ES2855228A1 - PROCEDURE FOR CELLULAR NANORCOVERING AND ITS IMMUNOISOLATION (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Procedure for cell nanocoating and its immunoisolation. The invention relates to an optimized process for the nanocoating of cells, such as pancreatic islets or insulin-secreting β cells, in order to cause their immunoisolation once transplanted into the recipient organism for cell therapy. Said procedure is based on cellular nanoencapsulation using the layer-by-layer technique with different layers of elastin-type recombiners (ELRs) modified to comprise azide or alkyne groups capable of reacting with each other by means of "click" chemistry, positive charges and cell adhesion sequences. . The cells thus coated remain viable and functional once implanted, and do not induce immune rejection by the recipient organism. In addition, this system provides a correct permeability that allows both the secretion of factors, such as insulin, from inside to outside the implant, as well as the entry of nutrients and oxygen to maintain cell viability. (Machine-translation by Google Translate, not legally binding)

Description

DESCRIPTION

PROCEDURE FOR CELLULAR NANORCOVERING SU

IMMUNOISOLATION

The present invention falls within the field of cell therapy and the procedures for the generation of cell products used in said therapies. In particular, the invention relates to a process for the nanocoating or nanoencapsulation of cells or cell groups, preferably pancreatic islets or insulin-secreting p cells, which are to be subsequently transplanted into the body for the structural and / or functional repair of the cells. tissues. Said nanocoating procedure, based on the layer-by-layer technique and the use of modified elastin-type recombinamers, manages to immunoisolate the cells, which continue to be viable and functional, so that once implanted they can exert their function without triggering an immune response. by the recipient organism.

BACKGROUND OF THE INVENTION

Multiple encapsulation systems for cells or cell groups have been described, such as pancreatic islets, including macro (hydrogels), micro (capsules) and nanoencapsulation (layers adhering to the cell membrane) systems, many of them used for the immunoisolation of islets. pancreatic drugs for the treatment of diabetes. Macroencapsulation consists, mainly, in embedding a large number of cells in reticular systems, such as hydrogels, and its main application is the administration of these cells in a specific tissue or organ to promote its regeneration, increasing the retention time of cells in that area by preventing their diffusion. On the other hand, microencapsulation consists of containing cells in microparticles or microfibers created by using microfluidic systems or by micro-molding. The small size of the microencapsulation systems improves, a priori, the diffusion of oxygen, nutrients and factors to the encapsulated cells. Its efficacy has been proven in the microencapsulation of different types of cells, including pancreatic islets, although there are major limitations, such as the poor stability of the biomaterials used or the lack of integration (vascularization) and biocompatibility of the same.

Regarding nanoencapsulation, the most used method to coat cells is the “layer-by-layer” (LbL) or, in Spanish, layer by layer, (Richardson JJ, et al., 2016, Chemical Reviews, 116 ( 23): 14828-67). In its most basic version, this procedure consists of adding layers that interact with each other electrostatically. In this way, using different polyelectrolytes (polycations and polyanions), it has been possible to coat pancreatic islets for the treatment of diabetes (Krol S, et al., 2006, Nano Letters, 6 (9): 1933-9). However, this system has not been tested in vivo and therefore its suitability in real application is not clear. Furthermore, the low biocompatibility of this type of polyelectrolyte could compromise the survival of the implant. In fact, there are several examples in the literature on nanoencapsulation of pancreatic islets using the layer-by-layer technique, all of them mentioning the disadvantages that this system has, such as the use of cytotoxic polyelectrolytes or those with low biocompatibility, in addition to problems derived from the lack of graft integration, mainly due to the absence of vascularization, which compromises the long-term viability and functionality of the islets, as occurs with microencapsulation systems (Kizilel S, et al., 2010, Tissue Engineering PartA ., 16 (7): 2217-28).

Very recently, nanoencapsulation of pancreatic islets with chitosan and poly (sodium styrene sultanate) has been described, achieving good in vivo performance, with a low presence within the islets after implantation and subsequent harvesting, also reporting moderate angiogenesis ( Syed F, et al., 2018, Nanomedicine: Nanotechnology, Biology and Medicine, 14 (7): 2191-203). In a similar recent example, the use of nanoencapsulation with heparin and polyethylene glycol (PEG) has been reported to isolate pancreatic islets, whose effectiveness has been demonstrated in non-human primates, not reporting, in principle, any abnormality in the treatment (Park H , et al., 2018, Biomaterials, 171: 164-77). Within the same field of diabetes treatment, several nanoencapsulation systems have also been used for insulin-producing p cells, although only in one case have they been implanted in vivo giving satisfactory results (Fukuda Y, et al., 2018, Biomaterials, 160: 82-91).

On the other hand, nanoencapsulation of other cell types has also been described, for example, mammalian cells in general, using the layer-by-layer technique with gelatin and PEG, demonstrating, for example, the protection of cells coated with this system against centrifugal force (Yang J, et al., 2017, Biomaterials, 133: 253-62). No However, it is doubtful that this system can be used for applications such as the coating of pancreatic islets for diabetes, due to its thickness, greater than 10 nm, which compromises the correct diffusion of factors from inside the cell (in the case of insulin), and vice versa. In another example, it has been possible to obtain functional and vascularized liver tissue using the layer by layer technique, although in this case it is a complex system, using different cell types in the same culture to give rise to the final tissue, which can be implanted (Sasaki K, et al., 2017, Biomaterials, 133: 263-74). However, this system has been introduced into immunosuppressed mice, so its effectiveness as an immunoisolation system has not been proven.

On the other hand, document EP2737913B1 describes elastin-type recombinamers (ELRs) modified to comprise a cell adhesion sequence (RGD) and the amino acid lysine, which allows it to be modified to form covalent bonds between the ELR and a culture surface. previously functionalized cell. In said document it is explained that the functionalization of a support ( scaffold) with alkyne groups and of the ELR with azide groups allows the ELR thus modified with said support to interact by "click" chemistry. This support / ELR system is proposed to carry out cell harvesting, since the cells adhere to the biopolymer attached to the support and subsequently these can be recovered by inducing a temperature change in the biopolymer, which is heat sensitive.

The generation of multilayer films based on chitosan-bound ELRs has also been described. The technique used to develop this system is layer-by-layer deposition (JS Barbosa, et al., 2009, Nanoscale Res Lett, 4: 1247-1253).

Rui R. Costa et al. They also generated coatings produced by the layer-by-layer technique based on chitosan bound to ELRs that also contained the RGD cell adhesion sequence. The cells thus adhere to the ELRs-RGD attached to chitosan which acts as a support (Rui R. Costa, et al., 2011, Small, 7, No. 18, 2640 2649).

