Process for the preparation of conductive PEDOT:PSS particles

Abstract

The invention relates to a process for the preparation of poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) particles comprising at least the following steps: a) providing a mixture comprising poly(3,4-ethylenedioxythiophene) and polystyrene sulfonate in at least an aqueous solvent; b) forming one or more PEDOT:PSS droplets by introducing the mixture from process step a) into an organic solvent A, wherein the aqueous PEDOT:PSS mixture forms the inside of the droplet and the organic solvent A forms the outside of the droplet; c) contacting the PEDOT:PSS droplets obtained from process step b) with a coagulation solution comprising a coagulation agent and at least one further solvent B, said coagulation solution having a density greater than the density of said organic solvent A and less than the density of the aqueous mixture of poly(3,4-ethylenedioxythiophene) and polystylene sulfonate; and d) coagulating the PEDOT:PSS droplets to PEDOT:PSS particles. Furthermore, the invention discloses spherical PEDOT:PSS particles which do not require further mechanical coagulation of the substance and the use of such particles, for example as cell culture microcarriers or as a suspended electrode.

Description

Technical Field

[0001] This invention relates to a process for preparing poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) particles, the process comprising at least the following steps:

[0002] a) Provide a mixture of poly(3,4-ethylenedioxythiophene) and polystyrene sulfonate comprising in a solvent containing at least water;

[0003] b) Forming one or more PEDOT:PSS droplets by introducing the mixture from process step a) into organic solvent a, wherein the aqueous PEDOT:PSS mixture forms the inside of the droplet and the organic solvent A forms the outside of the droplet;

[0004] c) Contacting the PEDOT:PSS droplets obtained from process step b) with a coagulation solution containing a curing agent and at least one additional solvent b, wherein the density of the coagulation solution is greater than the density of organic solvent a, but less than the density of the aqueous mixture of poly(3,4-ethylenedioxythiophene) and polystyrene sulfonate; solidifying the PEDOT:PSS droplets into PEDOT:PSS particles. Furthermore, this invention discloses spherical PEDOT:PSS particles that do not require further mechanical curing of the material, and the use of these particles, for example, as cell culture microcarriers or suspended electrodes. Background Technology

[0005] Many industries, such as food, cosmetics, and pharmaceuticals, require large quantities of complex biological materials. These materials are typically produced in stirred-tank reactors using cell cultures. Since most vertebrate cells are adherent, meaning they depend on a matrix for growth and proliferation, biocompatible carrier particles are often added to the culture medium. Poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) particles are the preferred choice here because their size is within the correct micron range, their surface structure facilitates cell anchoring to the particle surface, their particle density allows them to suspend in stirred-tank reactors with low energy input, and they are also biocompatible. PEDOT:PSS particles also offer a very high surface-to-volume ratio due to their specific porosity, providing a foundation for efficient, large-scale production of biological materials. Furthermore, PEDOT:PSS exhibits exceptional electrical properties as a material system. The particles can provide very fast charge / discharge kinetics and high energy and power densities because the charge is stored not only in the electron bilayer but also within the polymer matrix. These fundamental properties mean that these particles also represent a promising matrix in energy storage and energy conversion technologies.

[0006] To date, filled or porous PEDOT:PSS particles can only be produced in combination with composite or carrier materials, resulting in particles with inherent deficiencies, mechanical support, and moldability. The synthesis of these stabilized particles requires complex, multi-step processes, which provide only insufficient prerequisites for effective scalability, and in the case of mixed particles, the electrochemical properties of the particles deteriorate due to the presence of electrically inactive additive materials. Furthermore, there are no commercially available microcarriers or their processes based solely on a single, fully synthesized hydrogel.

[0007] Some methods for PEDOT:PSS microparticles can be found in patent literature.

[0008] For example, EP 28 311 83B1 describes a composite particle comprising: a single spherical core containing at least one inorganic oxide; and a polymer layer disposed on and defining the spherical core, the polymer layer comprising a cationic polymer and an anionic polymer.

[0009] In addition, EP 01 953 81B1 discloses a composite material of a porous material and a conductive polymer, wherein the surface of the pores is first coated with a conductive polymer layer obtained by treating the monomer with an oxidant, and a conductive polymer layer obtained by anodizing the monomer is coated thereon.

[0010] Another patent document, CN 110 233 061A, discloses a method for manufacturing a porous flexible PEDOT:PSS membrane with high conductivity. According to this method, polystyrene nanospheres are used as a matrix; a PEDOT:PSS dispersion and polystyrene nanospheres are mixed in situ; and a porous PEDOT:PSS membrane is obtained by vacuum suction filtration. The conductivity of the membrane is optimized by solvent post-treatment. The prepared PEDOT:PSS film has a porous structure and exhibits excellent electrochemical performance, such as relatively high conductivity, high charge-discharge stability, and high rate performance, making it suitable for high-performance thin-film electrodes or high-performance thin-film capacitors.

[0011] Such solutions, known from existing technologies, can offer further improvement potential, particularly in terms of the simplicity and reproducibility of the manufacturing process and the uniformity of the particles produced by the process.

[0012] Therefore, the object of the present invention is to at least partially overcome the disadvantages known in the prior art. In particular, the object of the present invention is to disclose a simple and repeatable manufacturing process that is easily scalable and provides highly precisely defined PEDOT-PSS particles within a short process time. Furthermore, the object of the present invention is to provide PEDOT:PSS particles that exhibit a very uniform shape and size distribution without the need for further polymer stabilization.

[0013] This task is accomplished by the features of the respective independent claims relating to the method of the invention, the particles of the invention, and the uses of the particles of the invention. Preferred embodiments of the invention are described in the dependent claims, the description, or the drawings, whereby other features described or shown in the dependent claims, the description, or the drawings may, individually or in any combination, constitute the purpose of the invention unless the context explicitly indicates otherwise.

[0014] According to the present invention, this problem is solved by a method for preparing poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) particles, the method comprising at least the following steps:

[0015] a) Provide a mixture of poly(3,4-ethylenedioxythiophene) and polystyrene sulfonate comprising in a solvent containing at least water;

[0016] b) Forming one or more PEDOT:PSS droplets by introducing the mixture from process step a) into organic solvent a, wherein the aqueous PEDOT:PSS mixture forms the inside of the droplet and the organic solvent A forms the outside of the droplet;

[0017] c) Contact the PEDOT:PSS droplets obtained from process step b) with a coagulation solution containing a curing agent and at least one additional solvent b, the density of which is greater than the density of the organic solvent a and less than the density of the aqueous mixture of poly(3,4-ethylenedioxythiophene) and polystyrene sulfonate; solidify the PEDOT:PSS droplets into PEDOT:PSS particles.

