Hybrid biomatrix and method for producing same
A hybrid biomatrix production method using supercritical CO2 and genipin stabilization, combined with chitosan nanoparticle coatings, addresses biocompatibility and antimicrobial issues in biological transplants, enhancing stability and durability.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- OBSHCHESTVO S OGRANICHENNOJ OTVETSTVENNOSTYU MNT
- Filing Date
- 2024-12-11
- Publication Date
- 2026-06-18
AI Technical Summary
Existing methods for producing biological transplants suffer from low biocompatibility and antimicrobial properties due to the use of toxic detergents and complex, multi-stage coating processes, leading to issues like infective endocarditis and calcification.
A hybrid biomatrix production method involving two-stage decellularization with supercritical CO2 and Tween-80, stabilization with genipin, and application of a chitosan nanoparticle coating with antimicrobial additives like silver or vancomycin under pressure, ensuring biocompatibility and antimicrobial properties.
The method enhances biocompatibility, stability, and durability of biological transplants by preventing calcification and providing an intelligent antimicrobial response, while using safe, natural compounds and simplifying the coating process.
Abstract
Description
[0001] Hybrid biomatrix and method for its production
[0002] AREA OF TECHNOLOGY
[0003] The invention relates to the field of biotechnology, medicine and cosmetology, in particular to a hybrid matrix used as a biological transplant used for the manufacture of heart prostheses.
[0004] LEVEL OF TECHNOLOGY
[0005] A coated graft and a method for producing it are disclosed in RLI 2519219 C1, published June 10, 2014, a prototype. The method for applying a chitosan coating to the pericardium of a biological heart valve prosthesis involves direct application from a 0.1-2% chitosan solution in water saturated with carbon dioxide under pressure.
[0006] The disadvantage of the technical solution disclosed above is the low biocompatibility of the surface of the biological transplant, due to the presence of unremoved donor cells and its weak antimicrobial properties, insufficient to resist the development of infective endocarditis.
[0007] In addition, a coated graft and a method for producing the same are known from the prior art, disclosed in RU 2796364 C2, published 05 / 22 / 2023. The method for processing a graft includes cleaning the graft and applying a polyelectrolyte coating, wherein cleaning is carried out using a hybrid method by preliminary exposure in an aqueous 1% SDS solution for 1 hour at a temperature of ~40 °C, followed by exposure in a special sealed autoclave in a medium saturated with carbon dioxide with a small additive equal to 1 vol. %, a three-component solution: Tween-80 - 2%, ethyl alcohol - 20%, water - 78%, in an autoclave at a temperature of 39 °C and a pressure of 15 MPa for 1 hour, in an autoclave; The coating was applied sequentially using a direct method in three stages: a) a 0.8% chitosan solution, b) a 0.1% antimicrobial agent solution, and c) a 0.5% hyaluronic acid solution. All stages of coating layer application were carried out using solutions in water saturated with carbon dioxide under pressure.
[0008] The disadvantage of the technical solution disclosed above is the low biocompatibility of the surface of the biological transplant, due to the use of the toxic detergent SDS at the pre-processing stage, as well as the difficulty of obtaining the matrix, due to the multi-stage production of a complex coating.
[0009] DISCLOSURE OF THE INVENTION
[0010] The objective of the claimed invention is to develop a hybrid biomatrix that ensures biocompatibility of the surface of a biological transplant, imparting additional pronounced antimicrobial properties due to a bioactive coating with the relative ease of obtaining such a coating, using natural safe compounds, the processing and coating of which does not violate the main advantages of biotransplants, as well as preventing its calcification.
[0011] The technical result of the invention is to increase the biocompatibility of the surface, stability and durability of the biological transplant, providing an antimicrobial response and preventing calcification.