A system, based on the use of the genipin agent, has also been disclosed where two ELRs containing two different cell adhesion domains (RGD and REDV, respectively) are crossed. The union between both ELRs is carried out by electrospinning in the presence of said crosslinking agent. A scaffold (scaffold) can be reabsorbed for use in tissue regeneration in cardiovascular disease, which comprises an inner layer formed of an ELR comprising RGD linking precursor endothelial cell and an outer layer formed by another ELR comprising REDV is thus achieved (M Putzu, et al., 2016, Biofabrication 8 045009).

Finally, the use of modified ELRs to incorporate L-lysines, azide groups or cyclooctin (alkyne) groups present in two different ELR molecules and cell adhesion RGD sequences has been described. Both ELRs cross-link each other through "click" chemistry thanks to the use of the electrospinning technique. Thus, bioactive microfibers capable of supporting cell growth are generated (Israel González de Torre, et al., 2018, Acta Biomaterialia, doi: https://doi.Org/10.1016/j.actbio.2018.03.027).

In summary, the idea of coating cells or cell groups, such as pancreatic islets, in order to improve their viability and achieve immunoisolation when they are transplanted into an organism, has been studied through different coating techniques, however, its Viability and functionality has not been optimal, due, in the case of alginate and other biomolecules, to the lack of chemical and mechanical stability of the biomaterial, in addition to its limited biocompatibility, which compromises the immunoisolation of the encapsulated cells. On the other hand, elastin-type biopolymers have been described in the literature that react with each other through a chemical reaction ("click") that is fast, efficient and biocompatible and that allows the coating of different surfaces (biomaterials).

However, there is still a need in the state of the art for optimized procedures that allow direct nanocoating or nanoencapsulation of cells, preferably pancreatic islets or insulin-producing p cells, so that efficient immunoisolation is achieved in vivo, so that, once transplanted into the recipient organism, there is no immune rejection. In turn, these procedures should give rise to a biocompatible, stable and homogeneous cell coating with adequate permeability to allow the passage of nutrients and oxygen from the outside to the inside of the cell, which ensures the maintenance and viability of the cells, as well. as from factors (for example, hormones such as insulin in the case of pancreatic islets) released by cells, which is essential to maintain their functionality during the corresponding therapeutic application.

DESCRIPTION OF THE INVENTION

The invention referred to herein solves the problem raised above by means of an optimized method for the immunoisolation of cells or cell groups, such as, for example, pancreatic islets or insulin-secreting p-cells. This procedure is based on cell nanoencapsulation or nanocoating with different layers of protein biopolymers, called elastin-type recombinamers (ELRs), a modified recombinant protein derived from natural elastin that adhere to cells using the layer-by-layer technique. This nanoencapsulation allows the subsequent use of said cells in cell therapy, since they continue to be viable and functional once coated and implanted, and do not induce immune rejection by the recipient organism. In addition, this system provides correct permeability that allows both the secretion of factors, such as insulin, from inside the implant to the outside, as well as the entry of nutrients and oxygen to maintain cell viability.

In the procedure described here, the layer-by-layer technique is used to achieve a coating at the molecular level or nanocoating, that is, the deposition of a single layer of ELRs at a time, from the cell surface with several layers of ELRs capable of interlinking with each other. that isolate the cells or cell groups thus encapsulated from the outside, protecting them from external stimuli such as, for example, the immune response of the recipient organism.

In order to improve the layer-to-layer method, taking into account that the electrostatic interactions that occur in said method are weak and can lead to an unstable coating, in the invention described here, the properties of chemistry have been used. Click for ELRs. In this way, covalent bonding between ELRs has been achieved, greatly improving the stability of the coating. For this, they have been incorporated into the ELRs used in the process of the invention, reactive alkyne or azide groups capable of reacting with each other by chemistry "click" orthogonally when found in a solution, which allows the formation of a cross-link through covalent bonds between a ELR molecule containing alkyne groups and another ELR molecule with azide groups present in the above layer, thus giving rise to what, in the present invention, is called a bilayer arranged on the cell surface.

In accordance with the foregoing, the process of the invention allows to precisely control the number of bilayers that adhere and cover the cell surface, creating a stable multilayer system and also being a fast process, as it is a "click" type reaction. instantaneous, which can also be carried out under conditions compatible with cell viability. Furthermore, it is a fully cytotic and biocompatible method, which makes it suitable for the subsequent use of cells in clinical applications, and results in a homogeneous cell coating.

The ELRs used in the process of the invention have also preferably been modified to comprise bioactive amino acid sequences or cell adhesion sequences, such as the tripeptide L-Arg-Gly-L-Asp (RGD), which promotes specific cell adhesion via membrane integrins, or the tetrapeptide L-Leu-L-Arg-L-Glu-L-Leu (LREL, SEQ ID NO: 5), with the capacity to bind to collagen, mainly type I. The incorporation of binding domains Collagen, such as the sequence SEQ ID NO: 5, has the advantage that it allows the ELR to interact with the extracellular matrix secreted by cell groups, which is involved in the maintenance of cohesion between cells, thus achieving an improved interaction with the cell surface. Said cell adhesion sequences present in ELRs facilitate their specific binding to the cell type to be coated. Similarly, taking into account that ELRs are composed of repeats of the pentapeptide L-Val-L-Pro-Gly-X-Gly (VPGXG, SEQ ID NO: 6), where X can be any amino acid except L-Pro, In the present invention, the composition of said pentapeptide has been modulated to include amino acids L-Lys (K), which provide a positive charge to the ELR, thus allowing electrostatic interaction in a non-specific way with the cell surface whose charge is negative. Therefore, the combination of these positive charges (amino acids L-Lys in the amino acid sequence of the ELR) with the cell adhesion sequences allows a dual interaction with cells, in a non-specific and specific way, respectively.

The procedure described here enables cell nanocoating directly, that is, without the need for the use of scaffolds or materials, such as chitosan, poly (sodium styrene sulfonate), heparin, gelatin or PEG, which act as cell attachment support. This procedure also avoids the use of polyelectrolytes or crosslinking agents that can be cytotoxic.

Furthermore, the conditions of the process of the invention have been optimized to result in a cellular nanocoating that provides adequate permeability while remaining stable and homogeneous. As mentioned above, the permeability of the coating is a key and determining aspect, since it is necessary that it not be permeable to proteins of different molecular size related to the immune response that would drastically decrease cell viability, from antibodies, of high weight. molecular, to proteins of the complement system, of relatively small size. However, excessive impermeability could hinder the exchange of nutrients, oxygen and other factors necessary to maintain cell viability. In addition, and this is a fundamental factor, the non-permeability of the coating would cause a low or no secretion of factors by the cells, from inside the implant to the outside, such as, for example, insulin in the case of pancreatic islets, which would cause a loss of cellular functionality. Thus, the optimal concentration of ELRs, the incubation time of the cells with the different solutions containing the ELRs, the number of necessary bilayers, the volume and the reaction temperature, have been optimized by the inventors to achieve a stable, homogeneous nanocoating. , viable and functional that also presents adequate permeability.