[0018] Surprisingly, the above method has been found to allow for the flexible preparation of fully porous PEDOT:PSS particles that possess mechanical stability even without further complexing or supporting materials. Because no further mechanical stabilizing materials are used, the inherent properties of the polymer system are preserved overall, avoiding unnecessary incompatibilities in applications. The two-step process according to the invention allows for cost-effective production and provides easy scalability. Different particle sizes and densities can be flexibly achieved, and highly reproducible, preferably circular, geometries can be obtained through this method. Another advantage is the ability to obtain a very uniform and narrow particle size distribution. After the reaction, the PEDOT:PSS polymer complex is a biocompatible porous hydrogel with stereochemical and mechanical properties comparable to the extracellular matrix, thus providing a good foundation for cell culture applications. The lack of further stabilizing polymers also has a positive impact on the electrical properties of the particles.

[0019] The method of the present invention is a method for preparing poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) particles. The method according to the invention provides PEDOT:PSS particles that consist of or contain a PEDOT-PSS matrix. In this respect, the particles can consist of or contain only these two monomers. Preferably, the matrix can contain only PEDOT and PSS. The particles have a closed, dense surface or a porous structure, in the case of a porous structure, where pores exist within the particles and / or on the particle surface. The pores can also provide continuous diffusion paths through individual particles. For example, the particle density can be adjusted by the solids content of the PEDOT-PSS solution used, thereby allowing a wide concentration range to be handled by this method. Preferably, the solids content of the aqueous PEDOT:PSS solution can be greater than or equal to 0.5% by weight and less than or equal to 10% by weight, more preferably greater than or equal to 1.0% by weight and less than or equal to 5.0% by weight. Even with relatively low solids content, sufficiently mechanically dimensionally stable particles can be produced by the method of the present invention. The porosity of the particles can vary over a wide range by the proportion of other solvents in the aqueous solution, and can preferably be greater than or equal to 0 volume% and less than or equal to 95 volume%, more preferably greater than or equal to 15 volume% and less than or equal to 60 volume%.

[0020] In step a), a mixture of poly(3,4-ethylenedioxythiophene) and polystyrene sulfonate is prepared in a solvent containing at least water. The starting material is an aqueous mixture of PEDOT and PSS, wherein a polymeric complex of positively charged PEDOT and negatively charged PSS is dispersed. In addition to water, this aqueous starting solution may also contain other solvents or dispersants, thereby allowing the porosity of the particles to be adjusted, for example, by the proportion and type of solvent. The term "solvent" as used herein is not used in the physical sense of producing a true "solution," but rather in the sense that these substances generally fall into the category of liquid solvents. If water is used "only" as a solvent or dispersant, non-porous particles will be produced. For example, a molar ratio of PEDOT:PSS greater than or equal to 1:6 and less than or equal to 6:1 may be present. For example, the weight concentration of PEDOT:PSS in the aqueous mixture may be greater than or equal to 1% by weight and less than or equal to 10% by weight.

[0021] In process step b), one or more PEDOT:PSS droplets are formed by introducing the mixture from process step a) into organic solvent A, wherein the aqueous PEDOT:PSS mixture forms the interior of the droplets and organic solvent A forms the exterior. The aqueous PEDOT:PSS dispersion is emulsified in the organic solvent to form an aqueous (PEDOT:PSS) emulsion in solvent A. The aqueous PEDOT:PSS thus forms the internal phase, and the organic solvent forms the external phase. Therefore, organic solvent A may not be completely miscible with water. Preferably, the water miscibility of organic solvent A at 20°C is less than or equal to 10 g / L, more preferably less than or equal to 5 g / L. Emulsification can be carried out purely by mechanical methods without further use of emulsifiers. Therefore, the use of mechanical stirrers, Turrax, or microfluidic devices with T-branch geometry may be suitable to obtain emulsions with the most uniform droplet size. However, the emulsion can also be obtained by ultrasonic treatment. The emulsion does not need to remain stable over a long period of time. Chemically stable organic solvent A is suitable for further processing. For example, these organic solvents can be medium-chain hydrocarbons without other reactive groups. For instance, C4-C10 hydrocarbons or alkanes can be used. Furthermore, organic solvents composed of hydrocarbons and one or more other functional groups can also be used. For example, medium-chain alcohols such as C5-C10 alcohols can also be used as solvent A.

[0022] Process step b) can be carried out, for example, within a coaxial droplet separation process, where the emulsion droplets are separated from the nozzle by the continuous phase. This transforms PEDOT:PSS into a spherical shape. Here, the continuous phase from solvent A completely encapsulates the PEDOT:PSS emulsion and acts as a shell to delay the curing process. The formation of monodisperse PEDOT:PSS emulsion droplets in the continuous phase can be achieved, for example, by inserting one or more sleeves concentrically into a slightly kinked tube. In this case, the continuous phase from solvent A is fed through the tube, and the PEDOT:PSS emulsion is fed through the sleeve. The protective sleeve composed of the continuous solvent A prevents the PEDOT:PSS emulsion from curing within the nozzle and clogging the equipment. This process cannot be performed without the external resistance of the droplets composed of solvent A as the continuous phase. Dies or other designed molds can also be used to form other molding geometries. By selecting the extrusion rate and speed, more or fewer elongated molded bodies, such as fibers, can also be produced.

