Patent Description:
Extracellular vesicles (EVs) are a heterogeneous mixture of vesicular structures containing bioactive substances, surrounded by a lipid bilayer. EVs, as vesicles secreted from the cell surface, are surrounded by a biological membrane with a composition analogous to the composition of the cell membrane of cells secreting them. Importantly, the general composition and surface charge as well as the structure of biological membranes are the same for various types of animal cells, including human ones, being based on a lipid bilayer consisting mainly of phospholipids and proteins with a balanced composition. Consequently, such similarity also exists between the membranes of the EVs secreted by various types of animal cells, including those of human origin. At the same time, the EVs membrane is the part that comes into direct contact with the cryoprotective agent, thus it determines the mutual interaction between the vesicles and the cryoprotectant. Therefore, the similar structure of EVs membranes from animal cells, including human cells, also determines similar activity of these cryoprotectants to vesicles of various origin, and thus the effectiveness of the cryopreservation process.

Due to their size, EVs can be classified as apoptotic bodies (><NUM>), microvesicles (<NUM> - <NUM>) or exosomes (<NUM> - <NUM>). These structures differ not only in size but also in chemical and biological composition. Importantly, an increasing number of literature reports indicate that bioactive factors present in EVs, including lipids, proteins (e.g. transcription factors, enzymes, receptors, signalling and adhesive molecules), as well as nucleic acids (mRNA, miRNA and other small non-coding RNAs), can be transferred by EVs to target cells, inducing functional changes in them. Thus, EVs enable intercellular communication and play an important role in many cellular processes. Of particular interest are EVs produced by stem cells (SCs), including mesenchymal SCs (MSCs) and induced pluripotent SCs (iPS), as they are an important element of paracrine activity and the ability of these cells to regenerate damaged tissues [<NUM>]. EVs have been shown to have a therapeutic effect by transmitting biological signals to cells at the site of tissue injury. Therefore, it is expected that the use of EVs will soon enable the development of new cell-free therapies that will minimize the problems associated with immunogenicity, low retention, and possible side effects that may accompany whole cell transplantation. It is expected that EVs will be also used as effective drug carriers, biomarkers as well as components of vaccines [<NUM>-<NUM>].

While the number of registered clinical trials on EVs is still limited, the current results are very encouraging. In order to enable the development of the therapeutic use of EVs, it is necessary to improve the methods of obtaining clinical quality formulations containing EVs and, taking into account their unique features, to develop standard procedures that enable their structural integrity and biological activity to be maintained during long-term storage required due to the practical reasons [<NUM>,<NUM>]. Commonly used methods for obtaining EVs are ultracentrifugation, including gradient ultracentrifugation, ultrafiltration and precipitation with polyethylene glycol (PEG). EV storage methods include freeze-drying, spray drying and the most promising method - cryopreservation - which is widely used in the storage of biological specimen. Depending on the needs, the formulations are frozen at temperatures ranging from -<NUM> to -<NUM> and stored at temperatures ranging from -<NUM> to -<NUM>.

The results of research on cryopreservation of EVs indicate that in this process they undergo unfavourable structural changes, including aggregation and degradation, which reduces their biological activity [<NUM>-<NUM>].

The main reason for this are the destructive effects of water freezing - the formation of ice crystals and changes in osmotic pressure. To reduce these problems, chemical compounds - so-called cryoprotectants - are used, whose role is to reduce the formation of ice crystals and changes in osmotic pressure, which prevents the decomposition and fusion of EVs and stabilizes the proteins present in them. Cryoprotectants both capable of penetrating the lipid bilayer of EVs (internal cryoprotectants) and unable to penetrate it (external cryoprotectants) are used. Penetrating cryoprotectants are low-molecular compounds with a molecular weight not exceeding <NUM> Da. They penetrate inside the EVs, stabilizing the biomolecules present in them. Non-penetrating cryoprotectants are compounds with molecular weights of about <NUM>-<NUM> Da. They stabilize EVs from the outside, preventing hydroosmotic lysis, increasing the viscosity of the aqueous environment, slowing down the process of ice crystal nucleation and controlling the kinetics of their formation. It has been shown that the best effects are achieved by using both types of cryoprotectants simultaneously and that some of them act both inside and outside of EVs. These include low molecular weight compounds such as glucose, lactose, sucrose, glycerol, cyclodextrin derivatives, propylene glycol and trehalose [<NUM>]. They interact with the polar groups of phospholipids, disrupt the hydration shell around EVs created with the participation of hydrogen bonds and replace it with an amorphous, glassy protective layer, preventing fusion of EVs and destabilization of proteins. Trehalose has a particularly interesting effect, stabilizing the colloidal suspension of EVs and limiting the formation of ice crystals inside the EVs. Dimethyl sulfoxide (DMSO) is also used, which modifies both the surroundings of EVs and their interiors. However, in vitro studies have shown that DMSO is toxic to cell cultures, therefore, it requires rapid washing from the suspension of EVs immediately after their thawing [<NUM>]. However, such a procedure of removing DMSO from EVs samples requires their re-ultracentrifugation, which not only significantly extends the time and cost of material preparation, but also may result in destabilization of the content of bioformulations. Additionally, there is a risk that the addition of this compound may generate measurement artefacts, thus affecting the correct interpretation of biological analyses. Some research experiments have been also carried out using other polymers such as PEG, polyvinyl alcohol (PVA), poly(vinylpyrrolidone) (PVP), and gelatin.

