they can up-regulate significantly the MHC II molecule expression on the surface of macrophages, whereas the free tuftsin shows no such effect. Our results demonstrate that the C60 conjugation enhances the immunostimulation activity of tuftsin, and NH2etuftsineC60 conjugate exhibits more effective stimulation activity than C60etuftsineCOOH does. Furthermore, both the two conjugates show no toxicity to macrophages in the tested concentrations, that paves the way for the potential use of C60 fullerene as the vehicle for the delivery of immunostimulants. Acknowledgments We acknowledge the fund from the National Basic Research Program of China (973 Program) (No. 2011CB933402, 2009CB930303, and 2011CB911004), as well as the China Natural Science Foundation (Significant project No. 10490180). References
Fig. 7. The viability of murine peritoneal macrophages after 24 h exposure to tuftsin and the conjugates.
NH2etuftsineC60 is more potent than that of C60etuftsineCOOH is reasonable. Nevertheless, comparing with the natural tuftsin, both of the conjugates significantly enhance the phagocytosis and chemotaxis activities of the murine peritoneal macrophages. In addition, tuftsin itself does not exhibit stimulating activity on the MHC II expression, while both the two conjugates significantly up-regulate MHC II on the surface of macrophages. A previous report has pointed out that, nanoparticles larger than 100 nm usually exhibit the adjuvant activity toward immune cells [33]. And the DLS measurement has shown that the sizes of the aggregated C60etuftsin conjugates in solution are ranging from 100 to 200 nm. We therefore speculate that the nanoparticulate nature of C60etuftsin conjugates may play an important role for the immune activities of the two conjugates. In the last two decades, various nanoparticulate adjuvants, such as polymeric nanoparticles, immunostimulating complexes, latex, silica and polystyrene, have been extensively investigated for antigen delivery and immune stimulation [34,35]. Nevertheless, other vaccine adjuvants are still worthy of developing. In this work, fullerene C60 is found to be an effective carrier for the tetrapeptide tuftsin, both in the aspects of stability and immunostimulating activity. The synthetic C60etuftsin conjugates, including NH2etuftsineC60 and C60etuftsineCOOH, cannot only protect tuftsin from LAP degradation, but also enhance the immunostimulating activities of the natural tuftsin. The good biocompatibility as well as the potent immunostimulating activity of the synthesized conjugates demonstrates that the C60etuftsin conjugates might be potential candidates as the immunomodulators and vaccine adjuvants.
5. Conclusions We have successfully conjugated fullerene C60 to the immunomodulating peptide tuftsin by covalently linking with the carboxyl terminal and the amino terminal respectively. The two compounds, NH2etuftsineC60 and C60etuftsineCOOH, were satisfactorily characterized by RP-HPLC and ESI-MS. The synthetic C60etuftsin conjugates show complete resistance to the leucine aminopeptidase degradation. At the same time, both of the conjugates show more potent stimulation activities than the free tuftsin in the
[1] Tabata Y, Ikada Y. Biological functions of fullerene. Pure Appl Chem 1999; 71(11):2047e53. [2] Friedman SH, De Camp DL, Sijbesma RP, Srdanov G, Wudl F, Kenyon GL. Inhibition of the HIV-1 protease by fullerene derivatives: model building studies and experimental verification. J Am Chem Soc 1993;115(15):6506e9. [3] Tokuyama H, Yamago S, Nakamura E, Shiraki T, Sugiura Y. Photoinduced biochemical activity of fullerene carboxylic acid. J Am Chem Soc 1993;5(15): 7918e9. [4] Wang I, Tai L, Lee D, Kanakamma PP, Shen CF, Luh TY, et al. C60 and watersoluble fullerene derivatives as antioxidants against radical-initiated lipid peroxidation. J Med Chem 1999;42(22):4614e20. [5] Yang ST, Wang HF, Guo L, Gao Y, Liu YF, Cao AN. Interaction of fullerenol with lysozyme investigated by experimental and computational approaches. Nanotechnology 2008;19(1):395101e8. [6] Foley S, Crowley C, Smaihi M, Bonfils C, Erlanger BF, Seta P, et al. Cellular localisation of a water-soluble fullerene derivative. Biochem Biophys Res Commun 2002;294(1):116e9. [7] Zakharian TY, Seryshev A, Sitharaman B, Gilbert BE, Knight V, Wilson LJ. A fullerene-paclitaxel chemotherapeutic: synthesis, characterization, and study of biological activity in tissue culture. J Am Chem Soc 2005;127(36): 12508e9. [8] Lu FS, Haque SA, Yang ST, Luo PG, Gu LR, Kitaygorodskiy A, et al. Aqueous compatible fullerene-doxorubicin conjugates. J Phys Chem C 2009;113(41): 17768e73. [9] Chen BX, Wilson SR, Das M, Coughlin DJ, Erlanger BF. Antigenicity of fullerenes: antibodies specific for fullerenes and their