[0023] In process step c), the PEDOT:PSS droplets obtained from process step b) are contacted with a coagulation solution containing a curing agent and at least one additional solvent B. The PEDOT:PSS droplets are then removed from the curing agent. Due to mechanical energy input, an emulsion containing PEDOT:PSS droplets is obtained, at least temporarily, from the organic solvent A and the aqueous PEDOT:PSS solution, with the droplets protected by the external solvent A phase. These encapsulated droplets are then transferred to the coagulation solution. The transfer of the encapsulated droplets to the coagulation solution can be done directly from, for example, the pipe used for feeding the continuous phase in process step b). The protective seal composed of the continuous phase is then rapidly separated from the PEDOT:PSS emulsion droplets by the density difference between the coagulation bath and the continuous phase, thereby bringing the aqueous PEDOT:PSS solution emulsion into contact with the coagulation solution containing at least one additional solvent B and the curing agent. Preferably, the curing agent can be uniformly dissolved or dispersed in the organic solvent B. The curing agent can be any substance capable of dissolving PSS from the PEDOT-PSS polymer composite and causing PEDOT crystallization. Preferably, the curing agent can be dissolved in organic solvent B. Possible curing agents can be selected from, for example, the group consisting of sulfuric acid, ionic liquids, high-concentration salt solutions, or mixtures of at least two curing agents from this list. Suitable organic solvent B can be selected from, for example, the group consisting of branched or unbranched C1-C10 alcohols or water, or mixtures of at least two of these solvents. The suitable organic solvent must have a density less than that of PEDOT:PSS to allow the separation of the protective solvent A shell, and preferably has high solubility for solvent A.

[0024] The density of the coagulated solution is greater than that of organic solvent A and less than that of the aqueous PEDOT:PSS mixture. Therefore, according to the present invention, the density of the coagulated solution containing organic solvent B and the curing agent must be greater than the density of organic solvent A, and in cases where the aqueous PEDOT:PSS mixture also contains other solvents, it must be greater than the density of those other solvents. This relationship between the different solvents can be controlled, for example, by the density of the coagulated solution. In principle, the density of the coagulated solution can be influenced by two different parameters. First, of course, the density of the coagulated solution can be determined by the selection of solvent B itself. On the other hand, the density of solvent B itself can be further adjusted by selecting the concentration of the curing agent in solvent B. Of course, the above relationship is obtained by comparing the densities of organic solvent A and organic solvent B, including the curing agent, under the same temperature conditions. If the coagulated solution contains other density-related additives or substances besides the curing agent and solvent B, the density of the coagulated solution should include these additives or substances. Since the volume of the coagulated solution is considered very large compared to the volume of the dripping solution, changes in the density of the coagulated solution due to emulsion dripping are not considered. Compared to the aqueous PEDOT:PSS mixture, the density of the coagulation solution of solvent B and the curing agent must also be lower. Preferably, the density difference between the aqueous PEDOT:PSS mixture, organic solvent A, and the coagulation bath containing organic solvent B + curing agent can be greater than or equal to 5%, more preferably greater than or equal to 10%. Within these density differences, the protective layer of PEDOT:PSS droplets composed of organic solvent A can be removed very rapidly, and very uniform particles can be produced. The absolute density difference is important, and as a function of the density difference sign, the solution can be introduced into the coagulation bath from above or below in one step. The solubility of solvent A in solvent B further facilitates the separation of the protective shell composed of solvent A.

[0025] By introducing a PEDOT:PSS emulsion encapsulated in solvent A into a coagulation solution, PEDOT:PSS droplets solidify into PEDOT:PS particles. The density relationship described above allows the shell surrounding the PEDOT:PSS droplets to slowly separate from the aqueous PEDOT:PSS mixture due to density differences and solubility in solvent B. The aqueous PEDOT:PSS mixture is then coagulated. By removing the protection of the internal aqueous PEDOT:PSS droplets from organic solvent A, coagulation of the PEDOT:PSS complex begins in the coagulation bath with a curing agent. The curing agent causes partial crystallization of the PEDOT:PSS droplets, while solvent B causes non-solvent-induced phase separation. Thus, due to the interaction with the coagulation bath, the PEDOT:PSS droplets completely harden into particles within a short time. In the case of porous particles, additional 1 to 40 μm emulsion droplets composed of solvent (e.g., solvent A) can still form within the PEDOT:PSS particles, thereby creating pores in the particles during the coagulation process in the coagulation bath. The residual droplets of organic solvent A still present in the particles can act as a scaffold for forming spherical structures, in addition to contributing to particle porosity. If other solvents that can be separated by density difference are present in the aqueous PEDOT:PSS mixture, they can form solidified pores. Since organic solvent B can also partially act as a solvent for organic solvent A, this structure not only ensures sufficient particle consolidation but also ensures that the particle pores are released from the other solvent.

[0026] In a preferred embodiment of the method, organic solvent A may be selected from the group consisting of branched or unbranched C5-C10 alkanes, branched or unbranched C5-C10 alcohols, or mixtures of at least two of these solvents. The aforementioned solvent group has proven particularly suitable for obtaining an emulsion sufficiently stable in the aqueous PEDOT:PSS mixture in organic solvent A. Sufficiently uniform small droplets can be generated with very low shear forces, and their solubility in water is not too high. Beyond theoretical considerations, it may also be advantageous that the surface porosity of the particles is particularly positively affected by this solvent selection. Furthermore, another advantage may be that, in the case of aqueous PEDOT:PSS mixtures containing other solvents to form pores, the release of pores from the solvent is particularly rapid due to the potentially good solubility in solvent B.

[0027] In a preferred aspect of this method, solvent B may be further selected from branched or unbranched C1-C5 alcohols or mixtures of at least two substances. This group of solvents B in the coagulation bath has been found to be sufficiently stable for many different curing agents. Furthermore, these solvents exhibit sufficiently high solubility for the most important curing agent, resulting in a homogeneous coagulation bath. Another advantage of this group is the preferential interaction with any solvent A emulsified in the droplets. Solvent A dissolves within an optimal time window, thus allowing sufficient time to stabilize the droplet shape during curing and to completely separate the surrounding organic phase from the particle interior within the same process step.

[0028] In another preferred embodiment of the method, solvent A may include octanol, and the coagulation solution in step b) may include isopropanol as solvent B and sulfuric acid as curing agent. This combination of solvent and curing agent has proven particularly advantageous in various process steps. Particularly stable and uniformly crosslinked PEDOT:PSS particles are produced, characterized by particularly uniform porosity. Beyond theoretical considerations, the synergistic advantage stems from the particularly favorable solubility of solvents A and B within and between each other. Droplets detached from the octanol shell contact the coagulation mixture and are fully cured through interaction with sulfuric acid as the curing agent. Thus, octanol droplets still present in emulsified form in the aqueous PEDOT:PSS solution or dispersion can particularly serve as a scaffold for forming spherical shapes and ensure the formation of controlled porosity. Since isopropanol is a partial solvent for octanol and may be a solvent for further emulsification, the coagulation mixture not only ensures particle curing by removing the droplet shell but also controls the release of pores from other solvents. Preferably, 1-octanol can be used as the octanol. The latter can contribute to the formation of particularly uniform droplets.