In the examples shown below, results of the nanocoating of pancreatic islets and insulin-secreting p cells differentiated from human induced pluripotent cells (hiPSCs) are presented following the procedure of the invention, and the homogeneity and integrity of the coating is demonstrated (see Figs 2, 3 and 9), the functionality of the cells thus coated to release insulin (see Figs. 5, 6 and 11) and the survival and immunoisolation once the cells thus encapsulated have been transplanted into mice in vivo (see Figs. 8 , 12, 14 and 15).

Thus, a first aspect of the invention refers to an in vitro process for the nanocoating, nanoencapsulation or immunoisolation of cells, or cell groups, also called "process of the invention", which comprises the following steps:

to. immerse, for a period of between 5 and 30 min, the cells in a first solution comprising at least one elastin-type recombinamer (ELR), where said ELR has been modified to comprise (i) azide groups or alkyne groups and (ii) positive charges,

b. immerse, for a time between 1 and 5 min, the cells of step (a) in a second solution comprising another ELR, where said ELR has been modified to comprise: (i) alkyne groups, when the ELR of step (a) comprises azide groups, or (ii) azide groups, when the ELR of step (a) comprises alkyne groups, thus forming a bilayer of ELRs, and

c. repeat steps (a) and (b) at least once more until at least two bilayers of ELRs are achieved,

where the ELRs of steps (a) and (b) are at a concentration between 5 and 25 mg / ml, in a volume of between 3 and 15 ml and where steps (a) and (b) are carried out at a temperature between 4 ° C and 50C.

The process of the invention is based on the layer by layer or "layer by layer" (LbL) technique, since the deposition of the different layers of ELRs on the cell surface is done one by one, following consecutive steps (a ) and (b).

In the present invention, "layer" is understood to mean the film or layer that adheres to the cell surface after contact of the cells with the first solution of step (a) or with the second solution of step (b). A "bilayer" is, therefore, the film or layer that adheres to the cell surface after contact of the cells with the first solution of step (a) and with the second solution of step (b). A bilayer will therefore be the result of the covalent bond between the ELR / s comprised in the first solution of step (a) and the ELR comprised in the second solution of step (b).

An "elastin-type recombinamer or biopolymer" (ELR or ELP) is an artificial amino acid sequence, that is, generated by genetic engineering techniques, comprising repeats of the pentapeptide VPGXG (SEQ ID NO: 6, where X can be any amino acid except L-Pro) which is present in natural elastin. It is non-toxic and therefore suitable for interaction with cells.

The ELRs of the invention are preferably produced by DNA technology recombinant, that is, by inserting the nucleotide sequence that encodes them into a suitable expression vector and its subsequent introduction and expression in a host cell to finally purify it. This recombinant production makes it possible to introduce, in the amino acid sequences of the ELRs, the modifications referred to in the present invention, in particular, the addition of cell adhesion sequences and positive charges, as well as the introduction of amino acids, such as lysine, which have amino groups that can be easily transformed to give azide or alkyne groups.

Azide groups or alkyne groups can be incorporated into ELRs by, for example, the introduction at the 4 (X) position of the VPGXG pentapeptide (SEQ ID NO: 6), in one or more repeats of said pentapeptide within the ELR, of amino acids, preferably lysine, whose amino groups in the gamma position are easily able to give rise to the substitution reaction with reactive azide or alkyne groups. Said amino groups are transformed to subsequently carry out the delation reaction type "click".

In a preferred embodiment, the positive charges of the ELR of step (a) are L-Lys (K, Usines) present (introduced) at position 4 (X) of the amino acid sequence of the pentapeptide (SEQ ID NO: 6, VPGXG ) in one, in several or in all its repetitions within the ELR.

In general, all cell types are capable of being coated with the method of the invention, either for their immunoisolation or as a bioengineering technique that allows a subsequent application of these cells in advanced systems, such as, for example, in the generation of tissue to through the interaction of different cell types arranged in different layers. Examples of cells that can be coated with the method of the invention are, but are not limited to, endothelial cells, neuronal cells, skeletal or smooth muscle cells, fibroblasts, insulin-secreting p cells, or pancreatic islets.

In another preferred embodiment, the cells are insulin-secreting p cells obtained by differentiation of human induced pluripotent cells (hiPSCs). This differentiation is obtained by incubating the hiPSCs with different culture media (supplemented with different growth factors), which are added and removing at different times, simulating, in this way, the natural process of development of pancreatic islets, in which the factors present in the environment are not always the same, being a dynamic process. When the method of the invention is applied to these cells in particular, preferably said method also comprises a step prior to step (a) that comprises seeding the cells in a cell strainer or "cell stainer", which allows to pass (submerge ) cells from one solution to another easily. Preferably, when the procedure is carried out on these cells, the ELR of step (a) further comprises a cell adhesion sequence, more preferably the RGD cell adhesion sequence.

The RGD domain is well known in the art and consists, as its name suggests, of the amino acids arginine, glycine, and aspartic acid. This domain is recognized by cell surface proteins of various cell types and functions as a cell adhesion domain.

The "cell adhesion sequence" used in the present invention can be any of those known in the state of the art capable of specifically binding to a protein or to a protein fragment present on the cell surface, such as integrins. The choice of the appropriate cell adhesion sequence will depend on the cell type that is intended to be coated with the method of the invention, being able to use, for example but without limiting us, the RGD domain, the LREL domain (SEQ ID NO: 5), sequences that include domains L-Arg-L-Glu-L-Asp-L-Val (REDV, SEQ ID NO: 7) for the coating of endothelial cells, or sequences comprising domains L-lle-L-Lys-L-Val- L-Ala-L-Val (IKVAV, SEQ ID NO: 8) for coating neuronal cells.

In another preferred embodiment, the ELR of step (a) comprises azide groups and the ELR of step (b) comprises alkyne groups.

When the method of the invention is carried out on insulin-secreting p cells obtained by differentiation of hiPSCs, preferably the ELR of step (a) comprises SEQ ID NO: 1, even more preferably SEQ ID NO: 1 comprising groups azide (this latter sequence will also be referred to herein as "ELR-RGD-azide"). More preferably, the ELR of step (b) comprises SEQ ID NO: 2, even more preferably SEQ ID NO: 2 comprising alkyne groups (this latter sequence will also be referred to herein as "ELR-alkyne").