characteristics. Proc Nat Acad Sci USA 1998;95(18):10809e13. [10] Baierl T, Seidel A. In vitro effects of fullerene C60 and fullerene black on immunofunctions of macrophages. Fullerene Sci Technol 1996;4(5):1073e85. [11] Tsao N, Kanakamma PP, Luh TY, Chou CK, Lei HY. Inhibition of Escherichia coliinduced meningitis by carboxyfullerene. Antimicrob Agents Chemother 1999; 43(9):2273e7. [12] Liu Y, Jiao F, Qiu Y, Li W, Qu Y, Tian CX, et al. Immunostimulatory properties and enhanced TNF-a mediated cellular immunity for tumor therapy by C60(OH)20 nanoparticles. Nanotechnology 2009;20(41):415102e11. [13] Masalova OV, Shepelev AV, Atanadze SN, Parnes ZN, Romanova VS, Vol’pina OM, et al. Immunostimulating action of water soluble fullerene derivatives as perspective adjuvants for new generation vaccine. Dokl Akad Nauk 1999;369(3):411e3. [14] Zhu JD, Ji ZQ, Wang J, Sun RH, Zhang X, Gao Y, et al. Tumor-inhibitory effect and immunomodulatory activity of fullerol C60(OH)x. Small 2008;4(8):1168e75. [15] Siemion IZ, Kluczy A. Tuftsin: on the 30-year anniversary of Victor Najjar’s discovery. Peptides 1999;20(5):645e74. [16] Bump NJ, Najjar VA, Reichler J. The characteristics of purified HL60 tuftsin receptors. Mol Cell Biochem 1990;92(1):77e84. [17] Fridkin M, Najjar VA. Tuftsin: its chemistry, biology, and clinical potential. Crit Rev Biochem Mol Biol 1989;24(1):1e40. [18] Ji ZQ, Sun HF, Wang HF, Xie QY, Liu YF, Wang ZJ. Biodistribution and tumor uptake of C60(OH)x in mice. Nanopart Res 2006;8(1):53e63. [19] Pellarini F, Pantarotto D, Da Ros T, Giangaspero A, Tossi A, Prato M. A novel [60]fullerene amino acid for use in solid-phase peptide synthesis. Org Lett 2001;3(12):1845e8. [20] Ichinose M, Hara N, Sawada M, Maeno T. A flow cytometric assay reveals an enhancement of phagocytosis by platelet activating factor in murine peritoneal macrophages. Cell Immun 1994;156(2):508e18. [21] Terashima M, Hara N, Badruzzaman M, Shimoyama M, Tsuchiya M. ADPribosylation of tuftsin suppresses its receptor-binding capacity and phagocytosis-stimulating activity to murine peritoneal macrophages. FEBS Lett 1997;412(1):227e32.
Y. Xu et al. / Biomaterials 32 (2011) 9940e9949 [22] Jia G, Wang HF, Yan L, Wang X, Pei RJ, Yan T, et al. Cytotoxicity of carbon nanomaterials: single-wall nanotube, multi-wall nanotube, and fullerene. Environ Sci Technol 2005;39(5):1378e83. [23] Sun RH, Gao P, Chen L, Ma DL, Wang JM, Oppenheim JJ, et al. Protein kinase C zeta is required for epidermal growth factor-induced chemotaxis of human breast cancer cells. Cancer Res 2005;65(4):1433e41. [24] Pantarotto D, Bianco A, Pellarini F, Tossi A, Giangaspero A, Zelezetsky I, et al. Solid-phase synthesis of fullerene-peptides. J Am Chem Soc 2002;124(42): 12543e9. [25] Nishioka K, Constantopoulos A, Satoh P, Mitchell W, Najjar VA. Characteristics and isolation of the phagocytosis-stimulating peptide, tuftsin. Biochim Biophys Acta 1973;310(1):217e29. [26] Fridkin M, Stabinsky Y, Zakuth V, Spirer Z. Tuftsin and some analogs: synthesis and interaction with human polymorphonuclear leukocytes. Biochim Biophys Acta 1977;496(1):203e11. [27] Nishioka K, Obeyesekere NU, McMurry JS. Enhanced phagocytosis activity of cyclic analogs of tuftsin. Biochem Pharm 1995;49(5):735e8. [28] Babcock GF, Amoscato AA, Nishioka K. Effect of tuftsin on the migration, chemotaxis, and differentiation of macrophages and granulocytes. Ann N Y Acad Sci 1983;419(12):64e74.
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[29] Gershonov E, Granoth R, Tzehoval E, Gaoni Y, Fridkin M. 1Aminocyclobutanecarboxylic acid derivatives as novel structural elements in bioactive peptides: application to tuftsin analogs. J Med Chem 1996;39(24): 4833e43. [30] Yang JZ, Alemany LB, Driver J, Hartgerink JD, Barron AR. Fullerene-derivatized amino acids: synthesis, characterization, antioxidant properties, and solidphase peptide synthesis. Chem Eur J 2007;13(9):2530e45. [31] Innocenti A, Durdagi S, Doostdar N, Strom TA, Barron A, Supuran C. Nanoscale enzyme inhibitors: fullerenes inhibit carbonic anhydrase by occluding the active site entrance. Bioorg Med Chem 2010;18(8):2822e8. [32] Pastorin G, Marchesan S, Hoebeke J, Da Ros T, Ehret-Sabatier L, Briand JP, et al. Design and activity of cationic fullerene derivatives as inhibitors of acetylcholinesterase. Org Biomol Chem 2006;4(13):2556e62. [33] Powell JJ, Faria N, Thomas-McKay E, Pele LC. Origin and fate of dietary nanoparticles and microparticles in the gastrointestinal tract. J Autoimmun 2010;34(3):J226e33. [34] Singh M, Chakrapani A, O’Hagan D. Nanoparticles and microparticles as vaccine-delivery systems. Expert Rev Vaccines 2007;6(5):797e808. [35] Peek LJ, Middaugh CR, Berkland C. Nanotechnology in vaccine delivery. Adv Drug Deliver Rev 2008;60(8):915e28.