[0029] In a preferred aspect of this method, the weight ratio of the curing agent to solvent B in the coagulation solution (expressed as the weight of the curing agent divided by the mass of solvent B) can be greater than or equal to 0.005 and less than or equal to 0.2. This ratio has been found particularly suitable for efficient and controlled curing while obtaining particularly uniform spherical PEDOT:PSS particles. On the one hand, the separation rate of the encapsulating solvent A (removal of additional solvent from the aqueous PEDOT:PSS mixture if necessary) and the subsequent contact time with the curing agent undergo a dynamic equilibrium, which, based on kinetic considerations, is particularly useful for forming more spherical particles. Therefore, a higher level of curing agent can contribute to the formation of more non-spherical particles. A lower level of curing agent may only result in insufficient curing of the PEDOT:PSS droplets or require a longer contact time in the coagulation bath. In a preferred embodiment, the lower limit of this range can be 0.01.

[0030] In another preferred feature of this method, the PEDOT:PSS mixture in process step a) may not contain any further mechanically reinforcing material. Surprisingly, it has been found that mechanically very stable particles can be obtained by the method according to the invention, which contain no further mechanically reinforcing material in the sense of a mechanical stabilizer, other than PEDOT and PSS. Commonly used curing materials are selected from the group consisting of polymeric additives or pure support or molded solids. These materials are also known by those skilled in the art as "template particles." In the field of polymeric additives, this means that the PEDOT:PSS solution used may not contain other monomers or polymers. The polymeric component may be, for example, a substance with a molecular weight greater than or equal to 2000 g / mol, which may be present in an aqueous PEDOT:PSS solution or may be formed during the production process. Furthermore, the PEDOT:PSS particles may not contain other mechanically cured materials, such as plastic microparticles, such as polystyrene microparticles, silica microparticles, or salt crystals, such as calcium carbonate. In this respect, the group of non-polymeric curing materials includes at least salt crystals, plastic microparticles, quartz microparticles, or mixtures thereof. In addition, the PEDOT:PSS solution used may contain other low-molecular-weight substances, which may affect or adapt the electrical performance of the PEDOT:PSS network.

[0031] In another preferred aspect of the method, the aqueous PEDOT:PSS mixture in process step a) may include organic solvent A as another solvent component besides water. It has been found that generating an emulsion of solvent A in an aqueous PEDOT:PSS solution using solvent A is simple and effective for forming controlled pores and mechanically stable particles. The amount of the substance involved remains low, and separation from the emulsion is rapid and substantially complete. Furthermore, the volume fraction of solvent A in the total volume of the aqueous PEDOT:PSS mixture can be greater than or equal to 15% and less than or equal to 60%. Within these volume fractions of organic solvent A in the aqueous mixture, mechanically very stable particles exhibiting a very uniform pore size distribution can be obtained in a very short processing time. Smaller proportions may be disadvantageous because, due to the presence of only isolated solvent A droplets, cross-linked pores cannot be formed, resulting only in isolated defects within the particles. Higher proportions only lead to insufficient mechanical stability of the particles.

[0032] Furthermore, the poly(3,4-ethylenedioxythiophene)polystyrene sulfonate particles according to the invention are spherical and do not contain any other mechanically cured substances other than PEDOT:PSS. Mechanically extremely stable PEDOT:PSS particles, characterized by a particularly uniform spherical shape, can be obtained even without further mechanically cured additives in the reactants or formed particles. The cured substance is defined above in conjunction with the method of the invention. The polymer component can be, for example, a substance with a molecular weight greater than or equal to 2000 g / mol, which can be present in an aqueous PEDOT:PSS solution or can be formed during the production process. In addition to the specific embodiment of spherical form, the particles can also have a particularly narrow spherical size distribution. For example, the spherical configuration can be mathematically determined by the sphericity of the particles, which, according to the invention, can be greater than or equal to 0.91. This sphericity range can be determined microscopically and generally describes the ratio of the surface area of ​​a sphere of equal volume to the surface area of ​​an existing object. The average value of at least 20 individual particles can be used for determination. Furthermore, the sphericity of the particles can be greater than or equal to 0.95 and less than or equal to 1. In addition, the particles may be free of emulsifiers, wetting agents, or other surfactants commonly used in the production of emulsions.

[0033] Furthermore, according to the present invention, PEDOT:PSS particles are produced by the method according to the invention. In addition to size distribution, mechanical stability, and porosity, other properties can be determined by the method of the present invention, which differ from the properties of methods produced according to the latest technology. For further advantages of these particles obtained by the method of the present invention, reference is made to the advantages described in conjunction with the method of the present invention.

[0034] In another preferred aspect, the particles may have an elastic modulus greater than or equal to 0.05 MPa and less than or equal to 15 MPa. The mechanical properties of the particles according to the invention can also be adjusted over a wide range without the addition of other mechanically active substances. Besides the porosity of the particles, crystallinity has a particularly significant impact on the elastic modulus. The degree of crystallinity of each chain segment can be affected, for example, by the concentration of an acid catalyst, thereby affecting the molecular arrangement between the chain segments. The elastic modulus (Young's modulus) of the particles can be determined by tensile testing of the ribbon-like particles. As described in the examples, the elastic modulus of the fibrous particles was determined in a moist state.

[0035] Based on preferred characteristics of the particles, the particles may be at least partially crystalline, exhibiting a wavelength of 4.3 (±0.2) nm in the XRD spectrum. -1 and 18.4 (+ / - 0.2) nm -2Bragg reflection at the location. Mechanically very stable particles, particularly characterized by high crystallinity, can be obtained by the method of the present invention. Specifically, high crystallinity results in particles with a high elastic modulus. The crystalline particles are characterized by a solid-state powder X-ray diffraction pattern showing visible reflection at the aforementioned location. Furthermore, the diffraction pattern can be observed at 8.6 nm. -1 Another peak is shown at 4.3 and 8.6 nm. -1 The peak height ratio at that point can be 1:2. These reflections can be attributed to the layered arrangement of individual chains with a periodicity of 1.5 nm within the particles. At approximately 18 nm... -1 The reflection at the point is likely caused by the regular π-π stacking of adjacent PEDOT chains. Conversely, fewer crystalline particles, such as those produced in a coagulation bath with low acid concentrations, exhibit small sizes with crystalline regions of only 1.8 nm. In these particles with low crystallinity, a distinct layered structure cannot be detected by the defined Bragg reflection.