When the method of the invention is carried out on insulin-secreting p cells obtained by differentiation of hiPSCs, preferably said method further comprises a final washing step, to remove excess ELRs. Such washing can be carried out in the presence of buffered saline solutions, such as, but not limited to, phosphate saline buffer (PBS) or Hank's balanced salt solution (HBSS).

When the process of the invention is carried out on insulin-secreting p cells obtained by differentiation of hiPSCs, preferably steps (a) and (b) are carried out in a volume of between 3 and 10 ml at a temperature of 4 ° C.

When the method of the invention is carried out on insulin-secreting p cells obtained by differentiation of hiPSCs, preferably steps (a) and (b) are repeated, according to step (c), at least two more times until get at least three bilayers of ELRs.

In another preferred embodiment, the cells are pancreatic islets.

When the method of the invention is carried out on pancreatic islets, preferably the first solution of step (a) comprises a mixture of two ELRs, where both ELRs comprise azide groups and where one of the two ELRs also comprises an adhesion sequence cell that comprises, preferably consists of, SEQ ID NO: 5. More preferably, the proportion of the two ELRs in said mixture is 10% for the ELR that does not comprise the cell adhesion sequence and 90% for the ELR that comprises the cell adhesion sequence, or 20% for the ELR that does not comprise the cell adhesion sequence and 80% for the ELR that comprises the cell adhesion sequence, or 30% for the ELR that does not comprise the cell adhesion sequence. cell adhesion and 70% for the ELR comprising the cell adhesion sequence. Even more preferably, the ELR that does not comprise the cell adhesion sequence comprises, more preferably consists of, SEQ ID NO: 3 and the ELR that comprises the cell adhesion sequence comprises, more preferably consists of, SEQ ID NO: 4 .

SEQ ID NO: 3 comprising azide groups will also be referred to in the present invention as "ELR-K-azide" and SEQ ID NO: 4 comprising azide groups will also be referred to in the present invention as "ELR-LREL-azide. ”.

SEQ ID NO: 5 is LREL, and it is a domain with collagen binding capacity, fundamentally type I, which is present in the extracellular matrix secreted by cells. The presence of this cell adhesion sequence in the ELR therefore allows its specific interaction with the cell surface.

When the process of the invention is carried out on pancreatic islets, preferably the ELR of step (b) comprises SEQ ID NO: 2, more preferably SEQ ID NO: 2 comprising alkyne groups ("ELR-alkyne").

When the process of the invention is carried out on pancreatic islets, preferably steps (a) and (b) of said procedure are carried out in a volume between 5 and 15 ml and at a temperature of 50C, and step ( a) is carried out for a time of 30 min.

When the process of the invention is carried out on pancreatic islets, preferably said process further comprises shaking the cells throughout the process on a 360 ° rotary shaker. This agitation prevents sedimentation and aggregation of the cells, facilitating a homogeneous coating. More preferably, this agitation is carried out at 3 rpm.

When the method of the invention is carried out on pancreatic islets, preferably said method further comprises an additional step, between steps (a) and (b), of a first centrifugation, washing and a second centrifugation. More preferably, these two spins are carried out at 1000 rpm, for 1 min each, at 50C. These centrifugation and washing steps serve to remove the first solution from step (a) and to remove ELR residues contained in the first solution that have not bound to the cell surface.

When the process of the invention is carried out on pancreatic islets, preferably the repetition of steps (a) and (b), according to step (c), gives rise to two bilayers of ELRs of the same composition. More preferably, after Repeating step (c), two identical ELRs bilayers are generated, each composed of an ELR-K-azide / ELR-LREL-azide layer joined to an ELR-alkyne layer.

When the process of the invention is carried out on pancreatic islets, preferably said process further comprises an additional step (d) in which steps (a) and (b) are repeated at least once more and in which the first solution of step (a) comprises an ELR modified to comprise azide groups and the cell adhesion sequence RGD and the second solution of step (b) comprises an ELR modified to comprise alkyne groups. More preferably, in this further step (d) the ELR of the first solution comprises SEQ ID NO: 1, even more preferably comprising azide groups (ELR-RGD-azide), and the ELR of the second solution comprises SEQ ID NO : 2, even more preferably comprising alkyne groups (ELR-alkyne).

In another preferred embodiment, steps (a) and (b) are repeated, according to step (c), until at least three, four, five, six, seven, eight or nine bilayers are achieved.

In another preferred embodiment of the method of the invention, the cells originate from a mammal such as, but not limited to, humans, rodents, ruminants, felines or canids. More preferably, the cells are derived from a rat or human, even more preferably from a human.

Another aspect of the invention relates to isolated cells or isolated nano-coated cell groups obtained by the method of the invention. In a preferred embodiment, said cells are insulin-secreting p cells obtained by differentiation of hiPSCs or pancreatic islets.

Another aspect of the invention relates to said cells or cell groups for use in medicine, preferably for tissue regeneration, both functional and structural, or cell therapy.

By "cell therapy" is understood the administration to a subject, individual or patient of living cells capable of exerting a therapeutic effect by curing, mitigating or improving the pathology or delaying or inhibiting the progression or course of the same.

Another aspect of the invention refers to the nanocoated cells or cell groups obtained by the method of the invention when these are insulin-secreting p cells obtained by differentiating hiPSCs or pancreatic islets, for their use in the treatment and / or prevention of diabetes.

Another aspect of the invention refers to a method for the treatment and / or prevention of diabetes in a subject, which comprises the administration of a therapeutically effective amount of the nano-coated cells or cell groups obtained by the method of the invention when they are Insulin-secreting p cells obtained by differentiation of hiPSCs or pancreatic islets.

The "therapeutically effective amount" is the amount or concentration of cells that produces the desired effect without causing adverse effects. The dose to achieve a therapeutically effective amount depends on a variety of factors such as, for example, the age, weight, sex or tolerance of the individual to whom the cells are to be administered. Therefore, said amount must be determined for each individual case.

Throughout the description and claims the word "comprise" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will emerge in part from the description and in part from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES

Fig. 1. Diagram of the most preferred coating of pancreatic islets with different bilayers of ELRs.

Fig. 2. Fluorescence microscopy images of rat pancreatic islets coated with different numbers of bilayers (BL) formed by an ELR-azide-Acetylene Fluor488 (green) and an ELR-alkyne-Cy5 (red). Stopover: 100 pm.