In an <CIT> tissue regenerative biological composition is disclosed. More specifically, a composition at least in part formed from bone marrow and a method of manufacture and use of said composition with an acellular mixture.

However, so far no such polyelectrolytes have been developed, the use of which as cryoprotectants of EVs secreted by human and animal cells would ensure adequate stabilizing effect, structural integrity of EVs and would guarantee the maintenance of the biological activity of EVs and their therapeutic abilities. The main problem was also the selection of cryopreservation conditions, the selection of the cryoprotectant itself and its concentration to ensure the maximum protection of EVs during their freezing and thawing while maintaining their biological properties.

Therefore, the aim of the invention was to develop the use of selected polyelectrolytes as cryoprotectants, which would be effective and biocompatible, and would ensure the possibility of storing EVs samples under defined, reproducible conditions, ensuring structural stabilization and maintaining the biological activity of EVs secreted by human and animal cells.

The object of the invention is therefore a use of polyelectrolytes as cryoprotectants of extracellular vesicles, wherein said polyelectrolyte is the one obtained by modification of a polymer of natural origin and/or a synthetic polyelectrolyte, said polyelectrolyte of natural origin is dextran modified by substitution of the hydroxyl groups with glycidyltrimethylammonium chloride and said homopolymer is a synthetic polyelectrolyte selected from the group comprising:.

Preferably, the extracellular vesicles are secreted by human and/or animal cells.

The invention also includes a method of cryopreservation of extracellular vesicles secreted by human and animal cells, involving the following steps:.

The cryopreservation method is preferably characterized in that in the step b) of coating, the polyelectrolyte is a modified polymer of natural origin and/or a synthetic polyelectrolyte including a cationic homopolymer and/or zwitterionic homopolymer and/or a block copolymer and/or a graft polymer as defined in the first object of the invention; moreover, preferably optionally in step c) of coating, polyelectrolyte is a synthetic polyelectrolyte as defined in the first object of the invention.

The invention is also shown in Figures, where:.

In order to obtain polymers for use in cryopreservation, a number of experiments aimed at controlled synthesis and characterization of polymers were performed, and compounds with optimal cryoprotective properties towards EVs samples were selected. The analysis of the obtained data clearly indicates that the variants of polymers were obtained capable of protecting the integrity of EVs during their storage, while maintaining their biological functions. The results showing the effectiveness of the tested polymers as EVs cryoprotectants are presented below.

The subject of the invention is illustrated in more detail in the non-limiting embodiments.

The cationic dextran derivative with an average molecular weight of <NUM> kDa was modified by substituting the hydroxyl groups with glycidyltrimethylammonium chloride (GTMAC) according to the procedure shown below. Thus, <NUM> of dextran was dissolved in <NUM> of distilled water and <NUM> mmol of NaOH was added. The solution was heated to <NUM> while mixing with a magnetic stirrer. Then, <NUM> of GTMAC was added and heated with stirring for <NUM>. The reaction mixture was cooled and transferred to a dialysis tube with MWCO of <NUM> kDa. Dialysis was carried out against water. The purified polymer, denoted DEX, with a substitution degree (DS) of <NUM>% was isolated by freeze-drying. <CHM>
<CHM>.

A series of PAMPSx homopolymers (where x - the degree of polymerization) was obtained by the RAFT method and then characterized.

AMPS (<NUM>-acrylamido-<NUM>-methyl-<NUM>-propanesulfonic acid) (<NUM>, <NUM> mmol) was neutralized with <NUM> NaOH (<NUM>) to a pH of <NUM>. V-<NUM> (<NUM>, <NUM> mmol) and CPD (<NUM>-cyano-<NUM>-(phenylcarbonothioylthio)pentanoic acid) (<NUM>, <NUM> mmol) were dissolved in a mixture of <NUM> of MeOH and <NUM> of water, and then added to the aqueous AMPS solution. The solution was degassed by purging argon for <NUM>. Polymerization was carried out at <NUM> for <NUM>. The conversion of AMPS as determined by <NUM>H NMR was <NUM>%. The reaction mixture was dialyzed against pure water for one day. PAMPS142 was isolated by freeze-drying (<NUM>, <NUM>%). The number average molecular weight (Mn(NMR)), the degree of polymerization (DP) determined by <NUM>H NMR and the dispersion index (Mw/Mn) determined by gel permeation chromatography (GPC) were <NUM> × <NUM><NUM>, <NUM> and <NUM>,<NUM>, respectively. The remaining PAMPSx homopolymers were prepared in an analogous manner.

For further studies on stabilization of EVs, the PAMPS18 homopolymer was selected with the number average molecular weight determined on the basis of the <NUM>H NMR spectrum equal to <NUM> Da and the dispersion index Mw/Mn = <NUM>).

A series of PMAPTACy homopolymers (where y - degree of polymerization) were obtained by the RAFT method, and then characterized.

