Scaffolds and methods for selectively removing preservation damage from biological materials

Abstract

The present invention relates to scaffolds, combinations and compositions thereof that selectively remove storage damage from biological materials, particularly preserved cells. The present invention also relates to a system that selectively removes storage damage from biological materials, such as preserved blood cells. Furthermore, the present invention relates to a method for selectively removing storage damage from biological materials, such as preserved blood cells. In the present invention, the nanofiber scaffold that selectively removes storage damage from preserved cells comprises a backbone polymer functionalized with a group selected from (i) anionic groups, (ii) cationic groups, (iii) a combination of anionic and cationic groups, and (iv) zwitterionic groups.

Description

[Technical field]

[0001] The present invention relates to scaffolds, combinations and compositions thereof for selectively removing storage damage from biological materials, particularly preserved cells. The present invention also relates to a system for selectively removing storage damage from biological materials, such as preserved blood cells. Furthermore, the present invention relates to a method for selectively removing storage damage from biological materials, such as preserved blood cells. [Background technology]

[0002] Blood transfusion is one of the most common procedures in intensive care units (ICUs), with approximately 50-70% of hospitalized patients receiving a blood unit transfusion at some point (Non-Patent Document 1). Typical indications for blood transfusion include sickle cell crisis, anemia, and massive blood loss.

[0003] Crisis of stored cells, especially stored red blood cell (RBC) units, has always been a significant problem. Despite blood shortage, for example, in India, one million blood units are discarded every year due to deterioration in blood quality in 42 days (D). One of the main reasons for discarding large amounts of collected blood is the deterioration in blood quality during the storage period of 35-42 days. Current US FDA regulations allow RBC units to be stored for up to 42 days. However, in cases of diseases like sickle cell disease, the average storage period of transfused units in one processing is about two weeks. This two-week period is also permitted for transfusion after cardiac surgery.

[0004] The deterioration of blood quality is attributed to storage damage due to the formation of damage-associated molecular patterns (DAMPs) in blood bags during storage. Residual white and red blood cells undergo biochemical, morphological, and structural changes within RBCs during the storage aging process, resulting in the formation of their storage damages. These biochemical changes cause 1) hemolysis (with a concomitant increase in extracellular free iron and heme), 2) accumulation of shed bioactive proteins and lipids, 3) neutrophil extracellular traps (NETs) corresponding to extracellular DNA generated by neutrophils within the RBC unit, 4) cell-free mitochondrial DNA (mtDNA), and 5) RBC-derived microparticles or microvesicles (MPs / MVs). The most studied DAMPs in this context are (i) extracellular free iron and free hemoglobin (Hb) generated by the lysis of RBS, (ii) bioactive lipids such as polyunsaturated fatty acids (PUFAs), (iii) extracellular DNA, and (iv) nucleosomes generated by neutrophils. DAMPs often lead to transfusion-related complications, such as altered oxygen affinity, infections, allergic reactions, transfusion-associated acute lung injury (TRALI), and in severe cases, sepsis. These are potential causes of post-transfusion sequelae in high-risk populations. Previous investigations in cardiac research have reported increased severe morbidity and mortality when RBCs stored for more than 2 weeks were used for transfusion, and these increased complications are attributed to the accumulation of DAMPs in blood stored over a period of time.

[0005] Based on the accumulation of DAMPs, which depends on the storage period, stored human RBC units are classified into young or fresh RBCs (RBCs stored for less than 14–21 days) and old RBCs (stored for 21–42 days). Transfusion of old stored whole blood causes pooling of storage damage, which is a major mediator in the pathophysiology of fatal diseases such as transfusion-associated acute lung injury (TRALI), deep vein thrombosis, and organ dysfunction. It has long been established that leukocyte-associated DAMPs in donor blood are potential immune triggers in recipients and are implicated in transfusion-related complications. To reduce the accumulation of leukocyte-associated DAMPs, RBCs are treated with leukocyte reduction filters before storage. Leukocyte reduction reduces the formation of DAMPs such as NETs. However, this technique does not state that it can eliminate all types of DAMPs, especially erythrocyte-derived DAMPs.

[0006] Therefore, instead of the leukoreduction process, previous studies have focused on improving storage conditions through alternative cryopreservation protocols (Non-Patent Documents 2, 3), anaerobic storage (Non-Patent Documents 4, 5), and the use of additive / rejuvenation solutions (Non-Patent Documents 6-8). In addition, washing stored RBCs with saline (0.9% NaCl) to remove accumulated bioactive factors is another established strategy approved for use in transfusion medicine (Non-Patent Documents 9, 10). Previous studies suggest that adding preservatives, storing RBCs in alkaline hypotonic solutions containing antioxidants, or using anaerobic storage conditions can extend the storage period up to 10-15 days (Non-Patent Documents 6-8). Furthermore, the leukoreduction process is expensive.

[0007] However, these techniques cannot prevent RBCs from generating DAMPs during storage or improve overall quality. Yet, these efforts are not solely focused on removing RBC and WBC-related damage and improving blood quality or extending shelf life. There appears to be no practical approach in the field that removes all classes of storage damage and is more economical.

[0008] Prior art such as Patent Documents 1 to 10 focus on anaerobic storage of blood and deal with removing oxygen from blood to enhance storage.

[0009] US Patent No. 5,399,633 relates to a novel additive solution useful for the storage of human RBCs under refrigerated conditions.

[0010] Another finding is that the application of polycationic nanofibers aims to adsorb anionic compounds such as heparin or nucleic acids. However, various charged lesions exist in the preserved RBCs. Free histones, heme, Fe 2+ These range from RBC-related damage to DNA and bioactive lipids. Each of these components can significantly degrade the quality of stored blood / cells. These efforts are not focused on removing the RBC-related damage and improving blood quality or extending shelf life.

[0011] "Polycation microfibers and methods for using same" (Patent Document 12) discloses polycation microfibers capable of isolating or removing specific molecules, complexes or supramolecular assemblies from liquids including body fluids and biological samples.

[0012] US Pat. No. 5,399,633 relates to a method for producing polycationic nanofibers by grafting cationic polymers onto electrospun neutral nanofibers, and the polycationic nanofibers produced by this method.

[0013] US Pat. No. 5,399,633 provides a red blood cell preservation system and methodology in which red blood cells are treated, for example, in oxygen and carbon dioxide depleted conditions and stored in an anaerobic environment to optimize transfusion preparation.

[0014] As no practical approach to remove all types of storage lesions has been reported in the field, a technology is needed to solve this unmet clinical need and address transfusion-related complexities, which relate to the electrostatic interactions of charged storage lesions with cationic and / or anionic or zwitterionic scaffolds and the removal of DAMPs using such scaffolds. [Prior art documents] [Patent documents]

[0015] [Patent Document 1] U.S. Patent No. 9,877,476 [Patent Document 2] China Patent Publication No. 107847395 [Patent Document 3] U.S. Pat. No. 5,624,794 [Patent Document 4] International Publication No. 1996 / 029864 [Patent Document 5] Supplementary Search Report for European Patent Application Publication No. 2608816 [Patent Document 6] Translation of Spanish European Patent No. 2793484 [Patent Document 7] U.S. Patent No. 9,067,004 [Patent Document 8] U.S. Patent No. 9,199,016 [Patent Document 9] Supplementary Search Report for European Patent Application Publication No. 3268015 [Patent Document 10] Mexican Patent Application Publication No. 2018014530 [Patent Document 11] International Publication No. 2018 / 119422 https: / / patentimages.storage.googleapis.com / 56 / 64 / 3f / 40a8cb518019e5 / WO2018119422A1.pdf [Patent Document 12] U.S. Patent No. 6,150,085 [Patent Document 13] U.S. Patent Application Publication No. 2017 / 0037544 [Patent Document 14] U.S. Patent No. 10603417

Non-licensed documents

[0016]

Non-patent document 1

Non-patent document 2

Non-patent document 3

Non-patent document 4

Non-patent document 5

[0017] The present invention provides a technique that solves an unmet clinical need for techniques to remove any type of storage damage from stored cells. [Means for solving the problem]

[0018] In one embodiment, the present invention provides a nanofibrous scaffold that selectively removes preserved damage from preserved cells comprising a backbone polymer functionalized with groups selected from anionic groups, cationic groups, a combination of anionic and cationic groups, and zwitterionic groups.

[0019] In another embodiment, the present invention provides a system for selectively removing preserved lesions from preserved cells, the system comprising a nanofibrous scaffold having a backbone polymer functionalized with anionic groups and a scaffold having a backbone polymer functionalized with cationic groups, where the preserved lesions are selectively removed when preserved cells are placed in contact with the scaffold.

[0020] In yet another embodiment, the present invention provides a system for selectively removing preserved lesions from preserved cells, the system comprising a nanofibrous scaffold having a backbone polymer functionalized with zwitterionic groups, wherein the preserved lesions are selectively removed when preserved cells are placed in contact with the scaffold.

[0021] In one embodiment of the present invention, the functionalized polymer in the scaffolds and systems is in electrospun form.