Fig. 3. Confocal microscopy images of sections of rat pancreatic islets under different conditions: uncoated, coated with non-fluorescent ELRs, coated by six bilayers of ELR-azide-Acetylene Fluor 488 (RGD green) and ELR-alkyne-Cy5 (VKV red), and covered by three bilayers of ELR-azide-Cy5 (RGD red) and ELR-cyclo-Cy5 (VKV red ) (both in red). Cell nuclei were stained with DAPI (blue).

Fig. 4. Fluorescence microscopy images of uncoated islets coated with six bilayers of fluorescently labeled ELRs (Cy5) after 48 h in culture in the presence of the cytokines TNF-a and IFN-y or in the absence of these (control) . The calcein images show living cells, while those of Pl correspond to dead cells.

Fig. 5. Insulin secretion from islets coated with three ELR-azide and ELR-alkyne bilayers (black line) and uncoated islets (control, gray line) in response to glucose exposure.

Fig. 6. Assay of insulin release by glucose stimulation in the case of islets coated with ELR-LREL-azide ELR-K-azide and ELR-alkyne (right), and not coated (left).

Fig. 7. Immunhistochemical staining of T lymphocytes (CD3 positive) inside allogeneic pancreatic islets coated with three ELR bilayers, three weeks after their transplantation into the renal capsule of immunocompetent mice.

Fig. 8. Allogeneic pancreatic islets implanted under the renal capsule of immunocompetent mice and extracted at three weeks in perfect condition, at small (left) and high magnification (right). Note the expression of insulin, which has been stained by immunohistochemical techniques.

Fig. 9. Fluorescence (left) and confocal (right) microscopy images of insulin-secreting p-cell spheroids, coated with three bilayers of ELR-azide-Cy5 (red) and ELR-alkyne-Cy5 (red), using one or the other as the first layer. Scale bar corresponding to 100 pm.

Fig. 10. Immunohistochemical staining of ELRs nanocoating (three bilayers) with an anti-elastin antibody, on coated (left) and uncoated (right) insulin-secreting p-cell spheroids.

Fig. 11. Insulin release by stimulation with different concentrations of glucose in uncoated insulin-secreting p cells (control) and coated with three bilayers of ELRs (RGD). C1 and C2 correspond to the glucose exposure cycles.

Fig. 12. Morphology and cell viability of insulin-secreting p cells obtained by differentiating hiPSCs coated with three bilayers of ELRs extracted after 7 (left) and 14 days (right) of in vivo implantation in mice. Live cells are displayed in bright pixels and dead cells in black pixels.

Fig. 13. Hematoxylin-eosin staining of uncoated insulin-secreting p-cell spheroids after implantation in mice and subsequent extraction.

Fig. 14. Hematoxylin-eosin staining of insulin-secreting p-cell spheroids coated with three bilayers of ELRs after implantation in mice and their subsequent extraction.

Fig. 15. Comparison between insulin-secreting p-cell spheroids coated with three bilayers of ELRs (left) and uncoated spheroids (right). In the latter case, note the presence of a large number of infiltrated immune system cells (higher pixel intensity, in black, corresponding to the staining of the cell nuclei), which greatly compromises the stability and functionality of the spheroids due to this immune attack. In contrast, coated spheroids show great integrity and stability, highlighting the protective activity of ELRs.

EXAMPLES

The invention will now be illustrated by means of tests carried out by the inventors, which demonstrate the effectiveness of the process of the invention in the encapsulation or nanocoating of cells for their immunoisolation.

EXAMPLE 1. OBTAINING, CHARACTERIZATION AND CHEMICAL MODIFICATION OF RECOMBINANT ELASTIN TYPE PROTEIN BIOPOLYMERS.

1.1. Design, production and characterization of elastin-type recombinant protein biopolymers.

The design and production of the synthetic nucleotide sequences that code for the amino acid sequences of the different biopolymers used were carried out as described in WO / 2010/092224. Likewise, the expression, purification and characterization of the biopolymers was carried out as described in WO / 2010/092224.

The following biopolymers were designed:

Biopolymer 1 of 699 amino acids

Nomenclature: ELR-RGD

Amino acid sequence (SEQ ID NO: 1): MGSSHHHHHSSGLVPRGSH-MESLLP - {[(VPGIG) 2 (VPGKG) (VPGIG) 2] 2 AVTGRGDSPASS [(VPGIG) 2 (VPGKG) (VPGIG) 2] 2]} 6-V

The theoretical Molecular Weight for biopolymer 1 is 60,661 Da and was estimated experimentally by polyacrylamide gel electrophoresis and by MALDI-TOF in a Q-Star model spectrometer, resulting in 60,525 Da. The transition temperature obtained by DSC in PBS was 32.2 ° C.

This biopolymer contains RGD cell adhesion sequences, with the particularity that an isoleucine amino acid of the pentapeptide in position 3 and of the pentapeptide in position 8 of the series of 10 pentapeptides that flank the RGD adhesion sequence, has been changed by another of lysine. , carrier of amino groups capable of being transformed into azide groups, remaining distributed along the chain of biopolymer 1.

Biopolymer 2 of 727 amino acids

Nomenclature: ELR

Amino acid sequence (SEQ ID NO: 2): MESLLPVGVPGVG [VPGKG (VPGVG ^{) 5] 23} VPGKG (VPGVG) 3 VPGV

The theoretical Molecular Weight for biopolymer 2 is 60,451 Da and was estimated experimentally by polyacrylamide gel electrophoresis and by MALDI-TOF in a Q-Star model spectrometer turning out to be 60,243 Da. The transition temperature obtained by DSC in PBS was 36.7 ° C.

This biopolymer can be considered inert as it does not contain bioactive sequences, being simply composed of pentapeptides derived from elastin. In addition, it has the particularity that five out of every six pentapeptides is substituted with an amino acid valine, while the remaining pentapeptide contains lysine, carrier of amino groups capable of being transformed into alkyne groups, being distributed along the chain of biopolymer 2 .

Biopolymer 3 of 367 amino acids

Nomenclature: ELR-K

Amino acid sequence (SEQ ID NO: 3): MESLLP (VPGKG) ⁷² V

The theoretical Molecular Weight for biopolymer 3 is 32,362 Da and was estimated experimentally by polyacrylamide gel electrophoresis and by MALDI-TOF in a Q-Star model spectrometer, resulting in 32,357 Da. Biopolymer 3 does not have a transition temperature due to its strong hydrophilic character that makes the molecule stay hydrated, at least in the temperature range in which water is in a liquid state (4-100 ° C).

This biopolymer can be considered fundamentally inert as it does not contain bioactive sequences, being simply composed of pentapeptides derived from elastin, all of them substituted with the amino acid lysine, carrying amino groups capable of being transformed into azide groups, remaining distributed throughout the biopolymer chain 3. On the other hand, unmodified amino groups are capable of nonspecifically interacting with the outer layer of the cell membrane.

