WO2010037878A1 - Utilisation of nanoparticles of noble metals as immunomodulators and immunomodulator composition - Google Patents

Abstract

The present invention has as its subject the utilisation of the immunomodulator effects of metal nanoparticles of silver functionalised with tiopronin [N-(2-mercaptopropionyl) glycine]. A second aspect subject of the invention is an immunomodulator composition including silver nanoparticles functionalised with tiopronin.

Description

 TITLE:

Use of noble metal nanoparticles as immunomodulators and immunomodulatory composition

SECTOR AND OBJECT OF THE INVENTION

Chemical, biochemical, immunological sector. Product for biomedical applications. Immunotherapy

One aspect of the present invention is the use of the immunomodulatory effects of silver metal nanoparticles functionalized with thiopronin [iV- (2-mercaptopropionyl) glycine]. A second aspect is an immunomodulatory composition that includes silver nanoparticles functionalized with thiopronin.

STATE OF THE TECHNIQUE In recent years, interest in the synthesis and characterization of different types of metal nanoparticles has increased, especially those that can be coupled to biomolecules. This interest has been enhanced by the expectations derived from these nano-bioconjugates in a wide range of applications in the design of new drugs, biomarkers, construction of nanodevices, or for use as analytical elements of special sensitivity [Blow, N. Nanotechnology in biology: big collaborations for a small world. NatMethods 5, 569-74 (2008) and Kogan, MJ. et al. Peptides and metallic nanoparticles for biomedical applications. Nanomed l, 287-306 (2007)]. Among the different types of nanoparticles, those that have a nucleus formed by a noble metal are especially interesting, mainly due to their plasmonic properties, which allow it to act as molecular markers [Gong, JL et al. Ag / SiO2 core-shell nanoparticle-based surface-enlianced Raman probes for immunoassay of cancer marker using silica-coated magnetic nanoparticles as separation tools. Biosens Bioelectron 22, 1501-7 (2007)], together with its signal amplification effects in RAMAN and SEIR spectroscopy. They are also used as contrast elements in electron microscopy [Murphy, CJ. et al. Anisotropic metal nanoparticles: Synthesis, assembly, and optical applications. J Phys Chem B 109, 13857-70 (2005)]. On the other hand there is a growing interest in its use as "nanocarriers" of chemotherapeutic agents or as naked nanoparticles as potential cytostatic or anti-angiogenic agents [Jain, KK Nanomedicine: application of nanobiotechnology in medical practice. Med Princ Pract 17, 89-101 (2008)]. There is also a concern due to the systematic ignorance of its potential toxic effects or those related to environmental problems [Linkov, L, Satterstrom, FK & Corey, LM Nanotoxicology and nanomedicine: making hard decisions. Nanomedicine 4, 167-171 (2008)]. Recently, works that explore the biological effects of nanoparticles, not only those that have been functionalized for a specific purpose, but of those called "naked" or naked, are taking on a significant boom. An example of basic functionalization, used as a platform for more complex functionalizations, is the silver nanoparticles functionalized with thiopronin (Figure 1). Thiopronin functionalization is a standardized technique since it serves as a platform for future functionalizations due to the free carboxyl group since it solubilizes silver nanoparticles that have previously been reduced from Ag to Ag in the presence of NaBH ₄ . The binding of thiopronin to silver is produced by its -SH group, its synthesis and characterization being very standardized [Song, Y., Huang, T. & Murray, RW Heterophase ligand exchange and metal transfer between monolayer protected clusters. J Am Chem Soc 125, 11694-701 and Huang, LW & Murray, RW Luminescence of tiopronin monolayer-protected silver clusters changes to that of gold clusters upon galvanic core metal exchange. J Phys. Chem. B 107, 7434-7440 (2003)]. In this context, the study of the effects of these nanoparticles functionalized with thiopronin (Ag @) on the immune system is proposed. In particular, their effects on the main pathogen detection system, that constituted by ToIl receptors (TLR). ToIl receptors recognize what is known as pathogen-associated patterns or PAMPs (from pathogen-associated molecular patterns [Akira, S., Uematsu, S. & Takeuchi, O. Pathogen recognition and innate immunity. CeIl 124, 783-801 ( 2006)] These TLRs thus form the main system of detection of what is known as innate immunity and in this sense they are fundamental to recognize the own thing of the alien in the organism human. For this reason, the modulation of responses induced by the activation of TLRs is suggested as a therapeutic target in infectious diseases, sepsis, inflammatory and / or autoimmune diseases or in the development of vaccines [Romagne, F. Current and future drugs targeting one class of innate immunity receptors: the Toll-like receptors. Drug Discov Today 12, 80-7 (2007)]. In any case, the characterization of the effects on critical responses in the immune system is fundamental in materials that will be part of biomedical applications. Within the cellular components responsible for innate immunity, the macrophage is a fundamental cell type and is therefore the cell type of choice in numerous studies leading to the identification of new immunomodulatory molecules [Trinchieri, G. & Sher, A. Cooperation of Toll-like receptor sign in innate immune defense. Nat Rev Immunol 7, 179-90 (2007)]. Taking into account these antecedents, the effects of Ag @ on TLR-mediated signaling in the macrophage Raw 264.7 cell line were studied in order to identify potential immunodulatory activities. To date, there is only one previous study on the effects of nanoparticles on signaling mediated by stimulation of TLRs [Lucarelli, M. et al. Innate defense functions of macrophages can be biased by nano-sized ceramic and metallic particles. Eur Cytokine Netw 15, 339-46 (2004)]. However, these studies are performed with ceramic nanoparticles (silica, SiO ₂ ; titanium, TiO ₂ ; zirconium, ZrO ₂ ), or cobalt nanoparticles. The disadvantage of these metallic or ceramic nanoparticles is that they produce an increase in the secretion of pro-inflammatory cytokines. On the other hand, these nanoparticles have a relatively heterogeneous size. In the present invention, special care has been taken to characterize the immunomodulatory effects with a material of high homogeneity in size (approximately 5 nm).