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Synthesis and immunomodulatory activity of [60]fullereneetuftsin conjugates Yingying Xua, Jiadan Zhua, Kun Xianga, Yuankai Lia, Ronghua Suna, Jie Mab, Hongfang Suna, *, Yuanfang Liua, c, ** a
Beijing National Laboratory for Molecular Sciences, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, PR China State Key Laboratory of Molecular Oncology, Cancer Institute, Chinese Academy of Medical Sciences, PUMC, Beijing 100021, PR China c Institute of Nanochemistry & Nanobiology, Shanghai University, Shanghai 200444, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 August 2011 Accepted 7 September 2011 Available online 19 September 2011
Immunomodulating peptide tuftsin (Thr-Lys-Pro-Arg) was covalently conjugated to fullerene C60 by two different ways to prepare NH2etuftsineC60 and C60etuftsineCOOH. The two new compounds were intensively characterized. The synthetic C60etuftsin conjugates were assayed for their stability against leucine aminopeptidase degradation. And the immunostimulating activities to murine peritoneal macrophages were investigated in vitro. Compared with the natural tuftsin, significant enhancement of phagocytosis, chemotaxis activities and major histocompatibility complex class II (MHC II) molecule expression were observed in macrophages stimulated by both of the conjugates. The two conjugates also exhibit complete resistance to enzymatic hydrolysis, and they are non-toxic to macrophages in the tested concentrations. On all accounts, these results suggest that the C60etuftsin conjugates can be used as potential candidates of immunomodulators and vaccine adjuvants. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Fullerene C60 Tuftsin Peritoneal macrophages Phagocytosis Chemotaxis MHC II
1. Introduction The nanomaterial fullerene C60 and its derivatives have exhibited many interesting activities in biomedical application, such as antitumor photodynamic therapy [1], antiviral activity against human immunodeficiency virus [2], DNA photocleavage [3], antioxidant activity [4] and enzyme inhibition [5]. Fullerene can penetrate through cellular membrane localizing within a cell, preferentially in the mitochondria [6]. And it can be used as a drug delivery vehicle, in particular for the anticancer drug delivery. For instance, C60 has been used to deliver anticancer drugs paclitaxel [7] and doxorubicin [8]. As a xenobiotic, when introduced to the organism, fullerenes will interact firstly with the immune system and may elicit a series of immune responses. Therefore, it is extraordinarily important to well understand the impact of fullerene and its derivatives on the immune systems, including both innate and adaptive immune response. Researchers have concerned about the immune activity of fullerenes since 1998, when Chen and his coworkers reported the
* Corresponding author. Tel.: þ86 10 62754127; fax: þ86 10 62755203. ** Corresponding author. Beijing National Laboratory for Molecular Sciences, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, PR China. Tel.: þ86 10 62757196; fax: þ86 10 62755203. E-mail addresses: [email protected] (H. Sun), [email protected] (Y. Liu). 0142-9612/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2011.09.022
production of anti-fullerene-specific antibody by immunization of mice with a C60 fullerene derivative conjugated to bovine thyroglobulin [9]. They found solubilized fullerene-bovine thyroglobulin conjugate can yield a population of fullerene-specific antibodies of the immunoglobulin G (IgG) isotype. Fullerene derivatives can regulate cytokine secretion [10e12], and some amino acidmodified C60 can enhance IgG production as an adjuvant [13]. Moreover, our previous studies have shown that polyhydroxylated C60, fullerol, can activate the innate immunity in mice and thereby inhibit the growth of tumor [14]. Tuftsin (Thr-Lys-Pro-Arg) is a naturally occurring tetrapeptide which possesses a wide range of biological activities, including stimulation of phagocytosis, motility and chemotaxis of phagocytes, e.g. neutrophils, monocytes and macrophages [15]. Tuftsin exerts its biological activities through binding with specific receptors existing on the surface of phagocytic cells [16]. It is demonstrated that tuftsin has a marked therapeutic potential as antibiotics and anticancer agent based on its immunostimulating activities [17]. However, the degradation of the peptide in serum, especially caused by leucine aminopeptidase, greatly limits its clinical use [15]. Therefore, it is crucial to develop potent and metabolically stable analogs of tuftsin. In this work, the rational of conjugating tuftsin with fullerene C60 is based on the following considerations. Firstly, the fullerene conjugation is expected to protect tuftsin from the enzyme degradation and consequently increase its serum stability. And
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thereby the formation of the C60etuftsin complex might prolong the blood circulation time of tuftsin. Secondly, on observing that fullerene derivatives preferentially enter the mononuclear phagocyte system due to their nanoparticulate nature [18], we therefore speculate that C60 might enhance the targeting of tuftsin to phagocytes and thus improve the bioavailability of tuftsin as well. Herein, we synthesized two conjugates of C60 and tuftsin, NH2etuftsineC60 and C60etuftsineCOOH, by covalently coupling C60 to the carboxyl or amino terminal of the tetrapeptide tuftsin, respectively. The C60etuftsineCOOH conjugate was prepared by covalently linking tuftsin with a pre-functionalized fullerene via a solid-phase peptide synthesis protocol, whereas the NH2etuftsineC60 conjugate was synthesized in a solution phase. Then the primary murine peritoneal macrophages were employed to evaluate the immunostimulating activities of the synthesized conjugates to phagocytosis, chemotaxis and MHC II molecule expression. 2. Materials and methods 2.1. Materials All solvents were dried before use. HOBT (1-hydroxybenzo-triazole), HBTU (OBenzotriazole-N,N,N0 ,N0 -tetramethyl-uronium-hexafluoro-phosphate), DIEA (diisopropylethylamine), EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) DBU
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(1,8-diazabicyclo[5,4,0]undecen-7-ene), TIS (triisopropylsilane) and TFA (trifluoroacetic acid) were purchased from SigmaeAldrich (USA). Wang resin, FmocArg(Pbf)eOH, Fmoc-ProeOH, Fmoc-Lys(Boc)eOH, and Fmoc-Thr(tBu)eOH were obtained from Chinapeptide Co. (Shanghai, China). The completely protected tuftsin, BocNH-Thr(tBu)-Lys(Boc)-Pro-Arg(Pbf)eOH, was customized by the same company. Electrospray ionization mass spectrometry (ESI-MS) analysis was performed on a Bruker APEX IV (USA) instrument.