[0022] In one embodiment of the invention, the backbone polymer is selected from the group consisting of poly(acrylic acid), poly(4-vinylpyridine), poly(4-vinylphenol), poly(allylamine), poly(vinyl alcohol), poly(methyl vinyl ether-alt-maleic anhydride) (PMVEMA), poly(vinyl chloride), poly(glycerin), poly(2-hydroxyethyl methacrylamide), poly(2-((allyloxy)methyl)oxirane), poly(vinylpyrrolidone), poly(4-vinylbenzyl chloride-alt-maleic anhydride) and poly(vinylimidazole). The backbone polymer is preferably poly(methyl vinyl ether-alt-maleic anhydride (PMVEMA).

[0023] In one embodiment of the invention, the backbone polymer is functionalized with an anionic group selected from 1,3-propane sultone, taurine, 1,3-dioxolan-2-one, 1,2-oxathiane 2,2-dioxide, phosphorochloric acid, (3-aminopropyl)phosphonic acid, valeric acid, 2-(methylamino)ethyl hydrogen phosphate, pentyl hydrogen phosphate, and 4-(butylamino)butyl sulfate, preferably functionalized with taurine. Preferably, the polymer is functionalized with taurine present in an amount of 8-50%.

[0024] In another embodiment of the invention, the polymer is functionalized with a cationic group selected from ethidium bromide derivatives, cyanine dye YO-1, proflavine derivatives, acridine orange, ethylene blue derivatives, ellipticine derivatives, phenanthridine derivatives, quinolinium derivatives, thalidomide derivatives, naphthalimide derivatives and quinoline derivatives, preferably with acridine. Preferably, the polymer is functionalized with acridine present in an amount of 3-35%.

[0025] In one embodiment of the present invention, the cationic polymer has intercalation and positive charge properties.

[0026] In another embodiment of the invention, the polymers are functionalized with zwitterionic groups to produce zwitterionic polymers. Zwitterionic groups have both cationic and anionic groups in the same molecule. These zwitterionic polymers are prepared by functionalizing the backbone polymer with zwitterionic groups or a combination of cationic and anionic groups, as described above.

[0027] In one embodiment of the invention, the nanofiber scaffold is in the form of a sheet, bead, bag, insertion device, tube, etc., and preferably in the form of a nanofiber sheet.

[0028] In one embodiment of the invention, the stored cells are selected from red blood cells (RBCs), white blood cells (WBCs), platelets and stem cells, preferably red blood cells (RBCs).

[0029] In yet another embodiment, the present invention provides a method of treating preserved cells, said preserved cells being placed in contact with the scaffold or system described above.

[0030] In yet another embodiment, the present invention provides a method for removing damage associated molecular patterns (DAMPs) from preserved cells, the preserved cells being treated by the scaffold or within the system.

[0031] In another embodiment, the present invention provides a method for removing damage-associated molecular patterns (DAMPs) from preserved cells, the preserved cells being treated by the scaffold or within the system.

[0032] In another embodiment, the invention provides a method for improving the quality and shelf life of preserved cells by removing DAMPs from the preserved cells, the preserved cells being processed by the scaffold or within the system.

[0033] In another embodiment, the present invention provides a method for improving cell membrane integrity of preserved cells by removing DAMPs from the preserved cells, the preserved cells being treated by the scaffold or within the system.

[0034] In one embodiment of the invention, the DAMPs are extracellular DNA, histones, nucleosomes, cell-free hemoglobin, iron, proteins and polyunsaturated fatty acids.

[0035] In another embodiment, the invention provides a use of said scaffold in processing of preserved cells, comprising passing preserved cells through said scaffold.

[0036] In another embodiment, the invention provides a kit comprising said scaffold for processing of preserved cells comprising passing preserved cells through said scaffold.

[0037] In one embodiment of the present invention, the preserved cells are red blood cells (RBCs), white blood cells (WBCs), platelets and stem cells, preferably red blood cells (RBCs).In another embodiment, the present invention provides a method for preparing a nanofibrous scaffold according to any one of claims 1, 4 to 13, comprising the steps of preparing a solution of a polymer functionalized with a group selected from anionic, cationic or zwitterionic groups, and electrospinning the solution to obtain a nanofibrous scaffold.

[0038] In one embodiment of the present invention, the polymer is rendered hydrophobic before being functionalized with groups selected from anionic, cationic or zwitterionic groups. [Brief description of the drawings]