As a result, a potential immunomodulatory effect of Ag @, specific and differential nanoparticles on certain TLRs has been identified. EXPLANATION OF THE INVENTION

It is a first aspect of the present invention the use of noble metal nanoparticles in the preparation of an immunomodulatory composition. Said nanoparticles have a silver core of size between 1 and 100 nm coated with a thiopronin monolayer.

Preferably, the nanoparticles have a silver core of size between 2 and 10 nm and even more preferably the nanoparticles have a 5nm silver core. The immunomodulatory composition acts on the TLR2, TLR2 / 6, TLR3 and TLR9 receptors and is used for the treatment of inflammatory pathologies produced by:

- bacterial infections, particularly meningitis.

- an overproduction of viral particles.

The immunomodulatory composition can be used ex vivo in immune cell therapies where cell transfer occurs and also as adjuvants in vaccination protocols.

Another aspect of the present invention is an immunomodulatory composition for the treatment of pathologies mediated by the TLR2, TLR2 / 6, TLR3 and TLR9 receptors. Said composition comprises nanoparticles with a silver core of a size between 1 and 100 nm coated with a thiopronin monolayer. Preferably, the nanoparticles have a silver core of size between 2 and 10 nm and even more preferably the nanoparticles have a 5nm silver core.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: A. Electron microscopy images of Ag @ nanoparticles obtained with the Philips-FEI CM200 miscroscope. B. Scheme of a thiopronin molecule adsorbed to an Ag nanoparticle (not to scale) indicating the atoms of the thiopronin molecule used in the interpretation of NMR spectra. Figure 2: Effect of Ag @ on the viability of Raw 256.7 measured as release of LDH (membrane integrity) or mitochondrial function (reduction of MTT) after 24 hours of cultivation at the indicated conditions. MG-32 is a high toxicity proteasome inhibitor that we use as a positive control. Figure 3: Differential regulation of IL-6 production stimulated by different TLR ligands in Raw 264.7 in the absence or in the presence of Ag @. A. Shows the location TLRs on the cell surface. B. Shows the TLRs located in the endocytic compartment.