MAPTAC (<NUM>, <NUM> mmol), V-<NUM> (<NUM>, <NUM> mmol), and CPD (<NUM>, <NUM> mmol) were dissolved in a mixture of MeOH (<NUM>) and water (<NUM>). The solution was degassed by purging argon for <NUM>. Polymerization was carried out at <NUM> for <NUM>. The degree of MAPTAC conversion estimated from <NUM>H NMR was <NUM>%. The reaction mixture was dialyzed against pure water for one day. PMAPTAC147 was isolated by freeze-drying (<NUM>, <NUM>%). Mn (NMR) and DP, estimated from <NUM>H NMR, and the dispersion coefficient (Mw/Mn), estimated by GPC were <NUM> × <NUM><NUM>, <NUM> and <NUM>, respectively. The remaining PMAPTACy polymers were prepared in an analogous manner.

The PMAPTAC16 homopolymer was selected for further studies on EVs stabilization.

The RAFT method was used to obtain a series of poly (<NUM>-(methacryloyloxy)ethylphosphorylcholine) homopolymers (PMPCz, where z is the degree of polymerization, DP) differing in molecular weight, which were then characterized. The polymers were prepared using a literature procedure [<NUM>]. <NUM>-cyano-<NUM>- (phenylcarbonothioylthio)pentanoic acid (CPD) was used as chain transfer agent (CTA). The molecular weight of the polymer was controlled by the synthesis time as proposed in the literature [<NUM>].

<NUM> of (methacryloyloxy)ethylphosphorylcholine (MPC) monomer was dissolved in <NUM> of distilled water, and then this solution was passed through a column filled with neutral alumina to remove the polymerization inhibitor. The column was rinsed with enough water to bring the volume of the monomer-containing solution to <NUM>. The solution was then degassed by purging argon for <NUM>. After this time, an initiator (<NUM>,<NUM>'-azobis(<NUM>-cyanopentanoic acid), V-<NUM>) was added and the solution was degassed again for <NUM>. <NUM> of CPD dissolved in <NUM> of ethanol was added to the thus prepared solution. The resulting solution was stirred and heated to a temperature of <NUM>. This temperature was maintained for a strictly defined time according to the table below. After this time, the reaction mixture was cooled to room temperature and transferred to a cellulose dialysis tube (MWCO of 3kDa). The solution was dialyzed against water for <NUM> days by changing the water every <NUM>. The polymer was then isolated from the resulting solution free from unreacted monomer and initiator using lyophilization. A series of <NUM> polymers denoted as PMPCz was obtained. All polymers were well soluble in water (solubility of the order of <NUM>/l).

The elemental composition of PMPCz polymers was determined on the basis of the results of elemental analysis (Table <NUM>).

The results of elemental analysis confirmed that the extension of the synthesis time resulted in the formation of polymers with higher molecular weights (the content of sulfur coming from CPD decreases with increasing chain length). The N/C value also increases, approaching the value characteristic of the monomer for the polymer with the highest molecular weight.

These results correlate with the molecular weight studies carried out using gel permeation chromatography (GPC) (<FIG>) performed with the following analysis parameters: Polysep-GFC-P-linear <NUM> column, eluent <NUM> NaCl and <NUM>% NaN<NUM>, mobile phase flow rate: <NUM>/min, polymer concentration <NUM>/L, sample volume <NUM>µL.

On the basis of the obtained chromatograms, the molecular weight and the degree of polymerization of the obtained polymers were determined (Table <NUM>).

<NUM> of PMPC33 was dissolved in <NUM> of distilled water. The solution was then degassed by purging argon for <NUM>. After this time, <NUM> of V-<NUM> initiator was added and the solution was degassed again for <NUM>. <NUM> of fluorescein o-acrylate and <NUM> of ethanol were added to the solution prepared in this way. The mixture was stirred and heated to <NUM>. This temperature was maintained for <NUM>. At this time, the reaction mixture was cooled to room temperature, filtered to remove undissolved monomer, and transferred to a cellulose dialysis tube with MWCO of <NUM> kDa. The solution was dialyzed against water for <NUM> days changing water every <NUM>. The polymer was isolated by lyophilization from the resulting solution free from unreacted monomer and initiator.

To confirm the effectiveness of the fluorescence labeling of PMPC33 with fluorescein, the emission spectra of the labeled and unlabeled polymer were recorded (<FIG>). This spectrum shows a band with a maximum at wavelength of <NUM>, which corresponds to fluorescein, which confirms successful labeling.

In addition, GPC chromatographic analysis of the fluorescently labeled polymer was performed. <FIG> shows that the signal from the fluorescein-labeled compound is clearly shifted from the unlabeled polymer signal towards shorter retention times, which confirms effective fluorescent labeling of PMPC33 with fluorescein chromophores and excludes the simultaneous formation of a fluorescein o-acrylate homopolymer.

For the purposes of the present research, a series of PEGm-b-PMAPTACn block polyelectrolytes (where w and v are the degrees of polymerization of the respective blocks) containing a neutral block of poly(ethylene glycol) (PEG) and a cationic block of poly((<NUM>-(methacryloylamino)propyl)trimethylammonium chloride) (PMAPTAC) was synthesized and characterized (GPC, NMR, FTIR, elemental analysis). The polymers were synthesized by a Reversible Addition-Fragmentation Chain Transfer Polymerization (RAFT) process.