[0039] [Figure 1]Synthesis and fabrication of hemocompatible and hydrophobic nanofiber scaffolds with poly-Tau and poly-Acr. Figure 1a shows a schematic showing the synthesis of anionic polymer (pTau) and cationic polymer (pAcr) from PMVEMA polymer containing 50% hydrophobic domain with taurine and acridine bromide binding of 25% and 11%, respectively. Figure 1b shows a field emission scanning electron microscope (FESEM) image of the scaffolds, which shows that the fibers are continuous and bead-free, and contact angle measurements confirm their hydrophobicity. Figure 1c shows that surface charge measurements confirm the cationic and anionic nature of pTau and pAcr. Figure 1d shows a FESEM image of the fibers after treatment, showing no adhesion to the scaffold in the presence of RBCs or their supernatant. Figure 1e confirms that hemolysis assays confirm the biocompatibility of pTau and pAcr with RBCs, with a hemolysis rate (%) of less than 3% (n=3). [Figure 1A] We show that hemocompatible charged nanofiber sheets remove storage damage and improve blood quality. Figure 1Aa shows that stored RBCs generate storage damage or damage-associated molecular patterns (DAMPs), such as DNA, nucleosomes, heme (Hb), iron, and polyunsaturated fatty acids (PUFAs). Transfusion of DAMPs containing RBCs can cause transfusion-related complications, including systemic inflammation and organ damage. The presence of DAMPs gradually reduces the quality of stored RBCs and limits their shelf life. All DAMPs consist of entities with positive or negative charges. Therefore, we prepared polyacridine and polytaurine, which are hemocompatible polymers composed of complementary cationic and anionic charges, respectively. Using these polymers, we prepared charged electrospun nanofiber sheets (Tau-NFS and Acr-NFS) to remove charged DAMPs from stored RBCs, thereby significantly improving the quality and shelf life of stored RBCs. [Diagram 2]We show that charged nanofiber scaffolds remove DAMPs from both old stored RBCs and their supernatants. Figure 2a is a schematic diagram of pRBCs collected from three volunteers (male and female), packaged, and stored for 42 days, and treated with pTau and pAcr scaffolds. Figures 2b, 2c, and 2d show that the combination of pTau and pAcr scaffolds removed significant amounts of extracellular DNA, nucleosomes, and heme from 42D (D stands for "day") RBC supernatants and packaged RBCs. Each scaffold with an area of ​​12 cm2 (total = 24 cm2) was used to wash 1 ml of supernatant and pRBCs (4°C), which showed charge-based interactions between negatively charged DAMPs (extracellular DNA and bioactive lipids) and pAcr, and positively charged DAMPs (free nucleosomes and Hb) and pTau. Figure 2e shows that removal of pRBC-generated bioactive lipids, arachidonic acid (AA), 5-hydroxyeicosatetraenoic acid (5-HETE), 12-hydroxyeicosatetraenoic acid (12-HETE), and 15-hydroxyeicosatetraenoic acid (15-HETE), demonstrated the broad-spectrum cleaning activity of the charged scaffold. Data are presented as mean ± standard deviation unless otherwise stated. [Diagram 3] We show that intermittent removal of DAMPs on days 21 and 28 improved the quality of RBCs on day 42. Figure 3a is a schematic diagram of intermediate washing of pRBC units and evaluation of the generation of DAMPs by the end of 42D. Figures 3b, 3c, and 3d show that intermediate washing on day 28 demonstrated improved RBC health by generating less extracellular DNA, heme, and nucleosomes by the end of 42D when compared to DAMP washing on day 21. 24 cm2 scaffolds (pTau and pAcr) were used to wash 1 ml RBC units at 4°C (n=3, different volunteers including males and females). Figure 3e shows that intermediate washing on days 21 and 28 significantly confirmed the reduced formation of bioactive lipids when compared to the untreated group. [Figure 4]Intermittent scaffold treatment maintained the structural integrity and osmotic fragility of RBCs. Figure 4a shows a schematic of scaffold treatment of pRBCs on days 21 and 38 and the assessment of RBC health and fragility by FESEM and osmotic fragility analysis. Figure 4b shows SEM images of samples confirming the presence of discoid red blood cells (healthy), whereas 42D RBCs are dominated by spherocalctic and acanthocytes (unhealthy). Figure 4c shows that intermittent treatment on day 28 confirmed an increase in the number of discoid red blood cells compared to day 21 treatment. Figure 4d shows that treatment on day 21 and treatment on day 28 increased the number of discoid red blood cells in RBCs by 2.5 and 3.5 times compared to the untreated group. Figure 4e shows that the dynamics of stored pRBCs during storage (0-42D) revealed an increase in MCF values ​​and a shift of the curve to the right. FIG. 4g shows that osmotic fragility tests of treated 42D pRBCs on days 21 and 28 of storage demonstrated higher resistance to lysis when compared to untreated RBCs. [Figure 5(1)] Intermittent removal of DAMPs from stored mouse blood delays aging and extends shelf life for stored RBCs. Figure 5a shows a schematic of packaging and washing mouse RBCs with pTau and pAcr scaffolds. Figure 5b shows that removal on days 5 or 10 (single) or both (twice) reduces the generation of free heme and free nucleosomes at 14D. Figures 5c and 5d show that 17D stored RBCs treated twice with scaffolds show a significant reduction in the exposure of phosphatidylserine in the outer leaflet of the cell membrane compared to the untreated group at 17D. Figure 5d shows that in the untreated group, biotin-labeled RBCs remained constant until 14D and then began to decrease, whereas in the treated group, the RBC population remained intact and healthy until 17D, suggesting good RBC quality and improved shelf life. [Figure 5 (2)] We show that intermittent removal of DAMPs from stored mouse blood delays aging and extends the shelf life of stored RBCs. [Figure 6] We show that twice treated 17D old mouse RBCs reduced dose-responsive proinflammatory cytokine responses, decreased RBC clearance, tissue iron delivery and circulating NTBI levels when compared to transfusion of untreated stored RBCs. All transfusion recipients were male / female C57BL / 6 mice (8-12 weeks old) housed in the inStem / NCBS animal house. Results are presented as mean (SD) unless otherwise specified. Figure 6a shows representative images of spleens obtained from mice 2 hours after transfusion with treated and untreated RBCs. Aliquots (200 uL) of 14D and 17D stored treated and untreated RBCs (n=3) were transfused. Total iron was measured in spleens obtained 2 hours after transfusion. Iron levels shown are compared to levels measured in controls transfused with fresh RBCs (n=3). Results are combined from three separate experiments. Figure 6b shows plasma cytokine levels were measured (as labeled) after sacrifice of C57BL / 6 mice (n=3) transfused with 14D and 17D stored unprocessed or processed RBCs 2 hours after transfusion. Figure 6c shows representative images of transfusion and recovery of old stored RBCs. 14D and 17D stored processed and unprocessed biotin-labeled RBCs (stored for 24 hours, n=3) were transfused (200 uL) and the viability of transfused RBCs was calculated by streptavidin-APC labeled flow cytometry tracking at 1 hour and 24 hours after transfusion. [Figure 7] We present NMR findings demonstrating that on PMVEMA, pTau has approximately 50% and 25% substitution of tetradecylamine and taurine, whereas pAcr has approximately 50% substitution of tetradecylamine and 11% substitution of Acr-hexyl bromide. [Figure 8A]We show that the generation of various classes of DAMPs increased significantly during storage of non-leukoreduced (NLR) RBC units. NLR units recorded a significant increase in extracellular DNA from 21 to 28 days. An overall spike of approximately 150-fold was observed in NLR blood from 0 to 42 days, which perfectly complemented the levels of other DAMPs. Free Hb increased up to 40-fold from 0 to 42 days (from approximately 0.03 g / dl to approximately 0.8 g / dl). All four nonpolar lipids were quantified by LCMS and showed a significant increase in levels from 28 to 42 days of RBC storage. af is the storage-induced generation of DAMPs as a function of time. RBCs stored at 4°C gradually produce DNA (a), free hemoglobin (Hb, b), and polyunsaturated fatty acids (PUFAs), such as AA (c), 5-HETE (d), 12-HETE (e), and 15-HETE (f). [Figure 8B] We provide comprehensive insight into the generation of DAMPs, such as free Hb and arachidonic acid, in leukoreduced (LR) RBC units over time. Leukoreduced RBCs (LR units) showed a stepwise increase in extracellular DNA production from 0 to 42 days, but it was 30-fold less compared to NLR. The increase in production at 42 days was approximately 2-fold higher than at 28 days, confirming the formation of RBC-associated lesions. A significant trend of up to 10-fold increase in free hemoglobin (Hb) production was observed, providing the idea that leukofilters cannot control the accumulation of RBC-associated DAMPs. [Figure 9]Optimization of scaffolds with respect to area and time shows that a combination of pTau and pAcr scaffolds with an area of ​​24 cm2 is sufficient to remove the maximum amount of DAMPs in 5 min. (A) Extracellular DNA and free Hb in the supernatant of 42D stored NLR blood were selected as markers to optimize the incubation time (5 min to 30 min) with individual scaffolds (pTau and pAcr). This parameter was considered to remove the maximum amount of DAMPs (without breaking the cold chain) at 4 °C. The combined use of cationic (pAcr) and anionic (pTau) scaffolds in the later removal experiments was based on the presence of negatively and positively charged lesions in pRBCs. The data suggested that individual 24 cm2 scaffolds were able to reduce extracellular DNA and Hb from 1 ml of supernatant within 5 min. (B) Area optimization showed that stepwise increases in surface area (>8 cm2) with 5 min incubation time had no significant effect on the removal of extracellular DNA by the pTau and pAcr scaffold groups. [Figure 10] It shows that no leaching phenomenon was observed for the (pAcr) scaffold during blood component processing. [Figure 11] The removal ability of the scaffolds shows remarkable results for the removal of RBCs associated with DAMPs. The effectiveness of the scaffolds, pTau scaffolds with an area of ​​12 cm2 each and pAcr scaffolds (total surface area of ​​24 cm2), was combined to treat both LR 42D RBCs and old supernatants. The washing of DAMPs by the scaffolds was performed at 40°C for 5 min. In the LR units, the reduction of free Hb was very evident in the post-treatment supernatant group, decreasing by up to 50%. The remarkable reduction in AA values ​​after treatment with the scaffolds provides a method to purify LR blood containing soluble bioactive lipids generated from RBCs. [Figure 12]We show that intermittent removal of DAMPs by treatment on day 28 during storage improves the health of LR RBCs on day 42. Extracellular DNA production in LR blood units was low as expected by the end of day 42. Intermittent treatment on days 21 and 28 did not significantly reduce its concentration. Treatment on day 21 did not affect the rate of lysis of RBCs in LR units, whereas treatment on day 28 limited the production of free Hb by 50% compared to the untreated group. AA production was significant, but intermittent treatment on days 21 / 28 significantly reduced its production by 10-fold. [Figure 13] The increase in mean cell fragility (MCF) values ​​indicates that LR promotes lysis of stored RBCs. (A) LR units show an increase in MCF values, which indicate the degree of RBC health damage, from approximately 0.35 to 0.5. (B) Intermittent treatment with LR units on days 21 and 28 reduced MCF values ​​and shifted the hemolysis curve to the left, or healthy, side compared to the untreated group. The decrease in MCF values ​​suggests that RBCs are healthier and more resistant to lysis after intermediate treatment. [Figure 14] We show that RBCs stored in mice exhibit similar DAMP formation as human blood. The data suggested that a large amount of free Hb was generated between 10 and 14 days of storage. Compared to day 10, the level of free Hb doubled on day 14. A two-fold increase in the generation of free nucleosomes was also observed. To identify the potential period of intermittent washing, we estimated the generation of DAMPs in mouse RBC units. [Figure 15]Figure 1 shows that twice treated 17D mouse RBCs showed a dose-responsive reduction in inflammatory cytokines. Mice (8-12 weeks old (male)) were infused with 14D and 17D stored RBCs with or without pTau and pAcr treatment, respectively. Mice were sacrificed 2 hours after transfusion and plasma cytokine levels were measured. (A) Representation of blots of cytokine arrays from all groups. (B) Cytokine levels such as IL-16, TIMP-1, CXCL-10, IL-1F3, and G-CSF were higher in the 17D untreated group but not detected in other treated groups where systemic inflammation was reduced after DAMP removal. [Figure 16] The treated group showed recovery of stored labeled RBCs within 17 days. To evaluate the extended shelf-life of the treated RBCs, male and female mice (8-12 weeks) were transfused with biotin. A few μl of stored blood of different shelf-life were collected from treated and untreated groups, and the percentage of labeled RBCs was quantified using flow cytometry. Compared to the untreated group, the treated RBCs remained in the circulation even after the shelf-life (14 days) had passed. They were as healthy as 0D / 7D fresh RBC units. [Figure 17] Freshly stored biotinylated RBCs were transfused into mice and blood was collected at 1, 24, 48, and 72 hours post-transfusion, labeled with streptavidin APCeFluor™, and quantified by flow cytometry. Flow cytometry profiles showed that over 75% of the labeled RBCs were in the circulation, suggesting good quality of the RBCs. [Figure 18] Complete blood counts of stored RBCs before and after treatment with Tau-AcrNFS are shown. a-e: Treatment of stored blood with Tau-AcrNFS does not cause cell loss. For quantification of RBCs (a), WBCs (b), neutrophils (c), lymphocytes (d), and platelets (e) before and after treatment with Tau-AcrNFS, data are mean ± standard deviation (n=3, from independent experiments). For a-e, P values ​​were determined by two-tailed Student's t-test with Welch's correction using GraphPad PRISM 9. ns=not significant. [Figure 19] We show that cryopreservation of human RBCs induced the generation of DAMPs, and that charged Tau-AcrNFS efficiently removed DAMPs from stored RBCs. a–f: Storage-induced generation of DAMPs as a function of time. In RBCs stored at 4°C, DNA (a), free hemoglobin (Hb, b), and polyunsaturated fatty acids (PUFAs), such as AA (c), 5-HETE (d), 12-HETE (e), and 15-HETE (f), were gradually generated. g–m: Incubation of 42-day-stored RBCs with accumulated DAMPs with anionic and cationic NFS, Tau-AcrNFS, at 4°C for 5 min effectively removed and significantly reduced the concentration of DAMPs. The relative concentrations of accumulated DAMPs, such as DNA (g), nucleosomes (h), Hb (i), and PUFAs (j–m), were significantly reduced before and after treatment with Tau-AcrNFS. Data are means ± standard deviations (n ​​= 3 from independent experiments). For a-f, P values ​​were determined by repeated measures one-way ANOVA; for (g-m), exact P values ​​are shown by two-tailed Student's t-test with Welch's correction using GraphPad PRISM 9. NS = not significant. [Figure 20] Figure 1 shows the removal of DAMPs using charged nanofiber sheets. a, b: RBCs stored for 42 days were incubated with Tau-NFS or Acr-NFS with different surface areas (8, 16, 24 cm2). c: RBCs stored for 42 days were incubated with Tau-NFS or Acr-NFS for different incubation times (5, 15, 30 min). Data are mean ± standard deviation (n=3 from independent experiments). P values ​​were determined by one-way ANOVA and Tukey's post-hoc test using GraphPad PRISM 9, and exact P values ​​are shown. [Figure 21]The synthetic scheme of charged polymers, polyacridine and polytaurine, is shown. Polyacridine and polytaurine polymers were synthesized using a solvent phase synthesis method. The reaction conditions were as follows: (i) anhydrous toluene, 120° C., 12 hours; (ii) tetradecylamine, anhydrous tetrahydrofuran, 80° C., 3 hours; (iii) anhydrous tetrahydrofuran, 80° C., 12 hours; (iv) anhydrous dimethylformamide, 50° C., 16 hours; (v) anhydrous tetrahydrofuran, 50° C., 16 hours. [Figure 22] 1 shows the synthesis of zwitterionic polymers from PMVEMA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] The present invention relates to scaffolds that selectively remove storage damage from biological materials, particularly preserved cells, said scaffolds being anionic and / or cationic, or a combination thereof, or zwitterionic.