[0012] The said technical result is achieved due to the fact that the method for producing a hybrid biomatrix includes the following stages: a) a two-stage preliminary purification of the biomatrix base for 0.75-6 hours, including decellularization of the xenopericardium in a 2-5 wt. % aqueous-alcoholic solution of Tween-80 saturated with CO2, at a temperature of 20-40 °C and a pressure of 10-30 MPa and subsequent decellularization of the xenopericardium in a multicomponent system containing a solution based on supercritical CO2 and an additive of 2-5 wt. % aqueous-alcoholic solution of Tween-80 in an amount of 1-1.5 vol. %, at a temperature of 20-40 °C and a pressure of 10-30 MPa; b) stabilization of the biomatrix base by treatment with a 0.1-0.5 wt. % aqueous buffer solution of genipin with a pH of 6.0-7.4 for 3-4 hours; c) Application of a coating of an aqueous solution of chitosan nanoparticles with a doping antimicrobial additive in the form of silver nanoparticles or vancomycin in a carbonic acid solution on a pre-cleaned biomatrix base under a pressure of 20-30 MPa at a temperature of 23-27°C for 1-6 hours.
[0013] A co-solvent, ethanol, is added to the multi-component system.
[0014] Decellularization in a multicomponent system is carried out in 2-3 cycles, each cycle lasting 40-50 minutes, which includes stages of pumping and releasing pressure from 17-20 MPa to 8-9 MPa at a rate of 5-20 ml / min.
[0015] The biomatrix base is stabilized by treatment with a 0.1-0.5 wt.% aqueous solution of genipin saturated with CO2 at a pressure of 10-30 MPa.
[0016] After stage b), additional treatment is carried out with a 0.1-0.5 wt.% aqueous solution of genipin for 5-7 days.
[0017] After stage b), decompression is carried out with a CO2 discharge rate of 5-20 ml / min.
[0018] The coating is formed in a solution of a suspension of chitosan nanoparticles in carbonic acid in an autoclave under pressure.
[0019] The formation of the said suspension of chitosan nanoparticles is carried out by the ion gelation method, in which 1 vol. % aqueous solution of sodium tripolyphosphate (STP) is first added to the said suspension of chitosan nanoparticles at a rate of 0.2-1 ml / min, with a ratio of the doping antimicrobial additive and sodium tripolyphosphate solution from 1:10 to 1:5 and a ratio of the tripolyphosphate solution and chitosan from 1:4 to 1:8, followed by mixing the suspension components at a speed of 500-750 rpm.
[0020] The said technical result is also achieved due to the fact that the hybrid biomatrix contains a base of xenopericardium, onto the surface of which a coating of chitosan nanoparticles is applied, containing a doping additive in the form of silver or vancomycin nanoparticles, wherein the base is pre-treated by the method according to any of paragraphs 1-8.
[0021] IMPLEMENTATION OF THE INVENTION
[0022] The claimed hybrid biomatrix, containing a pre-treated xenopericardium base, onto the surface of which a coating of chitosan nanoparticles is applied, containing a doping additive in the form of silver or vancomycin nanoparticles, is obtained as follows.
[0023] At the first stage, a two-stage preliminary purification of the biomatrix base is carried out for 0.75-6 hours, including decellularization of the xenopericardium in a 2-5 wt. % aqueous-alcoholic solution of Tween-80 saturated with CO2, at a temperature of 20-40 °C and a pressure of 10-30 MPa and subsequent decellularization of the xenopericardium in a multicomponent system containing a solution based on supercritical CO2 and an additive of 2-5 wt. % aqueous-alcoholic solution of Tween-80 in an amount of 1-1.5 vol. %, at a temperature of 20-40 °C and a pressure of 10-30 MPa. At the specified pressures, greater penetration of the detergent solution into the porous matrix of the transplant, including into small nano-sized pores, occurs, and the specified processing time is the optimal time to avoid the destruction of the tissue histoarchitecture with longer exposure to the detergent or insufficient degree of processing.Decellularization of xenopericardium into a solution based on supercritical CO2 away from the critical point ensures efficient extraction of non-polar components of the transplant matrix, since the solubility of carbon dioxide increases with increasing pressure and density of the medium. A detergent in the form of the specified pacTBopaTween-80 ensures the formation of a homophase medium, allowing for the extraction of xenogeneic cells and cellular components to proceed uniformly and isotropically throughout the volume of the solution. The specified Tween-80 solution has the following component ratios, in vol. %: water - 70-80; ethanol (EtOH) - 15-28; Tween 80 - 2-5. These ratios ensure the formation of stable micelles of the water-in-oil type in the solution [C. Prieto, L. Calvo, J. Appl. Chem. 2013, ID 930356, 10-13,], which have high penetrating ability and can enhance the interaction of the solution with both polar and non-polar components of the transplant tissue [S. Gupta, S. P. Moulik, and J.PCI7RU2024 / 000367.