The chain transfer agent (CTA), which was CPD, was prepared according to the procedure described in the literature [<NUM>]. PEG46-CTA macromonomer, i.e. poly(ethylene glycol) (PEG) with a degree of polymerization equal to <NUM>, terminated with a chain transfer agent (CTA), was prepared according to the procedure described in the publication [<NUM>]. MAPTAC (<NUM>, <NUM> mmol), initiator (<NUM>,<NUM>'-azobis (<NUM>-cyanopentanoic acid) (V-<NUM>), <NUM>, <NUM> mmol) and PEG46-CTA (<NUM>, <NUM> mmol) were dissolved in water (<NUM>). The solution was deoxygenated by purging argon for <NUM> hour. Polymerization was carried out at <NUM> for <NUM>. The reaction mixture was dialyzed against pure water for two days. The obtained polymer (PEG46-b-PMAPTAC52) was isolated by freeze-drying. <NUM> of product was obtained (<NUM>% yield). The structure of the polymer obtained is shown below.

The polymer has been characterized using spectroscopic and chromatographic methods. The GPC chromatogram of the obtained polymer (<FIG>) was measured at <NUM> using an aqueous <NUM> Na<NUM>SO<NUM> solution containing <NUM> acetic acid as eluent. The number average molecular weight of the polymer, Mn, determined on its basis, was <NUM> kDa. The polymer was characterized by a low dispersion index Mw/Mn = <NUM>. The number average degree of polymerization (DP) of the PMAPTAC block, determined by <NUM>H-NMR measurements, was <NUM>. The <NUM>H-NMR spectrum of the polymer in D<NUM>O is shown in <FIG>.

The characteristics of all obtained PEGw-b-PMAPTACv copolymers are presented in Table <NUM>.

Acryloyl chloride (<NUM>, <NUM> mol) was added over <NUM> minutes to <NUM> of a solution of <NUM>-aminoundecanoic acid (<NUM>, <NUM> mol) in <NUM> NaOH in water placed in an ice bath. The resulting solution was stirred for <NUM> at room temperature. After the reaction had taken place, the pH of the solution was adjusted to <NUM> with <NUM> hydrochloric acid. The precipitate was filtered off and washed twice with water. The product (<NUM>-(acrylamido)undecanoic acid) was purified by three recrystallizations from a mixture of acetone and n-hexane (<NUM>/<NUM> v/v) and dried at <NUM> in vacuo. Yield: <NUM> (<NUM> mmol, <NUM>%).

<NUM>-(Acrylamido)undecanoic acid was then neutralized with an equivalent amount of NaOH (<NUM>, <NUM> mmol) in methanol to give the salt precipitated with diethyl ether. The sample was dried at <NUM> under vacuum. <NUM> (<NUM>%) of sodium <NUM>-(acrylamido)undecanoate (AaU) were obtained.

AMPS (<NUM>, <NUM> mmol) was neutralized with a solution of NaOH (<NUM>, <NUM> mmol) in <NUM> of water. CPD (<NUM>, <NUM> mmol) and V-<NUM> (<NUM>, <NUM> mmol) were added to the resulting solution. The mixture was deoxygenated by purging argon for <NUM>. The polymerization was carried out at <NUM> for <NUM>. The obtained polymer was purified by dialysis against pure water for <NUM> week and isolated by freeze-drying. Yield: <NUM>, <NUM>%.

PAMPS-CTA macromonomer (<NUM>, <NUM> mmol), sodium <NUM>-(acrylamido)undecanoate (AaU, <NUM>, <NUM> mmol), V-<NUM> (<NUM>, <NUM> mmol) ) were dissolved in <NUM> of water. The solution was deoxygenated by purging argon for <NUM>. The polymerization was carried out at <NUM> for <NUM>. The obtained block copolymer was purified by dialysis against a NaOH solution at pH <NUM> for <NUM> week, which was changed twice daily. The product was isolated by freeze-drying. Yield: <NUM>, <NUM>%. The degree of polymerization of the PAMPS block (DPPAMPS = <NUM>), the number average molecular weight (Mn = <NUM> × <NUM><NUM>) and the dispersion coefficient ( <MAT>) were determined by gel permeation chromatography (GPC) using the mixture of water and acetonitrile <NUM>/<NUM> v/v containing <NUM> NaNO<NUM> as a mobile phase. The degree of polymerization of the PAaU block (DPPAaU = <NUM>) was determined from the <NUM>H NMR spectrum (<FIG>).

The structure of the resulting PAMPS75-b-PAaU39 block copolymer is shown below. <CHM>
m = <NUM>, <NUM>, <NUM>, and <NUM>.

The characteristics of the obtained PAMPSm-b-PAaUn copolymers are presented in Table <NUM>.

Poly(vinyl alcohol) (PVA, <NUM> kDa, degree of hydrolysis <NUM>%, <NUM>) was dissolved in <NUM> of dimethylformamide (DMF) at <NUM> under reflux with vigorous mixing using a magnetic stirrer. The temperature was then lowered to <NUM> and argon was purged through the mixture. After <NUM> of gas purging, a solution of benzoyl peroxide (BPO) in DMF (<NUM> BPO in <NUM> of deoxygenated DMF) was added. After <NUM>, <NUM> of (<NUM>-acrylamidopropyl)trimethylammonium chloride (APTAC, <NUM>% solution in water) was added gradually. The reaction was continued for <NUM> with argon flowing continuously through the system. After this time, the mixture was cooled to room temperature and dialyzed (MWCO of <NUM> kDa) first against DMF, then against DMF/water mixtures with decreasing amount of DMF, and finally against pure water. The purification was completed after several days when the conductivity of the water outside the dialysis tube dropped to <NUM>-<NUM>. The mixture was concentrated with an evaporator, centrifuged (<NUM>, <NUM>) and freeze-dried. The dry substance, which was a mixture of the desired product and the non-PVA-attached PAPTAC homopolymer, was washed twice with methanol and centrifuged (<NUM>, <NUM>). The product precipitate was dried in vacuo.