[0041] Additionally, the present invention relates to a method for selectively removing preservation damage from biological material, such as preserved cells.

[0042] The present invention discloses a technology that solves the unmet clinical need of removing all classes of storage damage from stored cells, including but not limited to RBCs, white blood cells (WBCs), platelets, stem cells, etc.

[0043] The present invention relates to scaffolds, combinations and compositions thereof that selectively remove storage damage consisting of DAMPs from biological materials, particularly stored red blood cells. Furthermore, the present invention relates to a method for selectively removing storage damage consisting of DAMPs from biological materials such as stored red blood cells.

[0044] The present invention relates to nanofibrous scaffolds that selectively remove preserved damage from preserved cells comprising backbone polymers functionalized with groups selected from anionic groups, cationic groups, a combination of anionic and cationic groups, or zwitterionic groups.

[0045] The present invention also relates to a system for selectively removing preserved lesions from preserved cells, the system including a nanofibrous scaffold having a backbone polymer functionalized with anionic groups and a scaffold having a backbone polymer functionalized with cationic groups, where the preserved lesions are selectively removed when preserved cells are placed in contact with the scaffold.

[0046] The present invention also relates to a system for selectively removing preserved lesions from preserved cells, the system comprising a nanofibrous scaffold having a backbone polymer functionalized with zwitterionic groups, wherein the preserved lesions are selectively removed when preserved cells are placed in contact with the scaffold.

[0047] In one embodiment, this technology improves the overall quality of stored RBC units to address transfusion-related complications and helps to extend the shelf life of RBCs to resolve blood crisis issues. The present invention discloses an ideal user-compliant technology, easy-to-use and non-obstructive biocompatible scaffolds, their combinations and compositions that can selectively remove the full spectrum of DAMPs, mainly cationic and anionic species. The scaffolds are nanofibrous scaffolds that selectively cleanse damage generated during storage to reduce the risk of transfusion-related complications for patients (Figure 1a).

[0048] The present invention discloses a strategy to eliminate the entire spectrum of DAMPs by developing a charged nanofibrous scaffold that selectively removes cell-free extracellular DNA, histones, DNA-histone complexes, cell-free hemoglobin, cell-free iron, proteins and bioactive lipids from old stored RBC units.

[0049] To remove neutrophil extracellular traps (NETs) and negatively charged bioactive lipids, nanofiber scaffolds have been developed from polymers made with cationic moieties that have intercalation and positive charge properties. Thus, such scaffolds can selectively bind negatively charged DNA and lipids with high affinity.

[0050] Furthermore, anionic nanofiber scaffolds have been developed from sulfonic acid-based polymers to capture positively charged histones within blood units, which mimic heparin sulfate glycoproteins (HSGPs) present on cell surfaces.

[0051] The present invention investigates the efficiency of selectively designed scaffolds that can remove cationic and anionic DAMPs. The main objective of this invention is to improve the quality and extend the shelf life of stored blood by removing these storage damages on specific days during the storage period (42 days). This specific removal reduces the overall accumulation of DAMPs at the end of storage on day 42.

[0052] The development of the scaffold was based on identifying a specific polymer to serve as the backbone, which can be easily functionalized and modified to exhibit hydrophobicity. Another requirement is that the polymer does not interact with the blood cells during the DAMP washing process. The backbone polymer was selected from the group of polymers shown in the table below. [Table 1(1)] [Table 1(2)]

[0053] In a preferred embodiment, high molecular weight polymethoxyvinylether-alt-maleic anhydride (PMVEMA) polymer was selected as the backbone polymer, and its hydrophobicity was enhanced by the binding of alkylamines with various numbers of hydrocarbon chains, such as tetradecylamine. Similarly, it was mixed with a solvent selected from tetrahydrofuran (THF) and stirred at 50-80°C until dissolved, then tetradecylamine was added in a ratio of 1:1-2:1, further stirred at 50-80°C for about 3-16 hours, and the solvent was removed to obtain a powdered hydrophobic polymer.

[0054] Additionally, the backbone polymer was functionalized with anionic and / or cationic moieties. The reaction conditions vary depending on the backbone polymer and the compounds used for anionic and cationic functionalization.

[0055] For anionic functionalization, the backbone polymer is mixed with an anionic compound selected from the group of compounds tabulated below. [Table 2]

[0056] For cationic functionalization, the backbone polymer is mixed with a cationic compound selected from the group of compounds tabulated below. [Table 3(1)] [Table 3(2)]

[0057] Additionally, zwitterionic polymers have been made by functionalizing the backbone polymer with zwitterionic groups, which have both cationic and anionic groups in the same molecule. These zwitterionic polymers are prepared by functionalizing the backbone polymer with zwitterionic groups or a combination of cationic and anionic groups, as previously described.

[0058] In a preferred embodiment, taurine and acridine hexyl bromide are used to impart anionic (pTau) and cationic (pAcr) moieties, respectively. The anionic polymer (pTau) is prepared by mixing taurine with a polar solvent, preferably selected from sodium bicarbonate, and then adding this solution to the backbone polymer and stirring at 50-80° C. for 12-16 hours. The polar solvent is selected from ethanol, methanol, tetrahydrofuran, dioxane, acetonitrile and dimethyl sulfoxide.

[0059] The cationic polymer (pAcr) is prepared by mixing acridine orange base in anhydrous toluene, adding 1,6-dibromohexane and refluxing at 120 °C overnight to prepare acridine hexyl bromide. N,N-dimethylethylenediamine (87 μl, 0.8 mmol) in THF (10 ml) is added to the solution of the backbone polymer and the mixture is stirred at 50-80 °C for 3-16 h. After its dissolution, a rotary evaporator is used to reduce the THF in the reaction mixture to a viscous solution. Dry dimethylformamide (DMF) is added to this viscous solution, followed by the addition of acridine (Acr) hexyl bromide. The resulting mixture is stirred at 50-80 °C for 12-16 h and the polymer is thoroughly triturated with deionized water to remove the unreacted Acr hexyl bromide and DMF.

[0060] These charged anionic and cationic polymers have been modified into nanofibers by electrospinning to solve the problems associated with the generation of DAMPs. The charged anionic and cationic nanofiber scaffolds are used individually or in combination. In another embodiment, the same backbone polymer is both positively and negatively charged.

[0061] The charged anionic and cationic nanofibrous scaffolds can be prepared in a variety of forms selected from the group consisting of sheets, beads, bags, insertion devices, tubes, etc. The size of the nanofibrous scaffold of a particular form varies depending on the blood volume.

[0062] In a preferred embodiment, the nanofiber scaffold is a sheet. Its size depends on the blood volume. Preferably, its size is between 8 and 30 cm. 2 is in the range.

[0063] 42-day-old (D) old pRBCs and their supernatants were treated with pTau and pAce scaffolds. The treatment helped to significantly reduce DAMPs from both pRBCs and supernatants within 5 min at 4°C. Intermittent treatment on days 21 and 28 of RBC storage improved the quality of human RBCs on day 42 (higher population of discoid red blood cells) compared to untreated, with treatment on day 28 proving to be better. In mice, stored RBC units were treated twice with scaffolds on days 5 and 10. Findings suggest that washing blood twice maintains RBC integrity and improves RBC functionality on day 14. Dual treatment extended the shelf life of mouse RBCs by 3 days (17 days). Recovery up to 72 hours without systemic inflammation was recorded for transfusion of 17-day old RBCs. The scaffolds were also tested for hemocompatibility and showed no interaction with blood cells and negligible leaching.