[0036] In another preferred embodiment of the particles, the surface of the particles may have a zeta potential less than or equal to 0 mV. Particularly suitable, mechanically stable particles can be obtained using the method of the present invention without the addition of additional polymeric backbone materials or mechanically effective fillers; the particles are also characterized by a favorable negative zeta potential. This negative surface charge can contribute to improved surface functionalization of the particles, particularly in the field of cell culture. Electronegative functionalization of the surface can be used in several steps, for example, by applying a positive charge, thereby subsequently obtaining improved cell adhesion. Thus, more biocompatible particles can be produced, exhibiting faster adhesion and cell proliferation on and within the particles. The zeta potential can be determined, for example, by a combination of optical / electrical measurements measuring the migration velocity of the particles as a function of the applied voltage. The influence of individual particle geometry on the measurement results is known to those skilled in the art, and these effects can be calculated. Preferably, the surface charge may be less than or equal to -10 mV, more preferably less than or equal to -15 mV. The lower limit of the potential may be, for example, -75 mV, more preferably -50 mV.

[0037] In a preferred embodiment of the particles, the particles may have a size distribution with a D50 quantile in the range of greater than or equal to 10 μm and less than or equal to 1000 μm. Particles obtained by the method according to the invention can be produced over a wide size range by selecting the nozzle size and the flow rate ratio between the continuous phase and the aqueous PEDOT:PSS dispersion. A very uniform size distribution is obtained, which may have a polydispersity index less than or equal to 1.2, more preferably less than or equal to 1.1. The polydispersity index can be determined by microscopic measurement.

[0038] In a preferred embodiment, the particles can be porous and have a porosity greater than 0% by volume and less than or equal to 95% by volume. It has been shown that a wide range of particle porosities can be obtained by the method according to the invention, and, particularly surprisingly, the particles exhibit sufficient mechanical strength even at high porosities, even in the absence of other stabilizing substances. Therefore, particles exhibiting very high specific surface areas and free from, for example, electrical interferences or inactive substances can be provided. Particle porosity can be determined, for example, by microscopy on freeze-dried particles.

[0039] Furthermore, according to the present invention, the particles can be used in various applications, including cell culture microcarriers, suspended electrodes, switchable redox absorber materials, catalyst supports, or combinations thereof. Due to the uniformity of particle size distribution, controllable porosity, and the fact that no other mechanically solidified and / or surfactants other than PEDOT:PSS need to be present on or within the particles, the particles are suitable for many different applications. In the case of use as suspended electrodes, the particles result in multiphase material systems having particles as active charge storage components. These can be suspended in ionic solutions or electrolytes. By weight, the electrolyte is the dominant component, facilitating the physical transport of the active material. The internal and surface porosity of the particles according to the present invention leads to improved electrochemical performance, including increased capacitance utilization and faster charge-discharge kinetics, thereby exhibiting high potentials for electrochemical energy storage in the form of supercapacitors or batteries. PEDOT:PSS particles are non-toxic and therefore cell-compatible, and exhibit excellent redox reversibility in addition to high mechanical stability in aqueous systems. Higher conductivity can be achieved compared to state-of-the-art polymer particles. Furthermore, as a material system, the PEDOT:PSS particles according to the invention exhibit very fast final charge dynamics, whereby charge can be stored not only in the surface double layer but also in the polymer matrix. The latter, in particular, leads to high energy and power density in the particles.

[0040] In preferred applications, the particles can be used as cell culture microcarriers, wherein prior to culturing, the surface of the particles is coated with one or more molecules selected from the group consisting of poly-L-lysine, laminin, collagen, fibronectin, hylocinin, or mixtures thereof. Excluding other carrier substances from the basic particle structure can produce highly biocompatible carriers, which also offer the possibility of subsequent electrostatic functionalization due to their surface charge. All extracellular matrix proteins exhibiting an isoelectric point <7 in aqueous solution can be coated onto the particles. Therefore, these compounds exhibit a negative charge and can thus adhere to, for example, poly-L-lysine. Other charged components of the extracellular matrix or synthetic polyelectrolytes, for example, can bind to the surface. These components can lead to faster and better adhesion, as well as higher cell division rates.

[0041] In another application embodiment, the surface of the particle can be first coated with poly-L-lysine, followed by coating with laminin. Continuous coating and bilayer coating can help improve the biocompatibility of the carrier. It can also process difficult-to-culture cell lines at high yields. For the coating, the surface of the particle can be treated with poly-L-lysine first. After sufficient absorption of poly-L-lysine, laminin can be absorbed onto the coated poly-L-lysine layer in a second step. Example

[0042] I-structure

[0043] The microfluidic co-flow apparatus for producing spherical PEDOT:PSS particles according to the present invention is made of a polyethylene tube with an inner diameter of 0.86 mm, a 30G disposable sleeve, and epoxy resin. The tube is bent at a 45° angle and fixed to a microscope slide with epoxy resin. The sleeve is then inserted into the tube at the bend. The sleeve is finally concentrically positioned in the tube and fixed to the microscope glass carrier with epoxy resin. Here, the end of the polyethylene tube facing the tip of the insert represents the subsequent device outlet, while the end facing away from the tip of the insert is the inlet of the continuous stage. The male threaded cap of the inserted 30G disposable sleeve is then connected to a female-to-female connector, which is connected to another polyethylene tube via a second 30G disposable insert. This polyethylene tube is used in the manufacturing process to deliver pure PEDOT:PSS dispersion (whole particles) or 1-octanol PEDOT:PSS emulsion (porous PEDOT:PSS particles).