The obtained product was subjected to physicochemical tests. The results of the elemental analysis are shown in Table <NUM>.

On the basis of the N/C ratio, the average number of APTAC groups attached to PVA per <NUM> mers of vinyl alcohol was calculated, amounting to approximately <NUM>. The modification of PVA was confirmed by measuring FT-IR spectra of the obtained product. The presence of the band at <NUM>-<NUM> originating from the C=O stretching vibrations of the amide group and the band at <NUM>-<NUM> originating from the N-H bending vibrations confirms the reaction (<FIG>).

Comparing the GPC chromatograms of PVA and PVA-graft-PAPTAC, it was confirmed that one well-defined product was obtained and that no degradation of PVA occurred during the reaction (<FIG>).

<NUM> of the previously prepared PVA-graft-PAPTAC was dissolved in <NUM> of dry dimethylformamide (DMF). The process was carried out at a temperature of <NUM> under a reflux condenser, mixing the system using a magnetic stirrer and purging nitrogen through the mixture. After dissolving the substrate, the mixture was cooled to room temperature. <NUM> of sodium hydride was added with continued stirring. After <NUM>, <NUM> of <NUM>-bromooctane was added. The reaction was complete after <NUM>. The mixture was dialyzed (MWCO of <NUM> kDa) first against DMF, then against a DMF/H<NUM>O mixture with increasing water content and finally dialyzed against pure water. Dialysis was completed when the conductivity of the water outside the dialysis tube had dropped to <NUM>. The mixture was centrifuged four times (<NUM>, <NUM>). The supernatant was lyophilized. The obtained dry product was washed 3x with diethyl ether and then the solvent was removed in vacuo.

The course of the reaction is shown schematically below:
<CHM>
<CHM>
or -H.

The obtained product was subjected to physicochemical tests. The results of the elemental analysis are presented in Table <NUM>.

Based on the N/C ratio, the mean degree of substitution of PVA-graft-PAPTAC with ionic groups was estimated. It is about <NUM>% (i.e. <NUM> PAPTAC groups per <NUM> mers of vinyl alcohol (VA).

The presence of octyl groups in the PVA-graft-PAPTAC-Oct structure was confirmed by recording the <NUM>H-NMR spectrum for this polymer in D<NUM>O (<FIG>). The signal present at <NUM> ppm originates from the protons of the terminal CH<NUM> group of the attached hydrophobic chain. Based on the integration of appropriate signals, the degree of substitution with hydrophobic groups was estimated to be approximately <NUM>%.

In order to investigate the cytotoxicity of the polymers (used for EVs cryopreservation) on eukaryotic cells in vitro, proliferation, cytotoxicity and apoptosis assays were performed on polymer-treated human osteoblasts (HOBs). A commercial ApoTox-Glo Triplex Assay kit (Promega) and the following polymers: DEX, PEG, PEG-PAMPS were used to perform the analysis. The addition of polymers at a dose corresponding to 1x, 10x and 100x the dose of the EVs formulation was tested. The analysis of the influence of polymers on cells was performed <NUM> hours after their addition into the culture medium.

The analysis showed no cytotoxic and pro-apoptotic influence of the polymers on the tested cells (<FIG>), which proves the safety of using these polymers for cryopreservation of EV formulations, that can be subsequently used in further functional studies in vitro.

First, EVs were isolated from the conditioned medium, i.e. harvested from the culture of appropriate human or animal cells, using the sequential centrifugation method, according to the scheme shown in <FIG>. The method involved several steps, including firstly the preliminary centrifugation of the culture medium (<NUM>, <NUM>, <NUM>) to remove cellular elements and larger cell debris. Thereafter, the supernatants were ultracentrifuged at <NUM> for <NUM> at <NUM> to pellet the EVs fraction. The obtained EV pellets were rinsed with phosphate buffered saline (PBS) free of calcium and magnesium ions and additionally filtered through pores with a diameter of <NUM>, and then again ultracentrifuged with the same parameters (<NUM> for <NUM> at <NUM>).

Importantly, in order to remove EVs from the components of the medium (including serum and platelet lysate), which could adversely affect the quality of the obtained results at later stages of the research, the culture media were also subjected to the ultracentrifugation procedure (<NUM>, <NUM> hours, <NUM>) before use. Thus, it was possible to isolate and then analyze only EVs secreted by cells. In addition, the conditioned medium was subjected to preparation immediately after harvesting it from cell culture, without freezing step, which eliminated the possible impact of the additional low-temperature storage step on the subsequent results. EV samples were mixed with polymer solutions immediately after the isolation step and then further analyzed.