2.2. Synthesis and characterization of NH2etuftsineC60 conjugate To conjugate C60 to the carboxyl terminal of tuftsin, we prepared compound 1 (Scheme 1) following the previously reported method in Ref [19]. Then EDC (14.3 mg, 0.075 mmol) was added slowly to a solution of compound 1 (40 mg, 0.05 mmol), BocNH-Thr(tBu)-Lys(Boc)-Pro-Arg(Pbf)eCOOH (75 mg, 0.075 mmol), HBTU (29 mg, 0.075 mmol), and DIEA (24 mL, 0.15 mmol) in DCM/DMF (5:1, 4.8 mL, DCM, dichloromethane; DMF, dimethyl sulfoxide). The mixture was stirred at room temperature for 2 h and the product was purified by silica gel chromatography (toluene/ethanol 3:1). The product was deprotected with TFA/TIS/H2O (95:2:3) for 1 h. The resulting NH2etuftsineC60 conjugate was purified by Sephadex G-15 with 1% acetic acid and then lyophilized. The conjugate was characterized by reverse phase high performance liquid chromatography (RP-HPLC) and ESI-MS. RP-HPLC equipped with YMC C4 analytic column was employed for purity analysis. A linear gradient of 30e100% B (A: 0.05% TFA in water; B: 0.05% TFA in acetonitrile) in 30 min at a flow rate of 1 mL/min was used for elution. The elution time was 14.5 min and the product purity was about 95%. The final yield was 52 mg (80%). ESI-MS: m/z, calculated 1288, found 1289.4[M þ H]þ. The particle size in aqueous solution determined with dynamic light scattering (DLS, Zetasizer, Malven Nano ZS90) was 161 19.2 nm.
Scheme 1. Synthesis of NH2etuftsineC60 and C60etuftsineCOOH conjugates.
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2.3. Synthesis and characterization of C60etuftsineCOOH conjugate
2.8. Chemotaxis assay
C60etuftsineCOOH conjugate was prepared with 50 mg Wang resin (0.6 mmol/ g). After swelling the resin in DCM for 15 min, Fmoc-Arg(Pbf)eOH in a 3-fold excess was coupled for 8 h using N,N-diisopropylcarbodiimide and HOBT in 1 mL DMF, as activating agents. For all subsequent couplings, a 3-fold excess of amino acid was used, and the reaction was activated with HBTU/HOBT/DIEA in 1:1:1:3 ratio in 2 mL DMF. Fmoc deprotection was performed using 20% perperidine in DMF solution. After the deprotection of the last residue (Thr), compound 2 [19] (Scheme 1) was coupled in a 1 mL mixture of DCM/DMF (1:1). The reaction was carried out for 8 h at room temperature. Fmoc deprotection was repeated twice with 2 mL of 5% of DBU for 2 min. The final product was removed from the resin using TFA/TIS/H2O (95:2:3) mixture. The crude fraction was washed with cold diethyl ether and lyophilized to remove excess TFA. Final purification was performed on Sephadex G-15 with 1% acetic acid. RP-HPLC equipped with YMC C4 analytic column was used for the purity analysis (eluant: A: 0.05% TFA in water and B: 0.05% TFA in acetonitrile, 30e100% B in 30 min at 1 mL/min). The elution time was 17.6 min and the product purity was about 98%. The final yield was 48 mg (63%). ESI-MS: m/z, calculated 1417, found 1418.4 [M þ H]þ, 710.2 [M þ 2H]2þ, 473.8 [M þ 3H]3þ. The particle size in aqueous solution determined with DLS was 132 27.2 nm.
Chemotaxis assay was performed as described by Sun et al. [23]. Briefly, peritoneal macrophages (5 106/mL) were suspended in a binding medium (DMEM lacking serum and containing 0.1% BSA and 25 mM HEPES). The upper chamber of the system was filled with the suspension of cells; the lower chamber was filled with the conjugates or tuftsin at tuftsin-equivalent concentration of 2, 10, 20 mmol/L. An adhesive polycarbonate filter with 8 mm pores (Corning, NY) was used to separate the two chambers. The binding medium was set as the negative control and was used for the dilution of the test substances. The cells were incubated at 37 C in a humidified atmosphere (5% CO2 and 95% air) for 90 min. The filter membrane was then rinsed, fixed and stained. The number of migrating cells was counted at 400 in three separate fields by the light microscope. Chemotaxis index is defined as the ratio of the migrating cell number in a chemoattractant gradient to the migrating cell number in a medium control.