[0044] II. Granulation Production

[0045] To prepare intact PEDOT:PSS particles, a 1.3 wt% aqueous PEDOT / PSS mixture (Haereus) is filled into a 10 mL disposable syringe. The syringe is then connected to the appropriate end of a co-flow apparatus via a 30G cannula. Another 10 mL disposable syringe is used to draw 1-octanol as solvent A. This syringe is also connected to the end of the apparatus via a 30G cannula. Finally, the two syringes are secured to the supports of two separate syringe pumps for delivering the respective phases. Preferably, the delivery rate of the PEDOT:PSS alkaline aqueous solution can be set to 0.01 mL / min, and the delivery rate of 1-octanol to 0.5 mL / min. These values ​​have proven particularly advantageous for droplet formation, as they ensure a sufficiently large droplet spacing in the tube and result in the desired droplet size. For solidification, the stationary liquid PEDOT:PSS droplets, along with the surrounding continuous 1-octanol phase, are introduced through the end of the tube into a coagulation bath consisting of 5 vol% sulfuric acid and 95 vol% isopropanol. The 1-octanol coating surrounding the PEDOT:PSS droplets prevents PEDOT:PSS from hardening in the sleeve and is then gently removed in the coagulation bath by density difference. As the 1-octanol shell is removed by separation in the coagulation bath, the solidification process of the PEDOT:PSS droplets begins, and due to their higher density, the PEDOT / PSS droplets deposit within the coagulation bath. During solidification, PSS is removed from the PEDOT:PSS polyelectrolyte complex due to complexation with H+ ions, causing the hydrophobic PEDOT to aggregate and crystallize through π-π interactions. Finally, fully solidified pure PEDOT:PSS particles can be picked up from the bottom of the coagulation bath container.

[0046] For the production of porous PEDOT:PSS particles, the experimental setup remained unchanged. However, instead of pure aqueous 1.3 wt% PEDOT:PSS solution, 1-octanol (solvent A) from the PEDOT:PSS emulsion was filled into a syringe. The emulsion was then emulsified for 1 minute using a Hielscher UP200S ultrasonic device at 0.5 cycles and 50% amplitude. The volume ratio of the 1.3 wt% PEDOT:PSS solution in the emulsion to other solvents in group A (e.g., 1-octanol) can be varied according to the desired porosity. Particles containing 30 vol% 1-octanol in 1.3 wt% aqueous PEDOT:PSS solution are suitable for cell culture experiments.

[0047] III. Measurement Methods

[0048] III.1 XRD Measurement

[0049] Powder X-ray diffraction (WAXS) was performed using an Empyrean apparatus obtained from Panalytical. Cu X-ray tube (12 × 0.04 mm) 2A line source provided CuKα radiation with λ = 0.1542 nm. The source and detector moved vertically around a fixed horizontal sample. After passing through a divergence slit of 1 / 8° and an antiscattering slit of 1 / 4°, the beam reached the sample at the center of the phi-chi-z table. In the Bragg Bretano geometry used, the beam was refocused at a secondary divergence slit of 1 / 4°. Finally, the signal was recorded as a function of the scattering angle 2θ using a pixel detector (256 × 256 pixels, 55 μm). The peak position was then calculated according to q = 2π / d = (4π / λ)sinθ, where q is the scattering vector. This detector was used to allow scanning geometry that allows simultaneous use of all rows. To reduce background, a 4 mm mask was used to control the diverging beam perpendicular to the scattering plane, which limited the beam width at the sample location to approximately 10 mm. Furthermore, vertical divergence was limited by the target slit with an angle ≤ 2.3°. The height of the (powder) sample was optimized for each new measurement. During scanning, the detector axis 2θ moves at twice the speed of the incident beam θ axis. Calibration is checked using a Si reference sample. The resolution of the entire device is determined by measuring a high-quality Si wafer, which yields a resolution-defined peak with a half-width of 0.026 degrees.

[0050] III.2 Mechanical Measurement

[0051] Tensile tests were performed on a custom-designed laboratory apparatus consisting of a micromanipulator linear arm (MM33). The system consisted of Wetzlar GmbH & Co. KG (Germany), a stepper motor (NEMA17, Stepperonline), and a high-precision balance (Mettler Toledo, Switzerland). PEDOT:PSS fibers were fixed to a C-shaped cardboard frame with an inner leg spacing of 10 mm. The cardboard frame ensured the initial length of the fibers and prevented elongation before the tensile test. The fixed PEDOT:PSS fibers were then immersed in DI water for 10 seconds, and the cardboard frame was then attached to the linear arm and balance via clamps. Finally, the PEDOT:PSS fibers were stretched to break at a tensile rate of 0.2 mm / s by cutting the cardboard legs. Strain and mass were recorded using a self-written Python script.

[0052] III.3ECM Molecular Coating

[0053] The adsorption behavior of polyelectrolytes (PEs) with different charges on the surface of PEDOT:PSS particles was investigated using FITC-labeled polyethylene. Positively charged poly-L-lysine (PLL) (15000–30000 Da, Sigma-Aldrich) and negatively charged polystyrene sulfonate (PSS) (Surflay Nanotec GmbH) (labeling level 10%) were dissolved at a concentration of 1 mg / mL in 0.1 M NaOH aqueous solution and stirred overnight at room temperature. Subsequently, the PEDOT:PSS particles were incubated in the corresponding PE solutions for 5 days under light-shielded conditions. Finally, the samples were rinsed twice in 0.1 M NaOH aqueous solution and imaged using a TCS SP8 Falcon confocal microscope (Leica, Germany). For MRC-5 cell culture, PEDOT:PSS particles were coated layer-by-layer with PLL and laminin (from human placenta, Sigma-Aldrich). At room temperature, the microcarriers were incubated in 0.1 mg / ml PLL solution on a roller apparatus for 24 hours. Subsequently, the samples were washed with Milliq water and then incubated for another 24 hours on a roller apparatus at 37°C in 40 μg / ml laminin solution.

[0054] III.4 Cell Culture

[0055] Cell maintenance: L929 mouse fibroblasts were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (4500 mg / L glucose, L-glutamine, sodium pyruvate, sodium bicarbonate) (Thermo Fisher Scientific); while MRC-5 human embryonic fibroblasts were cultured in minimally invasive medium (MEM) (1000 mg / L glucose, 1X non-essential amino acids, L-glutamine, sodium bicarbonate) (Sigma-Aldrich). Both cell cultures were supplemented with 10% (v / v) fetal bovine serum (FBS) and 1% (v / vv) penicillin / streptomycin. Cell lines were maintained at 37°C under 5% CO2 and 95% humidity. Regardless of cell line, passage times were limited to 30 passages.