The isolated EVs were suspended in <NUM>µl of PBS solution. The suspension was then divided into two parts of equal volumes. A solution of PEG<NUM>-b-PMAPTAC<NUM> in PBS was prepared at a concentration of <NUM>/ml. At room temperature, one part of the EVs suspension was mixed with the PEG<NUM>-b-PMAPTAC<NUM> solution in PBS solution by adding the EVs suspension (<NUM>µl) to <NUM>µl of the PEG<NUM>-b-PMAPTAC<NUM> solution and repeatedly pipetting. In the resulting mixture, the concentration of PEG<NUM>-b-PMAPTAC<NUM> was <NUM>µg/ml and the concentration of EVs was <NUM><NUM>/ml. An equal volume of PBS solution was added to the second part of the EVs suspension (control).

Then, the samples obtained in this way were divided into two parts, one of which was directly tested and the other was frozen in liquid nitrogen or at -<NUM>. After an appropriate storage period of the samples (days/months) under deep freeze conditions, they were thawed by heating in a <NUM> water bath until ice crystals disappeared and subjected to the tests described in the following examples.

The same procedure was also applied to the coating of EVs using the following polymers: PMPC<NUM>.

A <NUM>/ml solution of PEG<NUM>-b-PMAPTAC<NUM> polymer in PBS was prepared as in procedure A, and <NUM>µl of this solution was mixed at room temperature with the EVs suspension (<NUM>µl) in PBS solution. The concentration of PEG<NUM>-b-PMAPTAC<NUM> in the resulting mixture was <NUM>µg/ml and the concentration of EVs was <NUM><NUM>/ml. An equal volume of the PBS solution was added to the second part of the EVs suspension (<NUM>µl of control suspension).

A few minutes after the addition of PEG<NUM>-b-PMAPTAC<NUM>, the PAMPS<NUM> solution in PBS at a concentration of <NUM>/ml was mixed with the PEG<NUM>-b-PMAPTAC<NUM>-coated EVs solution and thoroughly mixed in the same way as in procedure A, i.e. by adding a suspension of EVs coated with PEG<NUM>-b-PMAPTAC<NUM> into PAMPS<NUM> solution and repeated pipetting. The concentration of EVs in the obtained mixture was <NUM><NUM>/ml and the concentration of both polymers was <NUM>µg/ml (thus the weight ratio of the polymers was <NUM>:<NUM>).

Then, the samples obtained in this way were divided into two parts, one of which was directly tested and the other was frozen in liquid nitrogen or at -<NUM> ° C. After an appropriate storage period (days/months) under deep freeze conditions, the samples were thawed by heating in a <NUM> water bath until ice crystals disappeared and subjected to the tests described in the following embodiments.

The same procedure was also applied to the coating of EVs using a combination of polymers selected from the polymers shown below for layers I and II:.

The coating of EVs by PEG46-b-PMAPTAC52 was confirmed by confocal microscopy (laser excitation at <NUM>, FITC channel) using a fluorescently labeled polymer PEG45-PMAPTAC51-<NUM> mol% Alexa Fluor <NUM>. The corresponding images are shown in Figs. The presence of fluorescent dots in Fig. 12A confirms the effective coating of EVs with the polymer PEG45-b-PMAPTAC51-<NUM> mol% Alexa Fluor <NUM> - no signal for EVs in the absence of polymer and no signal for polymer solution.

The influence of polymers on the integrity and phenotype of vesicle samples subjected to multiple freezing and thawing was tested. This is of particular importance in view of the necessity of frequent thawing of the same EV samples in laboratory practice. EV samples, after their isolation by ultracentrifugation, were mixed with polymers, and then frozen and thawed in <NUM>-fold or <NUM>-fold cycles. Measurement of the concentration and particle size distribution was performed with the NanoSight NS300 system (Malvern), using the nanoparticle tracking analysis (NTA) method. EVs samples coated with selected polymers were diluted appropriately with PBS in order to obtain a concentration range corresponding to the detection optimum of the NanoSight system, and then measured.

Table <NUM> presents the names of the samples used in the further part of the study together with their description.

The obtained results indicate that multiple freezing and thawing of the samples affects both the concentration of particles (<FIG>) and causes a slight decrease in their size (<FIG>, <FIG>).

The influence of selected polymers on the integrity and phenotypic characteristics of particles in cryopreserved EVs preparations was investigated using an Apogee A-<NUM> Micro flow cytometer (Apogee Flow Systems). The analysis was carried out on identical samples as in the case of measurements by the NTA analysis method (Table <NUM>).

Individual samples were prepared according to an optimized protocol, by subjecting them to immunofluorescence staining with antibodies directed against the selected surface antigens. For this purpose, CD81 tetraspanin was chosen, which is a marker for EVs, as well as an antigen highly expressed on cells from which EVs were obtained (variable depending on the cell type, e.g. CD90 in the case of mesenchymal cells). Additionally, in order to investigate the influence of polymers on the integrity of EVs structure, samples were stained with RNA Select dye (Thermo Fisher Scientific), which after penetration into undamaged EVs binds to RNA molecules, exhibiting green fluorescence. In order to correctly analyze and verify the specificity of the obtained results, a number of controls, including unstained and isotopically labeled controls were used in the cytometric analysis.

As with the results of the NTA analysis, changes in the signal level from RNA Select were observed (<FIG>). At the same time, the protective effect of the tested polymers against EVs formulations was observed.