[0024] Pharm. Sci. 97 (2008) 22-45; Z. Gao, H. Choi, H. Shin, K. Park, S. J. Lim, K. J. Hwang, C. Kim / / Int. J. Pharm. 161 (1999) 75-86].
[0025] Next, the biomatrix base is stabilized by treatment with 0.1-0.5 wt.% aqueous buffer solution of genipin with pH=6.0-7.4 for 3-4 hours.
[0026] At the final stage, a coating of an aqueous solution of chitosan nanoparticles with a doping antimicrobial additive in the form of silver nanoparticles or vancomycin in a carbonic acid solution is applied to the pre-cleaned biomatrix base under a pressure of 20-30 MPa at a temperature of 23-27°C for 1-6 hours.
[0027] Vancomycin or silver nanoparticles have activity against Staphylococcus aureus (S. aureus), which causes postoperative endocarditis [Mader, T. Stanton, T., 1999. Feeding High Moisture Corn. Beef Cattle Handbook. Product of Extension Beef Cattle Resource Committee. Iowa State University].
[0028] If necessary, a co-solvent, ethanol, is added to the multi-component system to increase the polarity of the medium in order to extract the polar components of the transplant tissue.
[0029] If necessary, decellularization in a multicomponent system is carried out in 2-3 cycles, each cycle lasting 40-50 minutes, which includes stages of pumping and releasing pressure from 17-20 MPa to 8-9 MPa at a rate of 5-20 ml / min in order to most effectively remove already dissolved tissue components.
[0030] If necessary, the biomatrix base is stabilized by treatment with a 0.1-0.5 wt.% aqueous solution of genipin saturated with CO2 at a pressure of 10-30 MPa, which ensures deeper penetration of the crosslinker solution into the porous matrix of the transplant, including into small nanosized pores.
[0031] After the stabilization stage, additional treatment is carried out with a 0.1-0.5 wt.% aqueous solution of genipin for 5-7 days for complete cross-linking (stabilization) of collagen fibrils with genipin.
[0032] After stabilization of the decellularized transplant tissue with genipin, it is preferable to expose the purified and stabilized matrix to a solution in carbonic acid under pressure with already formed chitosan nanoparticles impregnated with antimicrobial agents.
[0033] After the stabilization stage, decompression is carried out with a CO2 discharge rate of 5-20 ml / min.
[0034] The coating is formed in a solution of a suspension of chitosan nanoparticles in carbonic acid in an autoclave under pressure. This suspension of chitosan nanoparticles is formed by ionic gelation, in which 1 vol. of an aqueous solution of sodium tripolyphosphate is first added to the said suspension of chitosan nanoparticles at a rate of 0.2-1 ml / min, with a ratio of the doping antimicrobial additive to the sodium tripolyphosphate solution from 1:10 to 1:5 and a ratio of the tripolyphosphate solution to chitosan from 1:4 to 1:8, followed by stirring the suspension components at a rate of 500-750 rpm, to obtain the most stable chitosan nanoparticles.
[0035] It is preferable for the polymer (TPP and chitosan) concentration in solutions of water saturated with carbon dioxide under pressure to be in the range of 0.2-1.0% by weight to achieve complete coverage of the graft surface. Using higher polymer concentrations is not economically feasible.
[0036] It is preferable to use chitosan with an MM of 100 to 500 kDa to obtain the most stable nanoparticles.
[0037] It is preferable that the silver nanoparticles synthesized in the chitosan solution before adding the TPP as an antimicrobial agent impregnated into the composite polymer particles have a size of 5-20 nm.
[0038] In particular, the polymer exposure time (application of a chitosan coating) can be 1-6 hours, since longer exposure to deposition conditions can lead to deterioration of the mechanical characteristics of the transplant tissue (possibly due to the extraction of elastin), and shorter exposure times are insufficient to form a homogeneous and mechanically stable polymer coating on the transplant tissue.
[0039] In particular, the coating application should be carried out in the solution in which the nanoparticles were synthesized, without additional isolation of the nanoparticles, in order to apply both the formed polymer particles and individual chitosan macromolecules for a more uniform and complete coating of the collagen fibrils.
[0040] In particular, the autoclave decompression rate after coating application should be 10-20 ml / min. Longer decompression times can cause the coating components to be torn off along with the flow of carbon dioxide and water.