2.4. Stability analysis with leucine aminopeptidase Enzymatic hydrolysis of tuftsin as well as the synthesized conjugates by leucine aminopeptidase (EC 3.4.11.1, SigmaeAldrich Co., USA) was carried out at 37 C in 0.2 M phosphate buffer of pH 7.4. An aliquot of 50 mL hydrolyzed peptide solution was taken at the time 0, 5, 15, 30, 60, 90, 120, 180 and 240 min, respectively. The samples were intermittently added to 450 mL ethanol to denature the enzyme. The mixture was thoroughly stirred and then centrifuged at 8000 g for 10 min. The subsequent supernatants were filtrated with a 0.45 mm Nylon membrane filter and analyzed by HPLC (eluant: A: 0.05% TFA in water and B: 0.05% TFA in acetonitrile, 30% B at 1 mL/ min). The peak area of the peptide at time 0 is taken as the 100% control. 2.5. Detection of the endotoxin The Limulus Amebocyte Lysate test (LAL test, Xiamen, China) was performed to detect the endotoxin content of the water solutions of tuftsin, NH2etuftsineC60, and C60etuftsineCOOH within the tested concentrations. Lipopolysaccharide (LPS) was used as the standard, and the content was expressed as endotoxin unit (EU) per mL. 2.6. Macrophages Specific pathogen-free, male BALB/c mice (6e8 weeks) were provided by the Experimental Animal Center, Academy of Military Medical Sciences (Beijing, China). The mice were housed under the normal laboratory conditions with free access to standard rodent chow and water. Peritoneal macrophages were collected as described by Ichinose M. et al. [20]. Briefly, the peritoneal exudate cells were harvested by washing the peritoneum with cold phosphate-buffered saline (PBS) and pooled together. The collected cells were washed three times with cold PBS, then resuspended in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 mg/mL streptomycin and 100 unit/mL penicillin G. The cell viability determined by the trypan blue exclusion method is always greater than 98%. The animal experiments were conducted following the approval of the Institutional Animal Care and Use Committee of Peking University.
2.9. Flow cytometry Fluorescence-activated cell sorting (FACS) was employed to investigate the effect of the conjugates and tuftsin on the expression of MHC II on the surface of peritoneal macrophages. Macrophages were plated into a 24-well plate at the concentration of 8 105 cells per well with a medium of DMEM containing 10% fetal bovine serum, 100 mg/mL streptomycin and 100 unit/mL penicillin G. After 3 h incubation and following purification as described above, the cells were cultured overnight. Tuftsin, NH2etuftsineC60 and C60etuftsineCOOH at a tuftsin-equivalent concentration of 20 mmol/L was added respectively to the medium and the cells were cultured for 24 h. Macrophages treated with 100 ng/mL LPS and without LPS were set as the positive control and negative control, respectively. After stimulation, cells were harvested, and washed twice with FACS buffer. The surface Fc receptor was blocked by incubation with 200 mL of 5% normal rabbit serum (Dingguo, China) for 30 min on ice. After another twice-washing with cold FACS buffer, aliquots of 2 105 cells in 100 mL of cold FACS buffer were added into Falcon tubes. Ten mL of FITC-labeled mouse antibodies specific for MHC II and the mouse IgG1 isotypes (eBioscience, USA) were added to each tube, respectively. After 30 min on ice in the dark, cells were washed three times with FACS buffer and resuspended in 350 mL cold FACS buffer. Flow cytometry (FACS Calibur, BD Bioscience, USA) was employed to analyze the surface markers of each sample. 2.10. Toxicity assay The peritoneal macrophages were plated into a 96-well plate at a density of 1 105 per well and cultured overnight at 37 C in a humidified atmosphere (5% CO2 and 95% air). The cell viability was assayed by 2-(2-methoxy-4-nitrophenyl)-3-(4nitrophenyl)-5-(2,4-disulf-opheyl)-2H-tetrazolium monosodium salt (WST-8), using Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan). The conjugates and tuftsin were introduced to the cells at a tuftsin-equivalent concentration of 2, 10, 20 mmol/L, respectively. The cells cultured in the blank medium were taken as the control. After 24 h incubation, 10 mL CCK-8 solution was added to each well, followed by incubation for another 3 h. The absorbance value at 450 nm was measured with a plate reader (Varioskan Flash, Thermo Electron, Finland). 2.11. Statistical analysis At least three parallel experiments were conducted for each sample. The data were processed by Origin 8.0. Results were expressed as mean standard deviations (SD). Means of each treated group were statistically compared using the two-tailed Student’s t-test. p < 0.05 was considered statistically significant.