[0064] These (pTau and pAcr) scaffolds used in the present invention selectively remove charged extracellular DNA-histone complexes, hemoglobin (Hb) and bioactive lipids. It was reported that stored human blood, when treated with charged scaffolds, had a significant reduction in DAMPs at the end of 42 days. Scaffold treatment further slows down the aging process of stored pRBCs. Treatment with scaffolds on the 28th day of storage showed a smaller generation of DAMPs and maintained the membrane symmetry of RBCs on the 42nd day. Interestingly, for mouse RBCs treated twice with scaffolds (on the 5th and 10th days), not only did it improve the quality of stored blood but also extended the shelf life by 3 days. Recovery experiments in mice revealed that treated 17-day-old old RBCs had a longer residence time in the circulation. Thus, the blood washing technique of the present invention that removes DAMPs has great potential in the field of blood transfusion and can bring about a significant improvement to the currently accepted blood storage procedures.

[0065] Reference is made to the accompanying drawings which form a part hereof, in which are shown by way of illustration specific embodiments which may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the embodiments, but it is understood that other embodiments may be utilized without departing from the scope of the embodiments. While the invention will be described in conjunction with such embodiments, it is not limited to any embodiment. While only certain features of the invention have been illustrated and described herein, many modifications and changes will occur to those skilled in the art. EXAMPLES

[0066] Example 1: Synthesis and Fabrication of Hemocompatible and Hydrophobic Charged Polymer Nanofibers Materials: PMVEMA (polymethoxyvinylether-alt-maleic anhydride) with an average molecular weight of 330000 was used in all synthesis procedures. Tetrahydrofuran (THF), tetradecylamine, taurine, sodium bicarbonate, ethyl acetate, 3,6-bis(dimethylamino)acridine hemi(zinc chloride) salt, benzene, methanol, ammonium hydroxide, N,N-dimethylethylenediamine, 1,6-dibromohexane, sodium sulfate, toluene, dimethylformamide, Triton X-100, chamber slides (HI media), blood collection tubes (BD Vacutainer®), citric acid monohydrate, trisodium citrate dihydrate, sodium dihydrogen phosphate, dextrose, sodium chloride, anhydrous glucose, mannitol, adenine, APTMS (3-(aminopropyl)trimethoxysilane), glutaraldehyde, cacodylate buffer, osmium tetroxide, hexamethyldisilane, ethanol, double distilled water and deionized water were used. All chemicals were sourced from Sigma-Aldrich unless otherwise stated.

[0067] method Synthesis of anionic (pTau) and cationic (pAcr) scaffolds PMVEMA-C 14 Preparation of (50%): PMVEMA (500 mg, 3.2 mmol) was added to tetrahydrofuran (THF) (10 ml) in a pressure tube and stirred at 80 °C until dissolved. Then, tetradecylamine (345 mg, 1.6 mmol) prepared in THF (10 ml) was added. The reaction mixture was kept at 80 °C for 3 h. Finally, the solvent was removed using a rotary evaporator to obtain the polymer as a dry powder (800 mg, about 90% yield).

[0068] NMR data: 1H-NMR (CD3OD, 600MHz), δ: 3.0-3.51 (Br, 9H, -OCH3, -O-CH, PMVEMA and -NH-CH2 alkyl protons), 1.47 (Br, 2H, alkyl protons), 1.22 (Br, 22H, alkyl protons), 0.845 (t, 3H, alkyl protons). NMR data showed that about 50% of the PMVEMA polymer was replaced by tetradecylamine. FTIR (cm -1 ):3354, 2926, 1858, 1782, 1733, 1697.

[0069] PMVEMA-C 14 Preparation of (50%)-Tau(25%) (pTau): A solution of taurine (tau) (100 mg, 0.8 mmol) and sodium bicarbonate (84 mg, 1.0 mmol) was prepared in deionized water (1 ml). 14 (50%) (800 mg) in THF (20 ml) and stirred at 80 °C for 16 h. The reaction mixture was concentrated by rotary evaporation. It was then triturated successively with ethyl acetate and deionized water to remove residual tetradecylamine and taurine. The final polymer was obtained as a gel, which was lyophilized to a white powder (720 mg, approximately 75% yield).

[0070] NMR data: 1 H-NMR (CD3OD, 800MHz), δ: 3.9-4.1 (Br, 1H, taurine), 2.7-3.5 (Br, 5H, -OCH3, -O-CH, PMVEMA and -NH-CH2 alkyl protons), 1.95 (Br, 2H, PMVEMA protons), 1.4 (Br, 1H, alkyl protons), 1.1-1.2 (Br, 11.5H, alkyl protons), 0.8 (t, 1.5H, alkyl protons). According to the NMR data, PMVEMA has a molecular weight of approximately C 14 H 29 Approximately 50% of the -NH2 and approximately 25% of the taurine were replaced. FTIR (cm -1 ):3209, 3046, 2946, 1722, 1616, 1037.

[0071] Preparation of acridine hexyl bromide: 3,6-bis(dimethylamino)acridine hemi(zinc chloride) salt (10 g, 27.02 mmol) was dissolved in methanol (50 ml) and ammonium hydroxide (50 ml) was added to it. Acridine orange (AO) base was extracted from benzene and dried over sodium sulfate. AO base (5 g) was recovered from benzene using a rotary evaporator. AO base (2.65 g, 10 mmol) was dissolved in anhydrous toluene (100 ml) and 1,6-dibromohexane (7.7 ml, 50 mmol) was added to the solution and refluxed overnight. The bright red precipitate in the reaction mixture was filtered and dried (2.1 g, 41.2% yield).

[0072] NMR data: 1 H-NMR (CDCl3,600MHz)δ:8.7(s, 1H), 7.9(d, 2H), 7.08(m, 2H), 6.75(s, 2H), 5.0(t, 2) H), 3.4(t, 2H), 3.3(s, 12H), 2.048(m, 2H), 2.029(m, 2H), 1.96(m, 2H), 1.94(m, 2H).

[0073] PMVEMA-C 14 Preparation of (50%)-Acr(15%): A solution of N,N-dimethylethylenediamine (87 μl, 0.8 mmol) in THF (10 ml) was added to PMVEMA-C in a pressure tube. 14 A solution of 1,2-dichloro-1,3-diphenyl-2,4-tetrahydrofuran (50%) (800 mg) in THF (20 ml) was added dropwise to the reaction mixture. The mixture was stirred at 80° C. for 16 hours. After dissolution, the THF in the reaction mixture was reduced to a viscous solution using a rotary evaporator. Dry dimethylformamide (DMF) (10 ml) was added to the viscous solution, followed by the addition of acridine (Acr) hexyl bromide (243 mg, 0.48 mmol) in DMF (5 ml). The resulting mixture was stirred at 50° C. for 16 hours. The polymer was thoroughly triturated with deionized water to remove unreacted Acr-hexyl bromide and DMF. The polymer was then freeze-dried to obtain an orange powder (800 mg, ca. 80%).

[0074] NMR data: 1H-NMR (CDCl3, 800MHz), δ: 6.5-8.6 (Br, 0.77H, acridine protons), 3.0-3.5 (Br, 4H, PMVEMA protons), 0.98-1.7 (Br, 9.5H, alkyl protons), 0.87 (Br, 3H, alkyl protons). NMR data show that approximately C 14 H 29 Fifty percent of the -NH2 and approximately 11% of the acridine hexyl bromide were replaced.

[0075] Electrospinning conditions: Electrospinning solutions were prepared by dissolving 20% ​​(w / v) pTau and 15% (w / v) pAcr, respectively, in dry DMF at room temperature (RT). The two solutions were stirred overnight before electrospinning. A 2 mL plastic syringe (Dispo Van, India) and a blunt 22 G metal needle (Dispo Van, India) were used in the electrospinning procedure. A syringe pump delivered the solutions at a constant flow rate of 0.5 ml / h (pTau) and 0.3 ml / h (pAcr), respectively. Two electrospun nanofiber scaffolds were collected on an aluminum foil placed 15 cm away from the needle (a voltage of 15 kV was applied). All experiments were carried out at 25 °C and a relative humidity (RH) below 55%. The electrospun mats, measuring approximately 10 × 10 cm in length and width, were stored in a desiccant until further use.

[0076] Field Emission Scanning Electron Microscopy (FESEM): (pTau and pAcr) The morphology of electrospun scaffolds was investigated by FESEM (Carl Zeiss MERLIN VP compact). Dried electrospun mats were mounted on aluminum stubs and sputter coated with gold (Pelco® SC-7 Auto sputter coater). Gold-coated mats were imaged at a voltage of 2 kV. pRBC-treated scaffolds were imaged to investigate blood cell adhesion.

[0077] Contact angle measurements: The wettability of electrospun scaffolds was studied using the sessile drop method. The scaffolds were cut into uniform pieces and placed on glass slides before the measurements. A water drop (5 μL) with a diameter of approximately 2 mm was placed on the electrospun membrane and images were acquired. Images were processed using ImageJ software (VI.53).