[0041] It is preferable that the formed composite chitosan nanoparticles have the following characteristics: size 50-100 nm, zeta potential 20- 0 mV, to obtain the most uniform and stable coating /
[0042] Example 1
[0043] At the first stage, a two-stage preliminary cleaning of the biomatrix base is carried out for 0.75 hours, during which, at the first stage, decellularization of the xenopericardium is carried out in a 2% aqueous-alcoholic solution of Tween-80 (the ratio of components, in vol. %: water - 80; ethanol (EtOH) - 28; Tween 80 - 2), saturated with CO2, at a temperature of 20 ° C and a pressure of 10 MPa, and at the second stage - decellularization of the xenopericardium by feeding a multicomponent system containing a solution based on supercritical
[0044] CO2 and additives 2 wt.% in a water-alcohol solution of Tween-80 in an amount of no more than 1 vol.%, at a temperature of 20°C and a pressure of 10 MPa.
[0045] Next, the biomatrix base is stabilized by treatment with a 0.1 wt.% aqueous buffer solution of genipin with a pH of 6.0 for 3 hours.
[0046] At the final stage, a coating of an aqueous solution of chitosan nanoparticles with a size of 50 nm and a doping antimicrobial additive in the form of silver nanoparticles with a size of 5 nm in a carbonic acid solution is applied to the pre-cleaned biomatrix base under a pressure of 20 MPa at a temperature of 23°C for 1 hour.
[0047] Example 2
[0048] Example 2 differs from example 1 in that the first step involves a two-stage preliminary cleaning of the biomatrix base for 3 hours, wherein the first step involves decellularization of the xenopericardium in a 3 May. % aqueous-alcoholic solution of Tween-80 (component ratios, by vol. %: water - 75; ethanol (EtOH) - 22; Tween 80 - 3), saturated with CO2, at a temperature of 30 °C and a pressure of 20 MPa, and the second step involves decellularization of the xenopericardium in a multicomponent system containing a solution based on supercritical CO2 and an additive of 3 May. % aqueous-alcoholic solution of Tween-80 in an amount of no more than 1.25 vol. %, at a temperature of 30 °C and a pressure of 20 MPa; the biomatrix base is stabilized by treatment with 0.25 May. % aqueous buffer solution of genipin with pH=6.7 for 3.5 hours; application of a coating of an aqueous solution of chitosan nanoparticles with a size of 75 nm and a doping antimicrobial additive in the form of silver nanoparticles with a size of 10 nm in a carbonic acid solution under a pressure of 25 MPa at a temperature of 25°C for 3 hours onto a pre-cleaned biomatrix base.
[0049] Example 3
[0050] Example 3 differs from example 1 in that the first step involves a two-stage preliminary cleaning of the biomatrix base for 6 hours, during which the first step involves decellularization of the xenopericardium in a 5 May. % aqueous-alcoholic solution of Tween-80 (component ratios, by vol. %: water - 70; ethanol (EtOH) - 15; Tween 80 -5), saturated with CO2, at a temperature of 40 °C and a pressure of 30 MPa, and the second step involves decellularization of the xenopericardium in a multicomponent system containing a solution based on supercritical CO2 and an additive of 5 May. % aqueous-alcoholic solution of Tween-80 in an amount of no more than 1.5 vol. %, at a temperature of 40 °C and a pressure of 30 MPa; the biomatrix base is stabilized by treatment with 0.5 May. % aqueous buffer solution of genipin with pH=7.4 for 4 hours; application of a coating of an aqueous solution of chitosan nanoparticles with a size of 100 nm and a doping antimicrobial additive in the form of silver nanoparticles with a size of 20 nm in a carbonic acid solution under a pressure of 30 MPa at a temperature of 27°C for 6 hours onto a pre-cleaned biomatrix base.
[0051] Example 4
[0052] Example 4 corresponds to example 2, except that a doping antimicrobial additive in the form of vancomycin nanoparticles is used.
[0053] Decellularization with Tween-80 (without the use of toxic SDS), as well as supercritical CO2, ensures the death of donor cells and frees the tissue from immunogenic and calcification-inducing lipid components, preventing calcification in the recipient's body and increasing the biocompatibility of such transplants.