Figure 4: The previous exposure of Ag @ modulates the subsequent response of IL-6 production after stimulation with TLR ligands in Raw 264.7 A. It shows the localization TLRs on the cell surface. B. Shows the TLRs located in the endocytic compartment.

DETAILED DESCRIPTION AND MODE OF EMBODIMENT OF THE INVENTION

The TLRs can be divided into two groups according to their cellular location: TLRs 1, 2, 4, 5, 6 are mainly located on the cell surface and primarily recognize components of the battery wall, on the contrary the TLRs 3, 1, 8, and 9 are found in endocytic compartments and primarily recognize viral products. There are 13 paralogs identified to date in mouse and human genomes, however the ligands of some of them are not known for now. Binding of the ligand to TLR produces a secretion of pro-inflammatory cytokines such as the IL-6 chosen in the present invention [Akira, S., Uematsu, S. & Takeuchi, O. Pathogen recognition and innate immunity. CeIl 124, 783-801 (2006)].

Two types of approaches have been made:

1) Studies where Ag @ cells are treated in the presence and / or absence of different TLR ligands, and IL-6 secretion is monitored by ELISA in supernatants obtained after 24 hours. 2) Studies in which cells are treated in the presence or absence of Ag @ for 24 h, they are washed, and subsequently treated with different TLRs for another 24 hours. At the end of this period the secretion of IL-6 is monitored by ELISA in the obtained supernatants. This approach is interesting because it provides information about the state of hypo- or hyper-sensitivity in which the cells would be left when they are subjected to an Ag @ exposure and subsequently to an immune stimulus. Reagents

Silver nitrate (AgNO ₃ , 99.8%, Panreac, Lyon, France), _V- (2- mercaptopropionyi) glycma (thiopronin,> 98%) and NaBH ₄ , 98% come from Sigma-Aldrich, St. Louis, MO, USES. Water grade Milli-Q, Millipore, Billeríca, MA, USA.

Synthesis of Ag @

Ag @ thiopronin (Ag @) are prepared by reduction of AgNO ₃ using NaBH ₄ as a reducing agent, in an aqueous solution containing thiopronin (thiopronin / Ag molar ratio of 3: 1), according to procedures described previously [Song, Y ., Huang, T. & Murray, RW Heterophase ligand exchange and metal transfer between monolayer protected clusters. J Am Chem Soc 125, 11694-701 (2003) and. Huang, LW & Murray, RW Luminescence of tiopronin monolayer-protected silver clusters changes to that of gold clusters upon galvanic core metal exchange. J Phys. Chem. B 107, 7434-7440 (2003)].

Spectroscopic characterization

NMR spectra are obtained at 500 MHz on a Bruker AMX-500 spectrometer at room temperature in deuterated water. The HMBC (Heteronuclear Multiple Bond Coherence) studies are optimized for a J _H1C -S HZ and the TOCSY (Total Correlation Spectroscopy) studies are carried out with a DPFGSE sequence, 50 ms selective pulses and a 120 ms mixing time .

Transmission electron microscopy (TEM)

A Philips-FEI CM200 high resolution microscope (Hillsboro, OR, USA) was used. Infrared spectroscopy (FTIR)

A Bruker IFS 66 / s spectrometer with DTGS detector (Billerica, MA, USA) was used. 150 scans were acquired with a frequency of 2.5 Hz and resolution of 1 cm ^"1. All spectra are obtained in KBr tablets with 10 mg of nanoparticles.