[0056] Cytotoxicity of the particles was assessed on days 5 and 9 by comparing cell proliferation in fresh medium and in leachate medium using the Tetraazole-Based Cell Proliferation Kit II (XTT) (Roche Diagnostics GmbH). Leachate medium was prepared by culturing low- or high-crystallinity PEDOT:PSS microcarriers at 37°C for 5 days in RPMI and MEM media at a ratio of 10 particles per 100 μl of medium. The leachate medium was collected and stored at 4°C until use. Cells were seeded in TC-treated microtiter plates (96-well, Corning Life Sciences) at a seeding concentration of 1500 cells per well for L929 and 10000 cells per well for MRC-5. Cells were then held in fresh or leachate medium for 9 days, with the medium changed every 48 hours to ensure adequate nutrient supply. For cell viability analysis, cells were washed with PBS (1x) (Lonza, Switzerland) and held in the microplates before exposure to 150 μl of XTT solution at 37°C for 4 hours. Subsequently, 100 μl of each sample was transferred to a freshly treated TC microplate (96 wells), and absorbance was measured at 450 and 630 nm using a microplate reader (Synergy HT, BioTek). Cell viability was determined by the ratio of absorbance values ​​of cells cultured in leachate medium to those cultured in fresh medium (control).

[0057] Immunostaining: For morphological assessment, cell nuclei and F-actin were stained by treating samples with DAPI solution (abcam, UK) for 5 minutes and with phalloidin-iFluor 488 reagent (abcam) for 60 minutes. Prior to staining, all samples were fixed in 4% (v / v) paraformaldehyde (PFA) solution for 15 minutes and thoroughly rinsed in PBS (1x). Cell viability was visually analyzed using a live / dead cell double staining kit (Sigma-Aldrich). Cell samples were exposed to sterile PBS (1x) containing 0.1% (v / v) calcein AM and 0.2% (v / v) propidium iodide for 30 minutes at 37°C.

[0058] Further advantages and advantageous embodiments of the invention are illustrated by the figures or explanations in the following examples. It should be noted that the figures are merely illustrative and are not intended to limit the invention in any way.

[0059] Attached image:

[0060] Figure 1 FeSEM images of porous PEDOT:PSS particles prepared according to the present invention are shown.

[0061] Figure 2 FeSEM image showing porous PEDOT:PSS particles colonized with fibroblasts prepared according to the present invention.

[0062] Figure 3 Shows the PEDOT:PSS particle weight capacity as a function of porosity according to the present invention;

[0063] Figure 4 The redox kinetics of PEDOT:PSS particles according to the present invention are shown as a function of porosity.

[0064] Figure 5 This demonstrates the correlation between the particle size of the PEDOT:PSS particles according to the present invention as a function of porosity;

[0065] Figure 6 The size distribution of the PEDOT:PSS particles of the present invention prepared in an aqueous PEDOT:PSS mixture having a volume fraction of 30% 1-octanol is shown.

[0066] Figure 7 The pore size distribution of the PEDOT:PSS particles prepared according to the present invention is shown, wherein the volume fraction of 1-octanol in the aqueous PEDOT:PSS mixture is 30%.

[0067] Figure 8 The proliferation of L929 cells on particles according to the present invention is shown as a function of time and as a function of the crystallinity of the carrier material;

[0068] Figure 9 This demonstrates the effect of crystallinity on the aspect ratio of proliferating L929 cells according to the present invention;

[0069] Figure 10 This demonstrates the effect of particle crystallinity on the proliferation area of ​​L929 cells according to the present invention.

[0070] Figure 1 FeSEM images of porous PEDOT:PSS particles prepared according to the present invention are shown. The PEDOT:PSS particles were prepared aqueously using a PEDOT:PSS mixture (in which the volume fraction of 1-octanol was 30%). Since the PEDOT:PSS particles are hydrogels and therefore would collapse in an anhydrous environment, the particles were freeze-dried prior to optical analysis.

[0071] Figure 2FeSEM images of porous PEDOT:PSS particles colonized with L929 mouse fibroblasts are shown. Colonization of the microcarriers is shown after 4 days of culture at 37°C, 95% humidity, and 5% CO2. The culture medium was RPMI supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. The inoculum concentration was 10,000 cells / cm³. 2 Since PEDOT:PSS particles are hydrogels, they collapse in an anhydrous environment. Therefore, prior to optical analysis, the particles were dried with a series of ethanols (35%, 50%, 70%, 100%) and then treated in hexamethyldisilazane (HMDS).

[0072] Figure 3 The gravimetric capacitance of PEDOT:PSS particles is shown as a function of the 1-octanol volume fraction in the 1-octanol PEDOT:PSS emulsion and as a function of the sampling rate. A higher 1-octanol fraction indicates a larger ratio of pore volume to particle volume (porosity), and therefore a higher specific surface area. The capacitance as a function of scan rate was determined based on cyclic voltammetry measurements in a three-electrode setup. Since the specific surface area of ​​particles is proportional to particle capacitance, more porous particles exhibit higher gravimetric capacitance. Higher scan rates result in lower capacitance because faster voltage cycling means that the entire surface area of ​​the particles that contributes to capacitance is not used.

[0073] Figure 4 The current curves as a function of time are shown. The redox kinetics of the PEDOT:PSS particles, measured by chronoamperometry, are recorded as a function of the volume fraction of 1-octanol, and thus as a function of porosity in the 3-electrode setup. The reaction time of the particles decreases with increasing porosity, although the charge density increases with increasing porosity. The shortened reaction time is attributed to the high specific surface area and good accessibility of the pore system, which allows for rapid redox kinetics. Measurements were performed over nine cycles, with only one cycle shown in the figure.

[0074] Figure 5 The average particle size is shown as a function of the volume fraction of 1-octanol in the 1-octanol PEDOT:PSS emulsion, as a measure of particle porosity. All particles shown in the figure were prepared at a 1-octanol flow rate of 0.5 mL / min (continuous phase) and a PEDOT:PSS dispersion / emulsion flow rate of 0.05 mL / min. Regardless of particle porosity, the particle size is approximately 540 μm. The small standard deviation of the particle diameter is likely due to the monodispersity of the droplets in the co-flow apparatus. The small variation in particle size arises from very small differences in the separation kinetics of the protective 1-octanol shell in the coagulation bath.