[0054] The use of genipin as a cross-linking agent (instead of glutaraldehyde) will contribute to an increase in the biocompatibility of the matrix, due to its much lower toxicity (10 4 times lower than for glutaraldehyde).
[0055] Chitosan coating applied from carbonic acid under pressure allows for effective masking of free aldehyde groups of glutaraldehyde. Such shielding is known to significantly reduce the intensity of calcification processes on the surface of biological tissue [Kuribayashi R, Efficacy of the chitosan posttreatment in calcification prevention of the glutaraldehyde-treated porcine aortic noncoronary cusp implanted in the right ventricular outflow tract in dogs I Kuribayashi R, Chanda J, Abe T. / / Artificial organs. - 1996. - V.20(7). - P.761-766; Rodas A, Cytotoxicity and endothelial cell adhesion of lyophilized and irradiated bovine pericardium modified with silk fibroin and chitosan. I Rodas A, Polak R, Hara P, Lee E, Pitombo R, Higa O. / / Artificial Organs. - 2011. - V.35(5). - P.502-507].Moreover, when chitosan was applied by the claimed method to decellularized and genepin-cross-linked biotissue, there was a high degree of calcification suppression compared to the closest analogue, apparently due to the effective removal of potentially immunogenic calcification-promoting cellular debris prior to the application of the protective screen.
[0056] The claimed method of decellularization of implant tissue and application of a protective screen of solvents based on carbon dioxide under pressure (supercritical (sc) CO2 and carbonic acid), which after decompression spontaneously and completely transform into hypoallergenic components that are absolutely safe for the human body: water and carbon dioxide contribute to a decrease in the cytotoxicity and immunogenicity of the implants. Indeed, by changing the external pressure it is possible to avoid the problem of residual solvent during decellularization in scCO2, as well as the degree of saturation of water with carbon dioxide, and, accordingly, the pH of carbonic acid, by changing the degree of protonation of chitosan, which makes it possible to change the solubility of chitosan [Sakai Y, A novel method of dissolving chitosan in water for industrial applications I Sakai Y, Hayano K, Yoshioka H, Yoshioka H. / / Polym. J. - 2001. - V.33. - P.640-642].
[0057] The media in which decellularization and application of a protective screen to biological tissue are carried out have sterilizing properties. Indeed, at high pressure, carbon dioxide molecules dissolved in water or in a supercritical state diffuse into bacteria, bacterial spores, and viruses and biologically inactivate them [Ellis J, (2010) Supercritical CO2 sterilization of ultra-high-molecular weight polyethylene / Ellis J. / / J. Supercrit. Fluids. - 2010. - V.52. - P.235-240]. Due to this property of the media, additional sterilization of implants can be eliminated, thereby simplifying the technological process of their preparation for subsequent surgery.
[0058] Using carbonic acid as a chitosan solvent promotes a stronger bond with the collagen fibrils of the graft tissue, as well as greater coating stability, compared to chitosan bound to acetate ions when applied from acetic acid solutions. Indeed, due to the weak binding of carbonate anions to the polycationic chitosan macromolecules, the chitosan chains, initially protonated (positively charged) in carbonic acid, lose their charge when the pressure is released after application. Upon release of pressure and an increase in pH, the charges on the already adsorbed polyelectrolyte macromolecules will dissipate, and the coating will assume a stable, water-insoluble form. Thus, in the body's internal environment, the decharged polyelectrolyte chains will have no affinity for water, resulting in improved mechanical stability of the applied coating.
[0059] A coating based on composite chitosan nanoparticles will ensure the implementation of the effect of an "intelligent" antimicrobial response triggered by infection. Indeed, potentially cytotoxic antimicrobial agents encapsulated in chitosan nanoparticles are separated from the bloodstream by a polymer shell. Moreover, the polymer shell swells with a decrease in the pH of the medium due to the recharge of pH-dependent chitosan, which will be caused by the vital activity of the bacterial film [Tommeraas, Melander. I Biomacromolecules 2008. 9. 1535-1540; Albright et.al. P ActaBiomater. 2017. 61. 66-74.], which, in turn, will trigger the release of an antimicrobial agent that is toxic to microbes and can inactivate them.