Biopolymer 4 of 1201 amino acids

Nomenclature: ELR-LREL

Amino acid sequence (SEQ ID NO: 4): MESLLP {[VPGVG VPGSG VPGVG VPGKG VPGVG VPGSG VPGVG] 2 VAVTGRGDSPASSGGGGSGGGSGGGGS [VPGVG VPGSG VPGVG VPGVG VPGKG VPGVG VPGSG VPGVGS] 2 VAVTGRGDSPASSGGGGSGGGGSGGGGS [VPGVG VPGSG VPGVG VPGVG VPGGS VPGNGS VPGGVGS VPGGNGS VPG

The theoretical Molecular Weight for biopolymer 4 is 98,097 Da and was estimated experimentally by polyacrylamide gel electrophoresis and by MALDI-TOF in a Q-Star model spectrometer, resulting in 97,922 Da. Biopolymer 4 does not have a transition temperature due to its marked hydrophilic character that makes the molecule stay hydrated, at least in the temperature range in which water is in a liquid state (4-100 ° C).

This biopolymer contains RGD cell adhesion sequences, with the particularity that one amino acid valine of the pentapeptide in position 4 and of the pentapeptide in position 11 of the series of 14 pentapeptides that flank the RGD adhesion sequence, has been changed by another of Usina , carrier of amino groups capable of being transformed into azide groups, being distributed along the chain of biopolymer 4. In addition, a valine amino acid of the pentapeptides in position 2, 6, 9 and 13 has been replaced by another of serine, that maintains the hydrophilicity of the molecule thanks to the presence of alcohol groups that form stable hydrogen bonds with water. Finally, biopolymer 4 contains type I collagen binding sequences, preferably the so-called LREL (SEQ ID NO: 5), this type of collagen being present in the extracellular matrix of different cell groups, such as, for example, pancreatic islets. , improving interaction with them.

1.2. Chemical modification of elastin-type recombinant protein biopolymers.

Modification of biopolymer 1 to obtain biopolymer 1 ' (ELR-RGD-azide)

Obtaining biopolymer 1 'was carried out by modifying the amino group at the gamma position of lysine amino acids present in elastin pentapeptides. These amino groups are transformed to later carry out the "click" type delation reaction to form the different bilayers by "layer-by-layer". Specifically, in biopolymer 1 the amino groups were modified giving rise to azide groups in biopolymer 1 '.

To characterize biopolymer 1 ', a MALDI-TOF spectrometry was performed and from said analysis it was deduced that the molecular weight of the polymer was 62,211 Da, observing a logical increase of 1,686 Da with respect to biopolymer 1, by substituting the amino groups belonging to their Usinas by azide groups.

The transition temperature of biopolymer 1 'is 22.2 ° C and lower than that of biopolymer 1 of 32.2 ° C by 10.0 ° C. This behavior is due to the introduction of a strongly apolar group, such as the azide group, in this case, and the alkyne, in the case of biopolymer 2 '(see below), compared to the starting amino group. Therefore, this change in the transition temperature is a clear indicator of the modification of the biopolymers.

Biopolymers 2-4 were modified as described for biopolymer 1, giving rise to biopolymers 2'-4 ', respectively.

All the modified polymers were characterized in a similar way to T. Their molecular weights were determined by MALDI-TOF and their reverse transition temperature by DSC (except in the case of the 3 'and 4' biopolymer, since despite the modification no a transition temperature was observed), reaching conclusions similar to the previous ones.

Modification of biopolymer 2 to obtain biopolymer 2 ' (ELR-alkyne)

Experimental mean molar mass (MALDI-TOF): 62,271 Da.

Molar mass difference between biopolymer 2 and 2 ': 2,028 Da.

Transition temperature (DSC) in PBS: 18.9 ° C.

Transition temperature difference between biopolymer 2 and 2 ': 17.8 ° C.

Modification of biopolymer 3 to obtain biopolymer 2>' (ELR-K-azide)

Experimental mean molar mass (MALDI-TOF): 36,225 Da.

Molar mass difference between biopolymer 3 and 3 ': 32357.

Transition temperature (DSC) in PBS: N.A.

Transition temperature difference between biopolymer 3 and 3 ': N.A.

Modification of biopolymer 4 to obtain biopolymer 4 ' (ELR-LREL-azide)

Experimental mean molar mass (MALDI-TOF): 99.278 Da.

Molar mass difference between the biopolymer 4 and 4 ': 1,181 Da.

Transition temperature (DSC) in PBS: N.A.

Transition temperature difference between biopolymer 4 and 4 ': N.A.

EXAMPLE 2. ISOLATION OF PANCREATIC ISLETS AND OBTAINING INSULIN-SECRETARY CELLS FROM HUMAN INDUCED PLURIPOTENT CELLS (hiPSCs).

2.1. Isolation of pancreatic islets.

Pancreatic islets were obtained from a pancreatic biopsy, combining enzymatic digestion with collagenase P in Hanks culture medium with incubation at 37 ° C for 15 minutes, and subsequent density gradient with Histopaque 1077 to obtain islets with high purity. After obtaining, a part of each batch was used for morphological characterization by histology and immunohistochemistry. On the other hand, the hormonal levels in the islets were also studied, as well as the DNA content, the insulin releasing capacity and the expression levels of different genes by qPCR.

2.2. Obtaining human induced pluripotent cells (hiPSCs) and differentiation to insulin-secreting p cells.

Human induced pluripotent cells are obtained from the dedifferentiation of adult cells following well-established protocols (K Takahashi, et al., 2006, Cell, 126 (4), 663-676). Subsequently, they were cultured inside Matrigel hydrogels (Corning) with NutriStem XF medium (Biological Industries), which leads to the formation of spheroids, except when individual cells are needed, in which case the enzyme acutase is added, which breaks the cells. intercellular junctions. Once the spheroids were formed, they differentiated into insulin-secreting pancreatic p cells, specifically the cells that are part of the endoderm of the spheroid. For this, an established protocol was followed (Pagliuca et al., 2014, Cell, 159 (2), 428-439, and Vegas et al., 2016, Nature Medicine, 22 (3), 306-311) with slight modifications regarding the supplements used in the culture media. After 35 days of process, the cells were disaggregated from the spheroid and stained by immunofluorescence to determine the presence of insulin and / or glucagon, finding a much higher expression of the first than of the second, as desired for the treatment of the diabetes. The cells obtained from the differentiation of the hiPSCs secrete insulin in response to glucose, in a similar way to the p cells present in the pancreatic islets.