A cytometric analysis of the influence of selected polymers on the phenotype of particles was performed on EV samples subjected to multiple freeze-thaw cycles, using the EVs secreted by UC-MSCs cells (<FIG>). Samples were subjected into immunofluorescence staining with antibodies directed against selected surface antigens typical of EVs (CD81) and mesenchymal cells (CD90). The cumulative results of the analysis of the percentage of antigen-positive particles in individual types of samples indicate that the coating of EVs with ultra-thin polymer layers does not adversely affect the biological properties of the EV surface, in particular does not reduce the level of CD81 and CD90 surface antigens. The tenfold freeze and thaw EVs cycle has no significant effect on CD81 and CD90 levels. Freezing and thawing twenty times leads to a decrease in CD81 and CD90 levels, but this effect is considerably lower for CD90. The effect of the coating with polymers is slight, although generally positive.

The influence of polymers (PEG46-b-PMAPTAC52, DEX, PEG46-b-PMAPTAC52 + PAMPS18, PMPC16, PVA-graft-PAPTAC, PVA-graft-PAPTAC-Oct, PEG46-b-PMAPTAC52 + poly(AMPS/AmU)) on EVs sizes (description of samples is presented in Table <NUM>) was studied. The obtained results indicate that the freezing process influences the concentration and size distribution of particles in EV samples secreted by human and animal cells (<FIG>). At the same time, the cryoprotective effect of the polymers used on EVs is observed, and it varies depending on the type of parental cells secreting EVs. Thus, in the context of optimal cryopreservation, the key factor is the selection of the appropriate type of polymer to the type of frozen EVs, that are obtained from conditioned media from the culture of various types of cells or from other physiological fluids.

The influence of selected polymers on the integrity and phenotypic characteristics of particles in cryopreserved EV preparations was investigated using an Apogee A-<NUM> Micro flow cytometer (Apogee Flow Systems). The analysis was carried out on the same types of samples as in the case of measurements by the NTA analysis method (Table <NUM>).

Individual samples were prepared according to an optimized protocol, by subjecting them to immunofluorescence staining with antibodies directed against the selected surface antigens. For this purpose, CD81 tetraspanin was chosen, which is a marker for EVs, as well as an antigen highly expressed on cells from which EVs are obtained (variable depending on the cell type, e.g. CD90 in the case of mesenchymal cells). Additionally, in order to investigate the influence of polymers on the integrity of EVs structure, samples were stained with RNA Select dye (Thermo Fisher Scientific), which after penetration into undamaged EVs binds to RNA molecules, emitting green fluorescence. In order to correctly analyze and verify the specificity of the obtained results, a number of controls were used in the cytometric analysis, including unstained and isotopically-labeled controls, analogically to the previous analyses.

The analysis of the obtained results shows that the cryopreservation of the samples results in a higher percentage of RNA Select-positive objects, as compared to the unfrozen samples. This may indicate that the freeze-thaw procedure disintegrates non-vesicular objects (including debris) that are not stained with RNA Select. Thus, degradation of these objects increases the percentage of RNA Select-positive vesicles in the sample.

Importantly, the addition of polymer to cryopreserved EV samples significantly improves the stability of EVs. This is particularly evidenced by the fact that after sample thawing the percentage of particles positive for the RNA Select dye, as an indicator of the structural integrity of EVs, is higher in the samples with the polymer, compared to the control samples (<FIG>). Similarly, the increase in the percentage of CD81+ and CD90+ objects after thawing is also much higher in the polymer samples compared to the control samples (<FIG>). Thus, the polymer stabilizes EVs while promoting the disintegration of contaminating objects (e.g. protein aggregates).

Similarly, a cryoprotective effect was obtained for a number of tested polymers in the case of freezing both human (<FIG>, <FIG> and <FIG>) and animal (<FIG> and <FIG>) EVs.

Cryopreservation is one of the key steps in protecting EV formulations for their subsequent use, both in basic research and in biomedical applications. Many experimental systems have demonstrated the effect of EVs secreted by stem cells on selected in vitro functions of target cells. Therefore, the influence of EV cryopreservation with the use of selected polymers, on the functional effectiveness of these EVs on target cells was investigated, using human osteoblasts (HOB) as exemplary target cells. For this purpose, the polymer coated EVs and the control EVs (without polymers) were added to the HOB cell culture at an optimized dose. Both EVs from umbilical cord mesenchymal cells (UC-MSCs) and those isolated from adipose tissue (AT-MSCs) were tested. Both freshly isolated as well as cryopreserved EVs were tested. The influence of the vesicles on the proliferation, viability and metabolic activity of HOB cells was then analyzed at appropriate time points.

Measurement of proliferation was performed <NUM> and <NUM> hours after addition of EVs to the cells, using a commercial Cell Counting Kit <NUM> (Sigma Aldrich).

The obtained results show that the addition of polymers does not reduce the functional efficiency of EVs in the context of their influence on the proliferation of target cells (<FIG>). Additionally, in the case of EVs isolated from AT-MSCs cells, cryopreservation using selected polymers increased the positive effect of these vesicles on the proliferation of HOB cells.

In the next step, the influence of the addition of EVs cryopreserved with polymers on the viability of target cells was also investigated using the example of HOB cells. For this purpose, their viability was measured at selected time points after the addition of EVs to the cells (<NUM>, <NUM>, <NUM> hours), using the Caspase-Glo <NUM>/<NUM> kit (Promega).

The obtained results indicate that coating of EVs with polymers does not adversely affect the viability of target cells, and may even slightly increase the effectiveness of those EVs (<FIG>).