[0078] Hemolysis assay: A hemolysis assay was performed to evaluate the hemolytic properties of cationic and anionic (pTau-pAcr) scaffolds. Blood from human volunteers was collected in tubes containing the anticoagulant sodium citrate. The collected blood was centrifuged (500G, 10 min) and the supernatant was discarded. These washed RBCs were used to obtain a hematocrit of 2% in saline and were then transferred to chamber slides (8 cm) that had been pre-cleaned with double distilled water. 2 ) fixed scaffolds. RBCs were further resuspended in saline and pelleted. The RBCs were kept in an incubator shaker (37°C, 100 rpm) for 1 hour. After 1 hour, the hematocrit was transferred to a 2 ml centrifuge tube and centrifuged at 500G for 10 minutes at room temperature. 100 μl of the supernatant was taken and transferred to a 96-well plate. Thus, this procedure concerns the quantification of hemoglobin in the supernatant from lysed RBCs treated by scaffolds for 1 hour. 0.1% Triton-Xl00 (same volume) with a hematocrit of 2% was used as a positive control and pure 2% hematocrit was used as a negative control. 100% lysed samples of untreated RBC specimens were prepared with 0.1% Triton-Xl00 with a hematocrit of 2%. The concentration of Hb in 100% lysed RBC samples was measured by the cyanmethemoglobin (Drabkin) method, and this concentration was considered as 100%. A series of dilutions were then prepared from the lysed samples to generate a standard curve. The hemolysis level of pTau-pAcr-treated RBCs was then derived from the standard curve using the Drabkin method. For the Drabkin method, absorbance was recorded at 540 nm using a Varioskan LUX multimode microplate reader (Thermo Fisher, MA, USA). The percent hemolysis was calculated as follows:

number

[0079] Polymer leaching test: pAcr scaffold (8 cm 2 ) were fixed on chamber slides and incubated with double distilled (DD) water. The scaffolds were incubated for 30 min, followed by a short incubation for 5 min. The DD was removed after both incubations and lyophilized to recover the pAcr leached from the scaffolds. The lyophilized pAcr was dissolved in DMF and estimated using a fluorescence spectrophotometer (Horiba Flurolog QM, France). The fluorescence intensity was recorded as a function of time such that the excitation wavelength was 502 nm and the emission wavelength was 535 nm. The leaching rate (%) was determined by a pure pAcr polymer standard curve constructed at the excitation and emission wavelengths mentioned above.

[0080] Discussion: High molecular weight (Mw) PMVEMA (average molecular weight 330000) was selected to functionalize with anionic and cationic counterparts to remove soluble DAMPs generated in pRBCs. As the polymer is expected to remove DAMPs from aqueous media, it needs to be hydrophobic in nature. Hydrophobicity was imparted by conjugating tetradecylamine to PMVEMA via an amide bond. Following tetradecylamine conjugation, the polymer was functionalized with taurine and acridine derivatives to obtain charged hydrophobic polymers, namely, pTau and pAcr. The sulfonic acid in taurine imparts an anionic charge to pTau, whereas acridine imparts a cationic charge to pAcr. The anionic group provides a higher affinity for cationic species such as amines, guanidines, etc. Conversely, acridine acts as an efficient intercalating agent to selectively remove nucleic acids without interacting with cell membranes. This functionalization was carried out by one-pot synthesis. Fifty percent of the anhydride groups were conjugated to tetradecylamine (Poly-Cu-50%), followed by functionalization with taurine (anionic) and acridine hexyl bromide (cationic). Surface charge measurements confirmed the overall negative and positive charges of pTau and pAcr, respectively (Figure 1c).

[0081] 2926, 1782, 1732cm -1 FTIR peaks at 1000 nm confirmed the presence of alkyl chains, maleic anhydride and amide bonds. NMR findings demonstrate that pTau has approximately 50% and 25% substitution of tetradecylamine and taurine, whereas pAcr has approximately 50% substitution of tetradecylamine and 11% substitution of Acr hexyl bromide on PMVEMA. To develop a biomaterial platform, pTau and pAcr were then electrospun to engineer nanofibers at optimized concentrations of 20% and 15% (w / v). The polymer flow rate and voltage were maintained at 0.3 ml / h and 15–20 kV, and the needle-to-collector distance was fixed at 15 cm. The electrospun scaffolds were imaged with FESEM (Figure 1b), which confirmed that the fibers were intact, stable and free of beads.

[0082] Since the electrospun charged nanofibers are expected to be hydrophobic in nature, their degree of wettability was assessed by contact angle measurements. Water contact angles were recorded for pAcr (140.033 ± 4.41°) and pTau (144.33 ± 5.507°) (Figure 1b). The high contact angle (superhydrophobic domains) further confirms the incorporation of aliphatic chains into the polymer inducing hydrophobicity. Conferring hydrophobicity to the polymer was of utmost importance as it helps the nanofibers maintain their structural integrity in aqueous media (pRBC units).

[0083] To study the biocompatibility properties of the synthetic polymers, it was important to demonstrate that the polymer components would not leach into the blood during processing. To prove this, 8 cm 2 A nanofiber pAcr was weighed (6.75 mg) and incubated with 1 ml distilled water at room temperature for 30 min. The incubated water was collected after 30 min and lyophilized. The lyophilized product was processed by fluorescence spectroscopy (Figure 1) to quantify 0.015 mg (0.2%) of pAcr. Further successive incubation of pAcr with distilled water for 5 min confirmed 0.03% pAcr in water. Similarly, pTau was also examined and tested by NMR and FTIR spectroscopy, but no significant results were detected confirming leaching (data not shown). The leaching data proved the high stability of the nanofibers in aqueous media. To further eliminate the interference of the leached polymer with the pRBC units, the scaffolds were pretreated with heat-sterilized distilled water for 30 min.

[0084] From the viewpoint of biocompatibility, 24 cm 2The scaffolds pTau and pAcr were further investigated by incubating with 42-day-old human pRBCs and their supernatants for 30 min. After harvesting the RBCs and supernatants, the dried scaffolds were imaged with FESEM. The FESEM images showed that blood cells did not attach to the scaffolds (Fig. 1d). This experiment further verified that the scaffolds do not interfere with the membrane integrity / health of RBCs and are highly hemocompatible to proceed with the clearance studies. A hemolysis assay also established the viability of the scaffolds. The nanofiber scaffolds were incubated with human RBCs at 37 °C for 1 h (Fig. 1e) to evaluate hemolysis. Statistics revealed that the hemolysis of pTau was less than 1%, while the hemolysis of pAcr was less than 3%. These findings ensured the compatibility of the scaffolds with RBCs and ultimately enhanced the clearance ability of the scaffolds.

[0085] Example 1A: Charged nanofiber sheets remove DAMPs from both old stored RBC units and their supernatants Materials: The materials used were the same as in Example 1.

[0086] Methods: Synthesis of anionic (pTau) and cationic (pAcr) scaffolds: These were prepared in the same manner as in Example 1.

[0087] Fabrication of electrospun nanofiber sheets: Electrospinning solutions were prepared by dissolving 20% ​​(w / v) pTau and 15% (w / v) pAcr, respectively, in dry DMF at room temperature (RT). The two solutions were stirred overnight before electrospinning. A 2 mL plastic syringe (Dispo Van, India) and a blunt 22 G metal needle (Dispo Van, India) were used in the electrospinning procedure. A syringe pump delivered the solutions at a constant flow rate of 0.5 ml / h (pTau) and 0.3 ml / h (pAcr), respectively. The two electrospun nanofiber sheets (Tau-NFS and Acr-NFS) were collected on an aluminum foil placed 15 cm away from the needle (a voltage of 15 kV was applied). All experiments were carried out at 25 °C and a relative humidity (RH) below 55%. The electrospun mats, measuring approximately 10 × 10 cm in length and width, were stored in a desiccant until further use.

[0088] Field Emission Scanning Electron Microscopy (FESEM): The morphology of Tau-NFS and Acr-NFS was investigated as shown in Example 1.

[0089] Contact angle measurements: The wettability of Tau-NFS and Acr-NFS was studied using the sessile drop method. The scaffolds were cut uniformly and placed on a glass slide before the measurements. A water drop (5 μL) with a diameter of approximately 2 mm was placed on the electrospun NFS and images were acquired. Images were processed using ImageJ software (VI.53).

[0090] Hemolysis assay: A hemolysis assay was performed to evaluate the hemolytic properties of Tau-AcrNFS as shown in Example 1. Blood from human volunteers was collected in tubes containing the anticoagulant sodium citrate, centrifuged (500G, 10 min), and the supernatant was discarded. These washed RBCs were used to obtain a hematocrit of 2% in saline and were then transferred to chamber slides (16 cm) that had been pre-washed with double distilled water. 2) was added to Tau-AcrNFS immobilized on scaffolds. RBCs were further resuspended in saline and pelleted. The RBCs were kept in an incubator shaker (37°C, 100 rpm) for 1 h. After 1 h, the hematocrit was transferred to a 2 ml centrifuge tube and centrifuged at 500G for 10 min at room temperature. 100 μl of the supernatant was taken and transferred to a 96-well plate. Thus, this procedure concerns the quantification of hemoglobin in the supernatant from lysed RBCs treated with scaffolds for 1 h. 2% hematocrit in 0.1% Triton-Xl00 (same volume) was used as a positive control and pure 2% hematocrit as a negative control. 100% lysed samples of untreated RBC specimens were prepared with 2% hematocrit in 0.1% Triton-Xl00. The concentration of Hb in 100% lysed RBC samples was measured by the cyanmethemoglobin (Drabkin) method, which is considered as 100%. A series of dilutions were then prepared from the lysed samples to generate a standard curve. The hemolysis level of Tau-AcrNFS-treated RBCs was then derived from the standard curve using the Drabkin method. For the Drabkin method, absorbance was recorded at 540 nm using a Varioskan LUX multimode microplate reader (Thermo Fisher, MA, USA).