2.7. Phagocytosis assay Phagocytosis assay was carried out as described by Terashima M. et al. [21] with a few modifications. The collected macrophages were plated into a 48-well plate at a concentration of 3 105 cells per well. After incubation for 2 h at 37 C in a CO2 incubator, the cells were washed twice with PBS to eliminate the non-adherent cells, and cultured in DMEM. Fluorescent latex beads were incubated with 1% bovine serum albumin (BSA) for 30 min at 37 C followed by ultrasonication. The macrophages were washed twice with HEPES balanced bath solution (pH 7.5, 10 mM HEPES, 140 mM NaCl, 5 mM KCl, 2 mM CaCl2 and 1 mM MgCl2) and preincubated for 15 min at room temperature. After adding the fluorescein-labeled latex beads (beads to cells ratio 30:1) and the indicated concentration of conjugates or tuftsin, the cells were further incubated for 15 min at 37 C. The beads that were not phagocytosed were removed partly by discarding the culture medium and washing the cells with cold PBS for three times. Each macrophage preparation was fixed with 10% glutaraldehyde. The cells were observed under a fluorescent microscope (Leica DMI4000B, Germany), which permits examination with both visible and UV fluorescence light. The number of phagocytosed cells was counted at 400 in three separate fields. The cell surface-adhered particles and the phagocytosed particles can be distinguished by finely adjusting the focal depth in the microscopic observation [22]. The internalized particles usually locate in the same layer of macrophages, whereas surfaceadhered particles do not.
3. Results 3.1. Synthesis of C60etuftsin conjugates The synthesis of the conjugates of C60etuftsin is achieved according to Scheme 1. Namely, C60 is linked respectively to the carboxyl and amino terminals of tuftsin to obtain two conjugates, NH2etuftsineC60 and C60etuftsineCOOH. The NH2etuftsineC60 conjugate is synthesized in a solution phase, in which an equimolar combination of EDC and HBTU is employed as a coupling agent. The completely protected tuftsin [BocNH-Thr(tBu)-Lys(Boc)-ProArg(Pbf)eOH] is linked to the amino group of the C60 derivative, compound 1 (Scheme 1a). The resultant conjugate is highly hydrophobic and easily separated from the excess reactants by the silica gel chromatography. After deprotection process the NH2etuftsineC60 conjugate becomes highly hydrophilic. Then the crude product was purified by Sephadex G-15, and the red brown
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fraction was collected. Finally, the purity was tested by analytical YMC C4 HPLC, which shows a purity of 95% (Fig. 1a). On the other hand, the C60etuftsineCOOH conjugate was synthesized by a solidphase peptide synthesis protocol following previous work (Scheme 1b) [19,24]. The crude product was also highly hydrophilic and purified by Sephadex G-15 similarly. The resultant compound was analyzed by the RP-HPLC, which shows a purity of 98% (Fig. 1b). Both conjugates were characterized by ESI-MS, and Fig. 2 shows the correct spectra in accordance with their chemical structures. The DLS measurement shows the average size of the two conjugates are both in the range of 100e200 nm. The result demonstrates that the synthetic conjugates form homogenous aggregates in aqueous solution. Additionally, we assessed the endotoxin level of the aqueous solutions of the conjugates, as well as tuftsin with Limulus Amebocyte Lysate test (LAL test), because bacterial endotoxin LPS is an effective macrophages stimulator that might influence the following biological evaluation tests. The results show that at the highest concentration used in the following experiments (20 mM), the LPS content of the three samples are all lower than the detection limit. Therefore, these samples are considered free of endotoxin.
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carboxypeptidase B, pronase and subtilisin [25]. The LAP locating on the plasma membrane of human neutrophils, could remove the N-terminal threonine to yield a tripeptide Lys-Pro-Arg, which is an inhibitor of tuftsin activity [26]. Herein, the stability of the synthesized C60etuftsin conjugates as well as tuftsin per se against LAP degradation was investigated. The conjugates or tuftsin were incubated with LAP at 37 C, and the aliquots were taken at various time intervals. The amount of intact peptides was evaluated by the analytical HPLC. For tuftsin, there are two peaks in the HPLC chromatogram at 120 min. The right one at 1.86 min is the hydrolysate from tuftsin, which is probably the tripeptide Lys-ProArg [26]. While the C60etuftsin conjugates could not be recovered from the supernatant because they coagulated with the enzyme during the denaturing process by ethanol. Then we tried to quantify the degradation of the conjugates by the product generated during the hydrolysis. And it turns out that both of the conjugates show no such hydrolysate within 2 h incubation (Fig. 3a). Therefore, the results demonstrate that C60etuftsin conjugates have an excellent stability against LAP hydrolysis. Moreover, Fig. 3b indicates that the hydrolysis rate of tuftsin is quite fast with a half-life of about 16 min. After 1 h, the enzymatic hydrolysis reaches the equilibrium. 3.3. Stimulation of phagocytosis by C60etuftsin
3.2. Stability against leucine aminopeptidase (LAP) degradation One major complication for the clinical use of tuftsin is the instability against a battery of enzymes in serum. It has been reported that the biological activity of tuftsin is reduced by LAP,
Since the main target cells of tuftsin are phagocytes [16], we chose the murine peritoneal macrophages to investigate the immunomodulatory activity of the two synthesized conjugates. At first we assayed the phagocytosis activity of macrophages after
Fig. 1. RP-HPLC chromatogram of the purified conjugates. (a) NH2etuftsineC60, (b) C60etuftsineCOOH. The inset shows the UVeVis spectrum of the corresponding conjugate.