Similar results were obtained by measuring the metabolic activity of the target cells, measured using the ATPLite kit (Perkin Elmer) <NUM> hours after the addition of EVs to the medium. Similarly, the presence of the polymers improved the functional effect of EVs on the metabolic activity of HOB cells (<FIG>).

In the next stage of the research, the influence of EVs cryopreservation with selected polymers on their cytoprotective properties was also analyzed. For this purpose, target cells (HOB) were treated with staurosporine as an apoptosis inducer for <NUM> hours at a dose of <NUM>. Then, control EVs or those containing the polymers were added to the cells, both immediately after their isolation and after freezing. After another <NUM> hours of incubation, cell viability was measured using the Caspase-Glo <NUM>/<NUM> kit.

The obtained results indicate that coating of EVs with selected polymers improves the cytoprotective effect of EVs (<FIG>).

The influence of selected polymers (PEG46-b-PMAPTAC52 + PAMPS18 and PMPC16) on the size distribution of EVs subjected to long-term cryopreservation was investigated. Both unfrozen and frozen samples were analyzed, and then thawed at one-month intervals (the description of the samples is presented in Table <NUM>).

The obtained results indicate that the long-term freezing process did not cause significant changes in particle size, except for the samples containing PEG46-b-PMAPTAC52 + PAMPS18, where the mean particle size was larger than the control at each of the tested time points (<FIG>).

The influence of selected polymers on the integrity and phenotypic characteristics of particles in long-term cryopreserved EV formulations was investigated using an Apogee A-<NUM> Micro flow cytometer (Apogee Flow Systems). The analysis was carried out on the same types of samples as in the case of measurements using the NTA analysis method (Table <NUM>).

Samples were prepared according to an optimized protocol, by subjecting them to immunofluorescence staining with antibodies directed against selected surface antigens: CD81 and CD90. Additionally, in order to investigate the influence of polymers on EVs structural integrity, samples were stained with RNA Select dye.

The analysis of the obtained results shows that the freezing of the samples resulted in a higher percentage of RNA Select-positive objects when compared to the unfrozen samples (<FIG>). As with previous experiments, this result may indicate that the freeze-thaw procedure disintegrates non-vesicular objects (including debris), thereby increasing the percentage of RNA Select-positive objects in the sample.

Importantly, the addition of the tested polymers to EVs samples subjected to long-term cryopreservation significantly improves the stability of EVs, as evidenced by the higher percentage of RNA Select+ objects in the polymer samples, in particular PEG46-b-PMAPTAC52 + PAMPS18, compared to the control samples (<FIG>).

Likewise, the addition of polymers does not impair the availability of surface markers on EVs. Moreover, coating of EVs with the PEG46-b-PMAPTAC52 + PAMPS18 polymers causes an increase in the percentage of CD81-positive and CD90-positive targets after thawing of samples subjected to long-term cryopreservation, which also proves the EV stabilizing properties of the tested polymers (<FIG>).

The influence of selected polymers on the functional properties of EVs subjected to long-term freezing was investigated. For this purpose, the functional effectiveness of these EVs on target cells was tested, using human osteoblasts (HOB) as exemplary target cells. Changes in the metabolic activity of HOB cells after their incubation with EV formulations were analyzed.

UC-MSCs-derived EVs coated with polymers and control EVs (without polymers) were added to the HOB cell culture at an optimized dose. Both unfrozen EVs and vesicles subjected to long-term freezing were tested (the description of the analyzed samples is given in Table <NUM>). Then, <NUM> hours after the addition of EVs, the metabolic activity of HOB cells was analyzed using the ATPLite kit (Perkin Elmer).

Claim 1:
A use of polyelectrolytes as cryoprotectants preserving extracellular vesicles, wherein said polyelectrolyte is the one obtained by modification of a polymer of natural origin and/or a synthetic polyelectrolyte, said polyelectrolyte of natural origin is dextran modified by substitution of the hydroxyl groups with glycidyltrimethylammonium chloride and said synthetic polyelectrolyte is selected from the group comprising a homopolymer and/or a block copolymer and/or graft copolymer, wherein:
- a cationic homopolymer is selected from the group comprising polymers represented by the general formula PMAPTACy:
<CHM>
where y is a degree of polymerization ranging from <NUM> to <NUM>,
- an anionic homopolymer is selected from the group comprising polymers represented by the general formula PAMPSx:
<CHM>
where x is a degree of polymerization ranging from <NUM> to <NUM>,
- a zwitterionic homopolymer is selected from the group comprising polymers represented by the general formula PMPCz:
<CHM>
where z is a degree of polymerization ranging from <NUM> to <NUM>,
- a block copolymer is selected from the group comprising the copolymers represented by the general formulas PEGx-b-PMAPTACy:
<CHM>
- where x and y are degrees of polymerization in the range from <NUM> to <NUM> and from <NUM> to <NUM>, respectively; and "b" denotes "block"
and/or PAMPSm-b-PAaUn
<CHM>
where m and n are degrees of polymerization in the range from <NUM> to <NUM> and from <NUM> to <NUM>, respectively,
- a graft copolymer is selected from PVA-graft-PAPTAC
<CHM>
<CHM>
or -H
- and/or PVA-graft-PAPTAC-Oct
<CHM>
<CHM>
or -H