UV-visible spectroscopy

An Ocean optics spectrophotometer (Dunedin, FL, USA) with HR4000 detector was used. The characterization of Ag @ is summarized in the following two tables:

Table 1. Assignment of positions after the ¹ H-NMR spectra and comparison between free and functionalized thiopronin to Ag and Au [Kohlmann, O., Steinmetz, WE., Mao, XA et al. NMR Diffusion, Relaxation, and Spectroscopic Studies of Water Soluble, Monolayer-Protected GoId Nanoclusters. J Phys. Chem. B. 105, 8801-8809 (2001). The designation of protons as 1, 2, 3 corresponds to that shown in Figure 1.

Ag @ (present Au @ patent Thiopronin Group)

Methyl (1) 1.48 1.6 1.8

Metino (2) 3.65 4.3 4.2

Methylene (3) 4.01 4.0 3.9-3.7

Table 2. Comparison of the most intense FTIR peaks (cm ^'1 ) for Ag @ with Au @ [De la Fuente, JM, Berry, CC, Riehle, MO, Curtís, SG Nanoparticle Targeting at Cells. Langmuir 22, 3286-3293 (2006)] demonstrating a similar adsorption of thiopronin. Au @ 2925 2852 1722 1644 1531 1384 1199 1014

Ag @ 3257 1717 1638 1533 1075

3067 2900 1389 1212 1009

Stimulation of Toll-like receptors (TLR) in the Raw 264.7 cell line and quantification of IL-6

The cell line comes from the European Collection (ECACC, Gate Down, Wiltshire, UK). Cultivation conditions are the standards and previously used. Cultures are carried out in 12-well plates in a final volume of 2 mL. Macrophages are treated for 24 hours with specific TLR ligands at the concentration previously optimized in the absence or in the presence of 10 ppm of Ag @. The list of ligands and final concentrations used are indicated below: Lipopolysaccharide (1 μg / mL LPS. E. coli serotype 0127: B8, Sigma, St. Louis, MO, USA), Oligodeoxynucleotides with unmethylated CpG motifs (1 μg / mL CpG +. CpG-DNA 1668: 5'- TCCATGACGTTCCTGATGCT-3 ', TIB MolBiol, Berlin, Germany), polyinosinic acid: polycycidyl (50 μg / mL poly I: C), lipoteic acid (10 μg / mL LTA), lipopeptide synthetic bacterial Pam ₃ CSK4 (300 ng / mL), bacterial DNA (10 μg / mL DNA), synthetic mycoplasma lipoprotein (1 μg / mL FSL-I), peptidoglycan (10 μg / mL PGN), imiquimod (10 μg / mL IMQ) and ssRNA40 (0.25 μg / mL) were obtained from InvivoGen, San Diego, CA, USA. Supernatants are stored after 24 hours of treatment and the production of IL-6 is measured by conventional ELISA according to the manufacturer's instructions (OptEIA Mouse IL-6 set, BD Pharmingen, San Diego, CA, USA).

Cytotoxicity studies

The lactate dehydrogenase (LDH) enzyme detection system called Cytotoxicity Detection kit (Roche Basel, Switzerland) has been used. Proliferation is measured by quantifying the reduction of 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyl tetrazolium bromide (MTT) by the system provided by Roche Basel, Switzerland. . Results related to cytotoxic effects

Cytotoxic effects were characterized by quantifying the release of LDH in the medium and quantifying the MTT reducing capacity. Both parameters measure, respectively, the integrity of the cell membrane and the metabolic capacity of the cells and are compromised when the cell viability is reduced. Figure 2 shows that Ag @ (1-100 ppm) have no cytotoxic effects on Raw 264.7 cells, therefore the effects observed by

Ag @ are not due to a compromise of the viability of Raw 264.7 cells, but to a specific alteration of them in the TLR signaling system.