EXAMPLE 3. COVERING OF PANCREATIC ISLETS WITH ELRs THROUGH THE LAYER BY LAYER TECHNIQUE.

3.1. Coating method.

The coating of pancreatic islets was carried out using an optimized method to achieve coating of islets, both rat and human, without loss of viability, introducing them into the solution containing the first mixture of ELRs, containing 70, 80 or 90% of ELR-LREL-azide and 30, 20 or 10% ELR-K-azide, in 15 ml centrifuge tubes. Subsequently, these tubes were placed on a 360 ° rotary shaker to avoid sedimentation and aggregation of the islets and thus achieve a homogeneous coating. Once the process was completed, they were gently centrifuged to remove the solution with the first ELR and add washing solution to remove the remains of the same that have not been attached to the surface of the islet. The stirring and centrifugation process was repeated to add the solution containing the second ELR (ELR-alkyne), which reacts with the previous one to form a covalent bond and therefore form a stable bilayer (BL) by "layer-by-" layer ”. This whole process entailed an optimization of the different steps, modifying parameters such as the concentration of the ELRs, the volume of the solutions, the number of times the process is repeated (number of BLs), or the incubation time.

Specifically, an optimization of the composition of the first two BLs was made, these being decisive for achieving a complete and complete coating around the islets. For this, a mixture of an ELR with a high number of Usines that allow a nonspecific interaction with the cell surface, modified with azide (ELR-K-azide, SEQ ID NO: 3), in combination with a ELR incorporating the azide-modified type I collagen binding domain LREL (SEQ ID NO: 5) (ELR-LREL-azide, SEQ ID NO: 4), a protein present in the extracellular matrix of pancreatic islets. In this way a good interaction with the surface of the islets and the formation of a first integral and stable BL was achieved, repeating the process with the next one. Therefore, at this point it was necessary an optimization of the relationship between ELR-K-azide and ELR-LREL-azide (see Fig. 1). On the other hand, the BLs that settle on the first two bilayers are not so decisive, as they interact with the previous ones, providing a higher density to the nanocoating to achieve effective immunoisolation. The optimization was carried out as described in point 3.5.

3.2. Characterization of the homogeneity and integrity of the coating of pancreatic islets obtained by "layer-by-layer".

The characterization of the coating was carried out by marking the ELRs with fluorescent probes to be able to observe them by fluorescence microscopy. In this case, the observation of the fluorescent coating was carried out with rat islets, similar in composition to those found in humans, using different numbers of BLs. By fluorescence microscopy (Fig. 2) it was determined that the most homogeneous and continuous coating, avoiding the formation of aggregates, was achieved from the three bilayers. Furthermore, by confocal microscopy (Fig. 3) of sections of the coated islets, the integrity of the bilayers around the pancreatic islet was observed, which were coated with six BLs.

3.3. In vitro characterization of the permeoselectivity of the coating of pancreatic islets obtained by "layer-by-layer".

The impermeability to the cytokines TNF-a and IFN-y of the coating of six bilayers on human pancreatic islets was verified, which translates into greater cell viability when the islets are cultured in the presence of these cytokines (Fig. 4), confirming the effectiveness of the coating.

3.4. In vitro determination of insulin release from coated pancreatic islets.

The levels of insulin release in response to the addition of glucose were determined experimentally by means of a perfusion system, observing a similar release in the case of islets coated with up to nine bilayers of ELR-azide and ELR-alkyne, compared to the uncoated control islets, including the cases in which the conditions proposed in point 3.1 are used, that is, different ratios of combinations of ELR-K-azide and ELR-LREL-azide of the first layer of the first bilayer (10/90, 20/80 and 30/70%, respectively) (see Fig. 5 for the insulin release result with three bilayers and Fig.6 for the rest of the conditions).

3.5. Determination of the in vivo immunoisolation capacity of ELRs nanocoating on pancreatic islets.

For the determination of the stability and immunoisolation of the pancreatic islets thanks to the coating with ELRs, allogeneic islets coated with different numbers of bilayers were transplanted into mice. Subsequently, the islets were recovered after three weeks and their stability and the presence of insulin and glucagon releasing cells were verified by immunohistochemistry.

In a first case, mouse islets coated with three BLs composed of ELR-RGD-azide and ELR-alkyne were transplanted into the renal capsule of immunocompetent mice. After three weeks, the animals were sacrificed and the kidneys where the islets had been transplanted were recovered for immunohistochemical processing. Specifically, a specific staining of T lymphocytes (anti-CD3 antibodies) was performed, observing a large number of them in the transplanted islets (Fig. 7), which demonstrates an inefficient immunoisolation of the nanocoating with three bilayers.

In successive experiments, different amounts of BLs were tested, specifically three, six, nine and twelve, as well as incubation periods of the islets in the ELRs solutions of up to one hour, without achieving an improvement in immunoisolation.

This improvement was finally achieved by modifying the composition of the BLs, that is, the ELRs used in each layer, incorporating an ELR rich in positive charges in the first two bilayers for a better non-specific electrostatic interaction with the surface of the islets (ELR-K -azide, SEQ ID NO: 3), in combination with an ELR containing collagen-binding domains present on said surface (ELR-LREL-azide, SEQ ID NO: 4), which react with an ELR-alkyne to form the BLs by "layer by layer". This procedure is described in detail in section 3.1 and in Fig. 1. From In this way, a coating was achieved that allowed the immunoisolation of the pancreatic islets and, therefore, their non-recognition by the immune system of the transplant recipient organism (Fig. 8).

EXAMPLE 4. COATING WITH ELRs THROUGH THE LAYER BY LAYER TECHNIQUE OF INSULIN-SECRETARY P-CELLS OBTAINED THROUGH THE DIFFERENTIATION OF PLURIPOTENT INDUCED HUMAN CELLS

(hiPSCs).

4.1. Coating method.

For the coating method used for the insulin-secreting p cells obtained by the differentiation of human induced pluripotent cells (hiPSCs), hereinafter insulin-secreting p cells, the so-called "cell strainers" or cell strainer were used, which allows to pass cells from one solution to another easily. In the first place, the cells were seeded in said "cell strainer" and immersed in the first ELR-azide solution, to later pass it to a second solution that contained ELR-alkyne and that reacts with the previous one. This step was repeated to obtain as many BLs as necessary, to finish with a washing step to remove excess ELRs.

As in the case of pancreatic islets (Example 3), this procedure involved optimization to achieve perfect immunoisolation of insulin-secreting P cells. Specifically, the best protection was obtained with three bilayers of ELR-RGD-azide (SEQ ID NO: 1) and ELR-alkyne (SEQ ID NO: 2).