[0091] Polymer leaching test Acr-NFS (8cm 2) were fixed on a chamber slide and incubated with optisol (AS-5) solution [composition: sodium chloride (8.77 mg / mL), anhydrous glucose (8.18 mg / mL), mannitol (0.01% w / v), adenine (0.03 mg / mL)]. The sheets were incubated for 2 h. The optisol solution was removed after both incubations and freeze-dried to recover the polyacridine leached from the sheets. The freeze-dried polyacridine was dissolved in DMF and estimated using a fluorescence spectrophotometer (Horiba Flurolog QM, France). The fluorescence intensity was recorded as a function of time such that the excitation wavelength was 502 nm and the emission wavelength was 535 nm. The leaching rate (%) was determined by a pure polyacridine polymer standard curve made at the excitation and emission wavelengths mentioned above.

[0092] Discussion: Charged electrospun nanofiber sheets efficiently remove DAMPs. To synthesize polymers with anionic / cationic charges to generate electrospun nanofiber sheets, we selected poly(methyl vinyl ether-alt-maleic acid) [PMVEMA, average molecular weight: 330,000], in which 50% of tetradecylamine was functionalized to impart hydrophobicity to the polymer. Subsequently, 25% of taurine or 11% of hexyl acridine were functionalized to obtain anionic (pTau) and cationic (pAcr) polymers, respectively. In addition to imparting cationic charges, acridine can also bind to DNA by intercalation, thereby enhancing the ability of Acr-NFS to remove extracellular DNA. A detailed synthesis schematic is shown in Figure 21. Anionic electrospun nanofiber sheets (Tau-NFS) and cationic electrospun nanofiber sheets (Acr-NFS) were generated by conventional electrospinning of polytaurine and polyacridine, respectively (Methods). Tau-NFS and BAcr-NFS consist of nanofibers with widths of 100–200 nm and lengths of over 50–100 microns (Figure 1Ab). Surface charge measurements confirmed the overall negative and positive charges of Tau-NFS and Acr-NFS, respectively (Figure 1Ac). Furthermore, contact angle measurements showed that both NFSs are hydrophobic (Figure 1Ad), which is important to prevent absorption of aqueous liquids. A 2-hour incubation of Tau-NFS and Acr-NFS in optisol (AS-5) solution, used as a preservative for preserving RBCs, suggests that the NFSs are stable in the preservative solution, and quantification of the leached polymer indicates that there is no significant leaching (<0.2%) of either polymer. Scanning electron microscopy images of Tau-NFS and Acr-NFS after incubation with stored RBCs showed that RBCs or neutrophils did not adhere to NFS (Figure 1Ae), thus indicating no reduction in RBC numbers due to NFS treatment. Furthermore, to understand whether treatment with Tau-NFS and Acr-NFS results in cell loss, complete blood counts (CBCs) were performed before and after treatment with Tau-NFS and Acr-NFS.Quantifying the number of RBCs, leukocytes, platelets, neutrophils and lymphocytes before and after treatment with NFS (Figure 18) revealed no cell loss, suggesting that these nanofiber sheets do not cause cell death or cell capture. Furthermore, the hemolysis rate was measured after incubating RBCs with Tau-AcrNFS for 1 h at 37°C. Less than 1% hemolysis was observed for Tau-Acr-NFS (Figure 1Af). Collectively, these results suggest that Tau-AcrNFS is stable and does not cause significant hemolysis.

[0093] Prior to testing the DAMP removal effect of NFS, we quantified the time-dependent generation of DAMPs (extracellular DNA, Hb, and PUFAs) in human non-leukodepleted RBCs stored for up to 42 days. Various analytical techniques, such as PicoGreen assay, ELISA, Drabkin assay, and LC-MS, were used to quantify extracellular DNA, nucleosomes, Hb, and PUFAs, respectively (Methods). Quantitative elucidation of storage damage revealed that DAMPs, including extracellular DNA (Figure 19a), Hb (Figure 19b), arachidonic acid (AA, Figure 19c), and PUFAs such as hydroxyeicosatetraenoic acids (5-HETE, 12-HETE, and 15-HETE, Figures 19d-f), increased over time. For example, at day 42, the concentrations of extracellular DNA and Hb increased approximately 150- and 40-fold, respectively, compared to day 0.

[0094] First, we systematically optimized the required surface area of ​​nanofiber sheets and the incubation time for removing DAMPs. One milliliter of (stored for 42 days) RBCs was incubated on nanofiber sheets with different surface areas (8, 16, 24 cm). 2 ) sheet and incubated at 4 °C for 30 min. Quantification of extracellular DNA revealed that for both nanofiber sheets, 2 The sheet can efficiently remove DNA, and the 8cm 2 Over 24cm 2 The results suggest that the following sheet did not increase the removal capacity (Figure 20a). 2The optimal incubation time was identified by incubating with nanofiber sheets of Tau-NFS for 5, 15, and 30 min. The data suggest that Tau-NFS and Acr-NFS were able to remove DNA within 5 min of incubation, and longer incubation did not enhance the removal ability (Figure 20b).

[0095] To remove all DAMPs (extracellular DNA, nucleosomes, Hb, and PUFAs) from old RBCs, 1 ml of 42-day-stored RBCs was incubated with cationic nanofiber sheet / anionic nanofiber sheet (Tau-AcrNFS) at 4°C for 5 min. The data in Figure 19g-m suggest that Tau-AcrNFS is effective in removing DAMPs. After incubation, the nanofiber sheet removed 80% of DNA (Figure 19g), 50% of nucleosomes (Figure 19h), 45% of Hb (Figure 19i), and 75-80% of PUFAs including AA, 5-HETE, 12-HETE, and 15-HETE (Figure 19j-m). These results suggest that Tau-AcrNFS can efficiently remove and significantly reduce the concentration of DAMPs in old RBCs stored for 42 days. SEM images of Tau-NFS and Acr-NFS after treatment with preserved RBCs (Figure 1Ae) showed a sticky layer on the surface, which may be due to the accumulation of sequestered proteins and lipids in the nanofiber sheet.

[0096] Example 2: Removal of DAMPs from both aged stored RBC units and their supernatants by charged nanofiber scaffolds method Packaging of human RBCs (pRBCs) as non-leukocyte-reduced (NLR) and leukocyte-reduced (LR) units: NLR and LR RBC units were prepared from human whole blood of different blood types (both males and females, n=3) stored in 20 ml vacutainers containing citrate-phosphate-dextrose and adenine (CPD-A) (containing citric acid monohydrate 3.577 mg / mL, trisodium citrate dihydrate 29.972 mg / mL, sodium dihydrogen phosphate 2.496 mg / mL, dextrose 25.5 mg / mL). Whole blood was centrifuged at 500 G for 20 min and the supernatant was removed. After removing the supernatant, optisol (AS-5, preservative) (containing sodium chloride 8.77 mg / mL, anhydrous glucose 8.18 mg / mL, mannitol 0.01% w / v, and adenine 0.03 mg / mL) was added to obtain a hematocrit value of (approximately) 65%, which was referred to as NLR blood.

[0097] Similarly, LR RBCs were obtained from whole blood by an additional step of leukoreduction within 24 hours of collection with a leukoreduction filter (Acrodisc® PSF WBC filter, Merck). After leukoreduction, AS-5 preservative was added to obtain a hematocrit value of 65%, which was called LR blood. LR and NLR blood were then transferred into 2 ml tubes for packaging leaving a small space for residual air and stored at 4°C (in the dark) until further use.

[0098] In vitro efficacy of pTau and pAcr to remove DAMPs in human red blood cell units: 42-day stored pRBCs (both NLR and LR) and their supernatants, referred to as 42D-RBC and 42D-T, respectively, were treated with pTau and pAcr scaffolds, individually and in combination, to remove DAMPs up to 42 days, i.e., the shelf life of the RBCs. Also, for intermediate washing, NLR and LR blood were treated with pTau and pAcr, individually and in combination, on days 21 and 28 of storage. In both treatment conditions, the nanofibrous scaffolds were pre-wetted with distilled water for 30 min before adding the RBCs. In each experiment, the total surface area of ​​the scaffolds was 24 cm2 The RBCs and scaffolds were treated at 4°C for 5 minutes to maintain the cold chain storage. After the removal procedure, the RBCs were transferred again to a 2 ml Eppendorf tube that was heat sterilized to leave a small residual space and stored until day 42, when the supernatant was collected for further analysis including bioassay. The untreated group was not subjected to any treatment during or after the storage period, and the supernatant was collected on day 42 to compare with the treated group.

[0099] Quantification of free Hb removal experiments using the cyanomethemoglobin (Drabkin) method: Drabkin reagent lyses red blood cells and oxidizes all forms of Hb, except for sulfhemoglobin, which is present in trace amounts, to stable HiCN (Didar, TF et al. Improved treatment of systemic blood infections using antibiotics with extracorporeal opsonin hemoadsorption. Biomaterials 67, 382-392 (2015)). Supernatants of stored RBC units (NLR and LR, treated or not treated with scaffolds) were diluted (1:10) with Drabkin reagent (Sigma-Aldrich, St. Louis, MO, USA). Standards (0–40 mg / dl) were prepared and a calibration curve was created using human Hb diluted (1:10) with Drabkin reagent (Sigma-Aldrich, USA). Samples (LR and NLR) and standards were incubated for 15 min at room temperature in the dark. The absorbance was recorded at 550 nm using a multimode microplate reader (Thermo Fisher, Massachusetts, USA). The Hb concentration of each sample (LR and NLR) was calculated based on a standard curve of human Hb.