[0075] Figure 6The size distributions of high (left) and low (right) crystallinity PEDOT:PSS particles are shown, with 1-octanol comprising 30% by volume in the PEDOT:PSS aqueous mixture. For both cases, fairly narrow particle size distributions were obtained.

[0076] Figure 7 The pore-forming agents and pore size distributions of highly and poorly crystalline porous PEDOT:PSS particles prepared with 30% 1-octanol (volume fraction) are shown. Most pores are between 15 and 20 μm in size. Over 90% of the pores show a pore size between 10 and 30 μm.

[0077] Figure 8-10 Cell colonization results of the particles of the present invention are shown. For culture experiments, spherical PEDOT:PSS particles were prepared from a 30 vol% 1-octanol emulsion in PEDOT:PSS (1.1-1.3 wt%), which was droplet-disrupted in a continuous 1-octanol phase. The emulsion was obtained by ultrasonic homogenization (Hirschler UP100H). The two phases were added together by syringe pumps (Chemyx, NexusFusion 4000) at flow rates of 0.05 and 0.5 ml / min, respectively. The coagulation bath consisted of 5 vol% sulfuric acid in isopropanol, unless otherwise specified.

[0078] Figure 8 The results show the proliferation of L929 cells on the particles according to the invention as a function of time and as a function of the crystallinity of the carrier material. Porous particles with different crystallinities were prepared by using different amounts of acid for coagulation. Low-crystallinity particles were coagulated with 5% by volume, and high-crystallinity particles were coagulated with 95% by volume sulfuric acid. Particles with different mechanical properties were obtained. The performance results are as follows:

[0079]

[0080] The different mechanical properties clearly indicate that, despite having the same composition, the two samples have different structures. These differences in the properties of the spherical particles also lead to variations in their biological properties. Figure 8 The results showed a seeding density of 2600 cells / cm² on pure PEDOT:PSS microcarriers. 2 Cell proliferation results of L929 mouse fibroblasts with N=5 were presented, with viability quantified using the XTT proliferation assay. It is clear that the crystallinity of the support material affects cell proliferation. From day 5 onwards, the proliferation of highly crystalline samples (triangular) was significantly higher than that of low-crystallinity samples (circular).

[0081] Figure 9The effect of crystallinity on the aspect ratio of L929 cell proliferating particles of this invention was shown. Particle crystallinity also appears to affect the achievable morphology of the cell line used. Morphological assessment of DAPI / phalloidin-stained L929 cells was performed using confocal microscopy. One method for visualizing cell symmetry is to determine the aspect ratio of L929 cells. Different cell morphologies were observed on low-crystallinity and high-crystallinity particles; the cells in low-crystallinity particles were more rounded, while those in high-crystallinity particles were longer. The seeding density was 2600 cells / cm². 2 The 250 cells were measured the following day.

[0082] Figure 10 This study demonstrates the effect of particle crystallinity on the proliferation area of ​​L929 cells. Different crystallinities of the particles were obtained by subjecting spherical particles to different coagulation treatments. It can be seen that individual L929 cells colonize a significantly larger area on the crystalline particles. In contrast, cell diffusion on particles with low crystallinity is much more limited. Furthermore, this leads to cells on particles with low crystallinity potentially proliferating deeper into the particle interior. On the other hand, in the case of highly crystalline particles, the colonization density inside the particle appears to be reduced.

Claims

1. A method for preparing poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) particles, comprising at least the following steps: a) Provides a mixture of poly(3,4-ethylenedioxythiophene) and polystyrene sulfonate comprising in a solvent containing at least water; b) Forming one or more PEDOT:PSS droplets by introducing the mixture from process step a) into organic solvent A, wherein the aqueous PEDOT:PSS mixture forms the inside of the droplets and organic solvent A forms the outside of the droplets; c) Contact the PEDOT:PSS droplets obtained from process step b) with a coagulation solution containing a curing agent and at least one additional solvent B, the density of which is greater than the density of the organic solvent A and less than the density of the aqueous mixture of poly(3,4-ethylenedioxythiophene) and polystyrene sulfonate; solidify the PEDOT:PSS droplets into PEDOT:PSS particles.

2. The method according to claim 1, wherein the organic solvent A is selected from branched or unbranched C5-C10 alkanes, branched or unbranched C5-C10 alcohols, or mixtures of at least two of the above solvents.

3. The method according to any one of the preceding claims, wherein the additional solvent B is selected from branched or unbranched C1-C5 alcohols or mixtures of at least two of them.

4. The method according to claim 1, wherein solvent A comprises octanol, and the coagulation solution in step c) comprises isopropanol as solvent B and sulfuric acid as curing agent.

5. The method according to claim 1, wherein the weight ratio of the curing agent to solvent B in the coagulation solution (expressed as the weight of the curing agent divided by the mass of solvent B) is greater than or equal to 0.005 and less than or equal to 0.

2.

6. The method of claim 1, wherein the PEDOT:PSS mixture in step a) does not contain any further mechanically curing material.

7. The method according to claim 1, wherein the PEDOT:PSS mixture in step a) of the method further comprises, in addition to water, an organic solvent A as another solvent component.

8. The poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate particles prepared by the preparation method according to claim 1, characterized in that, The particles are spherical and contain no other mechanically cured substances other than PEDOT:PSS.

9. The particles of claim 8, wherein the particles are porous and have a porosity greater than 0% by volume and less than or equal to 95% by volume.

10. The particles according to any one of claims 8 or 9, wherein the particles have an elastic modulus greater than or equal to 0.05 MPa and less than or equal to 15 MPa.

11. The particles of claim 8, wherein the particles are at least partially crystalline and have a wavelength of 4.3 (+ / -0.2) nm in the XRD spectrum. -1 and 18.4 (+ / -0.2) nm -2 The Bragg reflection.

12. The particle according to claim 8, wherein the surface of the particle has a zeta potential of less than or equal to 0 mV.

13. Use of the particles according to any one of claims 8-12, wherein the use is selected from cell culture microcarriers, suspended electrodes, switchable redox absorbent materials, catalyst carriers, or combinations thereof.

14. The use according to claim 13, wherein the particles are used as cell culture microcarriers, wherein the surface of the particles is coated with one or more molecules selected from poly-L-lysine, laminin, collagen, fibronectin, hyalin, or mixtures thereof prior to culture.

15. The use according to claim 14, wherein the surface of said particles is first coated with poly-L-lysine and then coated with laminin.

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