4.2. Characterization of the homogeneity and integrity of the coating by "layerby-layer" of insulin-secreting p cells.

The coating was characterized by fluorescence and confocal microscopy, similar to that described in point 3.2 (Fig. 9).

On the other hand, an immunohistochemical staining was performed using an antibody anti-elastin, such that the coating around spheroids of insulin-secreting p cells could be observed (Fig. 10).

4.3. In vitro determination of insulin release by layer-by-layer Nano-coated insulin-secreting p cells with ELRs.

Measurement of the levels of insulin released by insulin-secreting p cells coated with three bilayers of ELRs showed a differential response, releasing more insulin by increasing the concentration of glucose in the medium (Fig. 11), as occurs in the same way. physiological.

4.4. Determination of the in vivo immunoisolation capacity of coated insulin-secreting p cells.

The viability of insulin-secreting p cells coated with three bilayers of ELRs was first determined after 7 and 14 days post-implantation in mice, demonstrating good viability (Fig. 12).

On the other hand, the presence of lymphocytes inside the spheroids of insulin-secreting p cells was studied after implantation in mice. A large infiltration of lymphocytes was observed in the case of the control spheroids (uncoated, Fig. 13), while no infiltrated lymphocytes were observed in the spheroids coated with three bilayers of ELRs (Fig. 14), demonstrating immunoisolation thanks to the coating.

In Fig. 15 a similar result can be observed, in which spheroids of insulin-secreting P cells coated by layer-by-layer with three BLs of ELRs (ELR-RGD-azide (SEQ ID NO: 1) and ELR-alkyne (SEQ ID NO: 2)) are in perfect condition, while the uncoated spheroids contain a large number of infiltrated cells of the immune system, so their stability is completely compromised.

Claims (30)

1. An in vitro procedure for the nanocoating of cells comprising the following steps: to. immersing, for a time of between 5 and 30 min, the cells in a first solution that comprises at least one elastin-type recombinamer (ELR), where said ELR has been modified to comprise azide groups or alkyne groups and positive charges, b. immerse, for a time between 1 and 5 min, the cells of step (a) in a second solution comprising another ELR, where said ELR has been modified to comprise: (i) alkyne groups, when the ELR of step (a) comprises azide groups, or (ii) azide groups, when the ELR of step (a) comprises alkyne groups, thus forming a bilayer of ELRs, and c. repeat steps (a) and (b) at least once more until at least two bilayers of ELRs are achieved, where the ELRs of steps (a) and (b) are at a concentration between 5 and 25 mg / ml, in a volume of between 3 and 15 ml and where steps (a) and (b) are carried out at a temperature between 4 ° C and 50C. The method according to claim 1, wherein the positive charges of the ELR of step (a) are L-Lys present at position 4 of the amino acid sequence of the ELR. 3. The method according to any one of claims 1 or 2, wherein the cells are insulin-secreting p-cells obtained by differentiation of human induced pluripotent cells. The method according to any of claims 1 to 3, further comprising a step prior to step (a) comprising seeding the cells in a cell strainer. The method according to any one of claims 1 to 4, wherein the ELR of step (a) further comprises a cell adhesion sequence. 6. The method according to claim 5, wherein the cell adhesion sequence is RGD. The process according to any one of claims 1 to 6, wherein the ELR of step (a) comprises azide groups and the ELR of step (b) comprises alkyne groups. 8. The method according to any of claims 1 to 7, wherein the ELR of step (a) comprises SEQ ID NO: 1. The method according to any one of claims 1 to 8, wherein the ELR of step (b) comprises SEQ ID NO: 2. The process according to any one of claims 1 to 9, further comprising a final washing step. The process according to any one of claims 1 to 10, wherein steps (a) and (b) are carried out in a volume of between 3 and 10 ml and at a temperature of 4 ° C. 12. The process according to any of claims 1 to 11, wherein steps (a) and (b) are repeated, according to step (c), at least two more times until at least three bilayers of ELRs are achieved. 13. The method according to any of claims 1 or 2, wherein the cells are pancreatic islets. The method according to claim 13, wherein the first solution of step (a) comprises a mixture of two ELRs, where both ELRs comprise azide groups and where one of the two ELRs further comprises a cell adhesion sequence comprising SEQ ID NO: 5. The method according to claim 14, wherein the proportion of the two ELRs in the mixture is 10% for the ELR that does not comprise the cell adhesion sequence and 90% for the ELR that comprises the cell adhesion sequence, or 20% for the ELR that does not comprise the cell adhesion sequence and 80% for the ELR that comprises the cell adhesion sequence, or 30% for the ELR that does not comprise the cell adhesion sequence and 70% for the ELR that it comprises the cell adhesion sequence. 16. The method according to any of claims 14 or 15, wherein the ELR that does not comprise the cell adhesion sequence comprises SEQ ID NO: 3 and the ELR that comprises the cell adhesion sequence comprises SEQ ID NO: 4. 17. The method according to any of claims 13 to 16, wherein the ELR of step (b) comprises SEQ ID NO: 2. 18. The process according to any one of claims 13 to 17, wherein steps (a) and (b) are carried out in a volume of between 5 and 15 ml and at a temperature of 50C, and where step (a) is carried out for a time of 30 min. 19. The method according to any of claims 13 to 18, further comprising shaking the cells throughout the process on a 360 ° rotary shaker. 20. The process according to claim 19, wherein the stirring is carried out at 3 rpm. 21. The method according to any of claims 13 to 20, further comprising an additional step, between steps (a) and (b), of spinning, washing and spinning. 22. The method according to claim 21, wherein the centrifugation is carried out at 1000 rpm, for 1 min, at 50C. 23. The process according to any of claims 13 to 22, wherein the repetition of steps (a) and (b), according to step (c), gives rise to two bilayers of ELRs of the same composition. 24. The process according to any of claims 13 to 23, further comprising an additional step (d) in which steps (a) and (b) are repeated at least once more and in which the first dissolution of the step (a) comprises an ELR modified to comprise azide groups and the cell adhesion sequence RGD and the second solution of step (b) comprises an ELR modified to comprise alkyne groups. 25. The method according to claim 24, wherein in further step (d) the ELR of the first solution comprises SEQ ID NO: 1 and the ELR of the second solution comprises SEQ ID NO: 2. 26. The method according to any of claims 1 to 25, wherein the cells originate from a human. 27. Isolated nano-coated cells obtained by the method according to any of claims 1 to 26. 28. Cells according to claim 27, which are insulin-secreting p-cells obtained by differentiation of human induced pluripotent cells or pancreatic islets. 29. Cells according to any of claims 27 or 28 for use in medicine. Cells according to claim 28 for use in the treatment and / or prevention of diabetes.