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Fig. 2. Electrospray mass spectra of the conjugates. (a) NH2etuftsineC60 (H2O), (b) C60etuftsineCOOH (H2O).
stimulation with the conjugates. It was reported that the natural tuftsin showed a bell-shaped activity profile in phagocytosis stimulation of macrophages [27], and the highest phagocytosis rate was observed at the concentration of 10 mmol/L. The same activity profile of tuftsin is observed in our experiment, but the two conjugates show different phagocytosis stimulation activity and concentration profiles (Fig. 4). NH2etuftsineC60 exhibits the similar activity profile as the natural tuftsin. Besides, NH2etuftsineC60 shows more potent stimulation ability than that of tuftsin at the same concentration. For C60etuftsineCOOH, the stimulation activity increases with increasing concentration, and at the same concentration the conjugate shows more potent activity than that of tuftsin too. 3.4. Chemoattractant effect of C60etuftsin Chemotaxis is the directed cell motility along a chemical gradient, and it is an important mechanism by which the inflammatory cells accumulate at the local sites. We conducted chemotaxis assay to evaluate the chemotactic activity of macrophages toward the C60etuftsin conjugates. As shown in Fig. 5, both NH2etuftsineC60 and C60etuftsineCOOH trigger a significant chemoattractant effect in the tested concentration 2e20 mmol/L. The activity increases with increasing concentration in both cases of the two conjugates. It was reported that tuftsin exerts chemotactic effect on granulocytes and monocytes in the concentration range of
103e102 mmol/L [28]. In our experiment, we also observe a significant chemotactic activity of tuftsin, especially at the concentration of 10 mmol/L. Whereas, the chemotactic index decrease slightly at 20 mmol/L. Comparing with tuftsin, the two conjugates exhibit significant enhanced chemoattractant activity, especially for NH2etuftsineC60. And the chemotactic index increases with increasing concentration for both the two conjugates, which is different from that of tuftsin.
3.5. Stimulation of MHC II molecule expression by C60etuftsin We also measured the MHC II molecule expression on the surface of macrophages. Macrophages are essential in the activation of the adaptive immune system via phagocytosing, processing and presenting of antigens. Since both NH2etuftsineC60 and C60etuftsineCOOH conjugates can up-regulate the phagocytic and chemotactic activities of macrophages, we speculate that they may also influence the presentation of antigens. MHC II is mainly expressed on the antigen-presenting cell surface, such as macrophages, dendritic cells and B cells. The main function of MHC II is to bind and present fragments of exogenous antigen to T cells. Our results in Fig. 6 show that both NH2etuftsineC60 and C60etuftsineCOOH can significantly up-regulate MHC II expression on the surface of murine peritoneal macrophages, whereas tuftsin per se does not influence the expression of MHC II.
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Fig. 3. Stability of tuftsin and C60etuftsin conjugates against enzymatic degradation by leucine aminopeptidase. (a) Representative HPLC chromatogram of aliquots at indicated time point (0 and 120 min). (b) Percentage of the intact peptide in aliquots taken at different time points.
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Fig. 4. Analysis of the phagocytosis of fluorescent latex beads by peritoneal macrophages in vitro. Data are presented as mean SD (n ¼ 3). *p < 0.05, compared with the control group.
3.6. Toxicity of the C60etuftsin conjugates To assess the biocompatibility of the synthesized conjugates, macrophages were exposed to tuftsin or conjugates at the concentration range of 2e20 mmol/L. As Fig. 7 shows, the cell viabilities of all the tested materials have no significant difference to the control, which indicates that they are non-toxic to macrophages. 4. Discussion Tuftsin has been discovered for several decades, however, practical use of tuftsin in cancer therapy and microbial infections still needs to develop more stable and potent tuftsin analogs. In years past researchers have investigated various approaches to improve the stability of tuftsin and preserve its biological activity as well. These approaches include substitution of the N-terminal threonyl residue, substitution of L-amino acids by D-amino acids and elongation of the tetrapeptide chain by the addition of further
Fig. 5. Chemotactic effect of peritoneal macrophages toward tuftsin or conjugates. Data are presented as mean SD (n ¼ 3). *p < 0.05, compared with control group.
amino acid residues [29]. With these approaches the metabolic stability of tuftsin can be enhanced in some extent, whereas the biological activity usually decreases or even be abrogated at all. In this work, we have established an effective approach by conjugating tuftsin with the nanocarrier C60. The stability assay have demonstrated that both the NH2etuftsineC60 and C60etuftsineCOOH conjugates are much stable than the natural tuftsin. Furthermore, the subsequent biological evaluation tests all suggest that the immunostimulating activities of tuftsin are well preserved and even significantly enhanced. Therefore, we are confident that the approach reported in this paper is rather promising for the further application of tuftsin in the clinic. Herein, to achieve high synthetic yield, different synthesis protocols were employed respectively for the preparation of NH2etuftsineC60 and C60etuftsineCOOH conjugates. It has been demonstrated that solid-phase synthesis is an useful and effective method for the construction of fullereneepeptide conjugates [24,30]. In the synthesis of C60etuftsineCOOH, a fulleropyrrolidinoglutamic acid (compound 2, Scheme 1) was used for the conjugation of C60 to tuftsin. The result illustrates that this fullerene amino acid is a good building block for the solid-phase synthesis of fullereneepeptide conjugate. On the other hand, solution phase synthesis is also a convenient route for the construction of fullereneepeptide conjugate. Using the activation agent EDC/HBTU, we obtained the NH2etuftsineC60 with a quite high yield. Moreover, we found that Sephadex G-15 size exclusion chromatography is an effective method for the purification of the C60etuftsin conjugates. At the beginning, we tried to use a semi-preparative