[0060] The claimed invention enables the creation of an "intelligent" protective shield formed by a single-step application of a coating based on composite chitosan nanoparticles impregnated with silver or vancomycin nanoparticles, formed in a carbonic acid solution, using a detergent solution saturated with CO2 under pressure. Information on such a method for treating biological transplants is absent in the scientific and technical literature. The claimed invention induces the death of donor cells and frees the tissue from immunogenic and calcification-inducing lipid components, prevents calcification in the recipient's body, improves the biocompatibility of such transplants, and enables the creation of a protective coating of nanoparticles that release antimicrobial agents during the development of an infectious infection.By selecting the right conditions for producing composite chitosan nanoparticles and applying them to a purified biomatrix from a carbonic acid solution under pressure, it is possible to create a stable coating of nanoparticles encapsulated with an antimicrobial agent. Thus, the proposed method for producing a biomatrix, compared to its closest analogue, differs in that the original biotissue is effectively pre-decellularized using a biocompatible method using significantly less toxic detergents (completely eliminating SDS) and, in a relatively simple, single-step process, a protective screen is created from a coating based on composite chitosan nanoparticles encapsulating the antimicrobial agent.
[0061] The invention has been disclosed above with reference to a specific embodiment. Other embodiments of the invention may be apparent to those skilled in the art without altering its essence as disclosed herein. Accordingly, the invention should be considered limited in scope only by the following claims.
Claims
CLAUSES OF THE INVENTION 1 . A method for producing a hybrid biomatrix comprising the following steps: a) two-stage preliminary purification of the biomatrix base for 0.75-6 hours, including decellularization of xenopericardium in a 2-5 wt. % aqueous-alcoholic solution of Tween-80 saturated with CO2, at a temperature of 20-40 °C and a pressure of 10-30 MPa and subsequent decellularization of xenopericardium in a multicomponent system containing a solution based on supercritical CO2 and an additive of 2-5 wt. % aqueous-alcoholic solution of Tween-80 in an amount of 1-1.5 vol. %, at a temperature of 20-40 °C and a pressure of 10-30 MPa; b) stabilization of the biomatrix base by treatment with 0.1-0.5 wt. % aqueous buffer solution of genipin with pH=6.0-7.4 for 3-4 hours; c) Application of a coating of an aqueous solution of chitosan nanoparticles with a doping antimicrobial additive in the form of silver nanoparticles or vancomycin in a carbonic acid solution on a pre-cleaned biomatrix base under a pressure of 20-30 MPa at a temperature of 23-27°C for 1-6 hours.
2. The method according to paragraph 1, characterized in that a co-solvent, ethanol, is added to the multi-component system.
3. The method according to paragraph 1, characterized in that decellularization in the multicomponent system is carried out in 2-3 cycles, each cycle is carried out for 40-50 minutes, which includes the stages of pumping and releasing pressure from 17-20 MPa to 8-9 MPa at a rate of 5-20 ml / min.
4. The method according to paragraph 1, characterized in that the stabilization of the biomatrix base is carried out by treatment with a 0.1-0.5 wt.% aqueous solution of genipin saturated with CO2, at a pressure of 10-30 MPa.
5. The method according to paragraph 1, characterized in that after step b), additional treatment is carried out with a 0.1-0.5 wt.% aqueous solution of genipin for 5-7 days.
6. The method according to paragraph 1, characterized in that after step b) decompression is carried out at a CO2 discharge rate of 5-20 ml / min.
7. The method according to claim 1, characterized in that the formation of the coating is carried out in a solution of a suspension of chitosan nanoparticles in carbonic acid in an autoclave under pressure.
8. The method according to paragraph 1, characterized in that the formation of said suspension of chitosan nanoparticles is carried out by the ionic gelation method, in which 1 vol. of an aqueous solution of sodium tripolyphosphate is first added to said suspension of chitosan nanoparticles at a rate of 0.2-1 ml / min, with a ratio of the doping antimicrobial additive and the sodium tripolyphosphate solution from 1:10 to 1:5 and the ratio of the tripolyphosphate solution and chitosan from 1:4 to 1:8, followed by mixing the suspension components at a speed of 500-750 rpm.
9. A hybrid biomatrix comprising a xenopericardium base, onto the surface of which a coating of chitosan nanoparticles is applied, containing a doping additive in the form of silver or vancomycin nanoparticles, wherein the base is pre-treated by the method according to any of paragraphs 1-8.