Results of the co-treatment studies

When co-treatment studies are carried out, it is observed that Ag @ are not pro-inflammatory agents since the baseline levels of IL-6 production were not affected (Figure 3). It is interesting to insist that Ag @ are a better option compared to other metal or ceramic nanoparticles that do produce an increase in the secretion of pro-inflammatory cytokines [Lucarelli, M. et al. Innate defense functions of macrophages can be biased by nano-sized ceramic and metallic particles. Eur Cytokine Netw 15, 339-46 (2004)]. However, Ag @ differently inhibit IL-6 secretion mediated by TLRs located on the cell surface (Figure 3A) or in the endocytic compartments (Figure 3B). The presence of Ag @ (10 ppm = 20 μg / 106 cells) significantly inhibited the secretion of IL-6 mediated by TLR2 (peptidoglycan) in 55% or TLR2 / 6 (lipoteic acid) in 77% compared to the values obtained in the presence of specific TLRs ligands in the absence of Ag @. The inhibitory effect is evident in the case of TLRs located in the endocytic compartment, although in a less accentuated manner (Figure 3B). The inhibition was 22.5% in the case of the TLR3 ligand (poly [I: C] acid) or 31% in the case of the TLR9 ligand. Results of pretreatment studies

When macrophages are exposed, in one case to Ag @ for 24 hours (10 ppm = 20 μg / 106 cells) or in the absence thereof as a control, and subsequently stimulated with the different TLR ligands, pretreatment with Ag @ increased IL-6 production in response to ligand Pam3CSK4 (stimulation of TLR2 / 1) and ligand FSL-I (TLR2 / 6), by 40% and 31%, respectively (Figure 4A). That is, pretreatment with nanoparticles produces an increased susceptibility state to the same immune stimulus. Likewise, stimulation with TLR2 ligands produces after pretreatment with Ag @ a 37% decrease compared to untreated controls (Figure 4A). It is noteworthy that pretreatments produce a diminished response in the case of TLR3 or TLR9 ligands (Figure 4B).

Claims

1.- Use of noble metal nanoparticles in the preparation of an immunomoductor composition, characterized by the fact that the nanoparticles have a silver core of between 1 and 100 ranks coated with a thiopronin monolayer. 2. Use of noble metal nanoparticles in the preparation of an immunomoductor composition according to claim 1, characterized in that the nanoparticles have a silver core of size between 2 and 10 nm. 3, - Use of noble metal nanoparticles in the preparation of an immunomoductor composition according to claim 2, characterized in that the nanoparticles have a 5nm silver core. 4, - Use of noble metal nanoparticles in the preparation of an immunomoductor composition according to claims 1 to 3, characterized in that the immunomoductor composition acts on the TLR2, TLR2 / 6, TLR3 and TLR9 receptors. 5. Use of nanoparticles of noble metals in the preparation of an immunomoductor composition according to claim 4, for the treatment of inflammatory pathologies caused by bacterial infections, particularly meningitis. 6. Use of nanoparticles of noble metals in the preparation of an immunomoductor composition according to claim 4, for the treatment of inflammatory pathologies caused by an overproduction of viral particles. 7. Use of noble metal nanoparticles in the preparation of an immunomoductor composition according to claims 1 to 3 as modulators ex vivo in immune cell therapies where a cell transfer occurs. 8. Use of noble metal nanoparticles in the preparation of an immunomodulatory composition according to claims 1 to 3 as adjuvants in vaccination protocols. 9.- Immunomodulatory composition for the treatment of pathologies mediated by the TLR2, TLR2 / 6, TLR3 and TLR9 receptors, characterized in that said composition comprises nanoparticles with a silver core of a size between 1 and 100 nm coated with a thiopronin monolayer. 10. Immunomodulatory composition for the treatment of pathologies mediated by the TLR2, TLR2 / 6, TLR3 and TLR9 receptors according to claim 9, characterized in that the silver-core nanoparticles coated with a thiopronin monolayer have a size between 2 and 10 nm. 11. Immunomodulatory composition for the treatment of pathologies mediated by the TLR2, TLR2 / 6, TLR3 and TLR9 receptors according to claim 9, characterized in that the silver-core nanoparticles coated with a thiopronin monolayer are 5 nm in size.