C4 RP-HPLC column, whereas almost all the product retained on the column and could not be eluted due to the interaction between C60 and the stationary phase of RP-HPLC. Then the Sephadex G-15 chromatography was used instead. And the results turn out that a high yield and purity have been achieved. The stability of the synthetic conjugates is a main concern in our present work, since it is an obstacle for the potential clinical use of tuftsin. Fortunately the above degradation test with leucine aminopeptidase (LAP) has shown that both of the conjugates are fully resistant to the LAP enzymatic hydrolysis. The inhibition effect of fullerene derivatives to various enzymes has been investigated [5,31,32], however the interaction between fullerene and LAP has not been reported. We speculate that there might be steric hindrance effect of C60 toward LAP, hence results in the inhibition of C60etuftsin conjugates degradation. Anyhow, further study on this issue is needed to clarify the inhibition mechanism. It is noticed that, there are somewhat differences lying between the two conjugates. In the phagocytosis assay, NH2etuftsineC60 exhibits a highest phagocytosis activity at 10 mmol/L concentration, while for C60etuftsineCOOH, the phagocytosis rate increases with increasing concentration. In the chemotaxis assay, the chemoattractant activity of the both conjugates increases along with increasing concentration. And at all the three tested concentrations, NH2etuftsineC60 exhibits more potent chemoattractant effect than C60etuftsineCOOH. Additionally, in the MHC II expression assay, the stimulation activity of NH2etuftsineC60 is also more potent than that of C60etuftsineCOOH. The different activities of NH2etuftsineC60 and C60etuftsineCOOH are probably determined by their chemical structures, since the C60 conjugation may influence the interaction of tuftsin and its receptor on the surface of macrophages. Based on the previous structureefunction studies of tuftsin, elongation on the amino terminal may decrease the activity, since the integrity of the free N-terminus of the Thr1 residue is found crucial for the manifestation of biological activity [17]. In the synthesis of NH2etuftsineC60, C60 is conjugated to the carboxyl terminal of tuftsin, presumably the activity of tuftsin is not affected. Therefore, in most of the cases the biological activity of
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Fig. 6. (a). Flow cytometry analysis of MHC II molecule expression of peritoneal macrophages after 24 h stimulation by tuftsin or conjugates at tuftsin-equivalent concentration of 10 mmol/L. The shadows and solid lines represent isotype control and FITC-labeled mouse antibodies specific for MHC II stained cells respectively. The percentage of positive cells is shown in the middle of each histogram. (b). Comparison on the percentage of positive cells for MHC II molecule expression on peritoneal macrophages induced by tuftsin and conjugates. Data are presented as mean SD (n ¼ 3). *p < 0.01 compared with negative controls.
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phagocytosis and chemotaxis of murine peritoneal macrophages. Moreover, they can up-regulate significantly the MHC II molecule expression on the surface of macrophages, whereas the free tuftsin shows no such effect. Our results demonstrate that the C60 conjugation enhances the immunostimulation activity of tuftsin, and NH2etuftsineC60 conjugate exhibits more effective stimulation activity than C60etuftsineCOOH does. Furthermore, both the two conjugates show no toxicity to macrophages in the tested concentrations, that paves the way for the potential use of C60 fullerene as the vehicle for the delivery of immunostimulants. Acknowledgments We acknowledge the fund from the National Basic Research Program of China (973 Program) (No. 2011CB933402, 2009CB930303, and 2011CB911004), as well as the China Natural Science Foundation (Significant project No. 10490180). References
Fig. 7. The viability of murine peritoneal macrophages after 24 h exposure to tuftsin and the conjugates.
NH2etuftsineC60 is more potent than that of C60etuftsineCOOH is reasonable. Nevertheless, comparing with the natural tuftsin, both of the conjugates significantly enhance the phagocytosis and chemotaxis activities of the murine peritoneal macrophages. In addition, tuftsin itself does not exhibit stimulating activity on the MHC II expression, while both the two conjugates significantly up-regulate MHC II on the surface of macrophages. A previous report has pointed out that, nanoparticles larger than 100 nm usually exhibit the adjuvant activity toward immune cells [33]. And the DLS measurement has shown that the sizes of the aggregated C60etuftsin conjugates in solution are ranging from 100 to 200 nm. We therefore speculate that the nanoparticulate nature of C60etuftsin conjugates may play an important role for the immune activities of the two conjugates. In the last two decades, various nanoparticulate adjuvants, such as polymeric nanoparticles, immunostimulating complexes, latex, silica and polystyrene, have been extensively investigated for antigen delivery and immune stimulation [34,35]. Nevertheless, other vaccine adjuvants are still worthy of developing. In this work, fullerene C60 is found to be an effective carrier for the tetrapeptide tuftsin, both in the aspects of stability and immunostimulating activity. The synthetic C60etuftsin conjugates, including NH2etuftsineC60 and C60etuftsineCOOH, cannot only protect tuftsin from LAP degradation, but also enhance the immunostimulating activities of the natural tuftsin. The good biocompatibility as well as the potent immunostimulating activity of the synthesized conjugates demonstrates that the C60etuftsin conjugates might be potential candidates as the immunomodulators and vaccine adjuvants.
5. Conclusions We have successfully conjugated fullerene C60 to the immunomodulating peptide tuftsin by covalently linking with the carboxyl terminal and the amino terminal respectively. The two compounds, NH2etuftsineC60 and C60etuftsineCOOH, were satisfactorily characterized by RP-HPLC and ESI-MS. The synthetic C60etuftsin conjugates show complete resistance to the leucine aminopeptidase degradation. At the same time, both of the conjugates show more potent stimulation activities than the free tuftsin in the
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