[0100] Quantification of free DNA by Pico green assay: RBC supernatants (NLR and LR) from scaffold-treated and untreated groups were thawed on ice. The supernatants were diluted with PicoGreen® reagent and Tris-EDTA (TE) buffer (Quant-iTTM PicoGreen TM The solution was diluted (1:50) in dsDNA assay kit (Invitrogen, USA) and incubated for 5 min in the dark. Free DNA estimation was performed using a fluorescence spectrophotometer (Horiba Flurolog QM, France). PicoGreen® avoids the contribution of interfering substances and has an emission maximum at 530 nm when explicitly bound to dsDNA (unbound PicoGreen® reagent shows minimal fluorescence in solution) (Land, WG Transfusion-related acute lung injury: The work of DAMPs. Transfus. Med. Hemotherapy 40, 3-13 (2013)). DNA was quantified by a standard curve in the range of 0-10 ng / μL.

[0101] Nucleosome quantification: The supernatant of stored RBCs was diluted (1:5) in PBS. Anti-histone biotin monoclonal antibody (clone H11-4) and anti-DNA-POD-monoclonal antibody from mouse (clone MCA-33) were used to quantify nucleosomes by ELISA (catalog number 11920685001, Cell Death Detection Kit, Roche, Indianapolis, IN) according to the manufacturer's instructions.

[0102] Estimation of bioactive lipids by LCMS Chemicals and standards: Eicosanoids, 5(S)-HETE, 12(S)-HETE, 15(S)-HETE and arachidonic acid (AA) were purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). 5(S)-HETE-d8 (Cayman Chemical Co.) was used as the internal standard (IS) in this study. Acetonitrile (Baker analyzed® LC-MS reagents), water (Baker analyzed® LC-MS reagents), formic acid, ethanol, ethyl acetate (EtOAc) and glacial acetic acid were used to prepare samples for LC-MS / MS measurements.

[0103] Preparation of stock solutions: 5-(S)-HETE, 12(S)-HETE, 15(S)-HETE, AA and the internal standard 5(S)-HETE-d8 were prepared in ethanol by the provider. 5(S)-HETE, 12(S)-HETE and 15(S)-HETE were further processed and serially diluted to obtain solutions ranging from 0.05ng / ml to 100ng / ml. The range of AA solutions was 0.5ng / ml to 1000ng / ml. The working concentration of pure IS was 1000ng / ml.

[0104] Sample preparation: Lipids were extracted from the supernatant of stored RBCs by gently mixing the samples with 0.5 mL of EtOAc containing 0.13% acetic acid (v / v). 5 μl of internal standard (IS) was added to all samples. After 10 min of vigorous shaking (1500 rpm), the samples were centrifuged (10000 rpm, 10 min, 4°C) and 450 μl of the organic layer was transferred to a new tube and evaporated at 4°C in a centrifuge (Scan Vac, Labogene, Denmark). The dry residue was reconstituted in 25 μL of nitrogen-purged EtOH and then directly injected (2 μL) into the LC-MS / MS system.

[0105] LC-MS / MS Conditions: HPLC analysis was performed using a Shimadzu Prominence HPLC system. The instrument comprises a communication module (CBM-20A), a degassing unit (DGU-20A5R), a solvent supply unit (LC-30AD), a column oven (CTO-20AC), and an autosampler (SIL-30AC). The temperature of the column oven was maintained at 40° C., and the temperature of the autosampler was set at 4° C. An Acquity UPLC® BEH C18 1.7 μm column (2.1×50 mm) was used to separate lipids. The mobile phase included solvent A, i.e., water with 0.1% formic acid (FA), and acetonitrile with 0.1% FA as solvent B. The flow rate was maintained at 0.2 ml / min, and the injection volume was 2 μl. The gradient (20.10 min) of lipid elution was set as 50% A and 50% B for 1 min. A linear gradient to 20% A and 80% B was then set for 8 min to elute 5HETE, 12HETE, and 15HETE. A linear gradient to 100% B was then set for 2 min and held for 5 min to elute AA. Finally, the column was equilibrated at 50% A and 50% B for 4 min before the next injection. LC-MS analysis was performed using a QTRAP 5500 (Sciex, Framingham, MA, USA) triple quadrupole mass spectrometer. The operating parameters of the mass spectrometer were: declustering potential (DP): -209.8 V, entrance potential (EP): -5.7 V, collision cell exit potential (CXP): -16.9 V, ion spray voltage: -4500 V, source temperature: 500 °C, curtain gas: 35 psi, ion source gas 1: 40 psi, ion source gas 2: 50 psi. All spectra of lipids were recorded and quantified in multiple reaction monitoring (MRM) mode. Negative electrospray ionization was used for all lipids and deuterated internal standards. Ion Q1 / Q3 transitions and other parameters are shown in the table below. [Table 4]

[0106] Discussion: US regulations state that pRBCs can be stored for up to 42 days (D) for transfusion. After 42D, increasing storage damage (DAMPs) poses a fatal risk to patients. Storage damage (extracellular DNA, nucleosomes, free Hb, bioactive lipids) can collectively or individually trigger immunomodulatory responses.

[0107] First, extracellular DNA, free hemoglobin, and bioactive lipids were measured from the supernatants of human RBCs stored for up to 42D (NLR-non-leukocyte-reduced units, LR-leukocyte-reduced units). First, extracellular DNA is usually present in the form of neutrophil extracellular traps (NETs). NETs are loose complexes of methylated histones and DNA. NLR units recorded a significant increase in extracellular DNA from 21D to 28D (~2.31 to ~7.3ng / μl) and from 28D to 42D (~7.3ng / μl to 13.3ng / μl) (Figure 8A). In NLR blood, an overall spike of ~150-fold was observed from 0D to 42D, which fully complemented other reports (Cancelas, JA et al. Additive solution-7 reduces the red blood cell cold storage lesion. Transfusion 55, 491-498 (2015)).

[0108] In contrast, leukodepleted RBCs (LR units) showed a stepwise increase in extracellular DNA production from 0 to 42 days (from about 0.05 ng / μl to about 0.3 ng / μl), but 30-fold less than NLR (Figures 8A and 8B). This was expected, since neutrophils were the main source of extracellular DNA and were initially removed using a leukofilter (Rapido, F. et al., Prolonged red cell storage before transfusion increases extravascular hemolysis., J. Clin. Invest. 127, 375-382). (2017) (Figures 8A and 8B). A similar trend was observed for the generation of free hemoglobin (Hb) in NLR storage units. Free Hb increased up to 40-fold from 0D to 42D (from approximately 0.03g / dl to approximately 0.8g / dl) (Figure 8A). Similarly, in LR units, hemolysis was limited (approximately 0.03g / dl to approximately 0.45g / dl), but was less pronounced than in NLR units (Figures 8A and 8B). This raises the idea that removal of leukocytes (by leukocyte reduction filters) reduces the formation of leukocyte-associated DAMPs but ultimately fails to control the accumulation of RBC-associated DAMPs.

[0109] Concomitantly, accumulation of lipids such as arachidonic acid (AA), 5-HETE, 12-HETE, and 15-HETE in the supernatants (LR and NLR) has been previously identified and reported (Yoshida, T., Prudent, M. and D'Alessandro, A. Red blood cell storage lesion: Causes and potential clinical consequences. Blood Transfus. 17, 27-52 (2019)). All of these lipids are recorded to reach a relative maximum concentration at day 42 of storage (Fuchs, TA et al. Neutrophils release extracellular DNA traps during storage of red blood cell units. Transfusion 53, 3210-3216 (2013)). In our study, all four non-polar lipids were quantified by LCMS and showed a significant increase in levels from 28 to 42 days of RBC storage (LR and NLR). AA, one of the most common omega-6 PUFAs involved in cell signaling and inflammation, increased exponentially in NLR units during storage from 28D (~66ng / ml) to 42D (~258ng / ml) (Figure 8A). The rapid production of AA is also due to the high activity of phospholipase A2, which releases AA present at the sn-2 position of membrane phospholipids (Vlaar, APJ et al. Accumulation of bioactive lipids during storage of blood products is not cell but plasma derived and temperature dependent. Transfusion 51, 2358-2366 (2011)). In LR units, the increase in production at 42D (~111ng / ml) was about two-fold higher than that at 28D (~63ng / ml), confirming the formation of RBC-associated lesions (Figure 8B).The oxidation products of AA are known as eicosanoids, a type of bioactive lipids that are mainly produced by enzymatic activity (Silliman, CC et al. Identification of lipids that accumulate during the routine storage of prestorage leukoreduced red blood cells and cause acute lung injury. Transfusion 51, 2549-2554 (2011)). Cyclooxygenase converts AA to prostaglandins, while lipoxygenase (LOX) activity leads to the production of HETE and leukotrienes. It has also been reported that cytochrome P450 (CYP) assists in the production of HETE (D'Alessandro, A. and Zolla, L. Biochemistry of red cell aging in vivo and storage lesions. Haematologica 7, 389-396 (2013)). In our study, we found that HETE increased significantly with the storage time of human RBCs, consistent with AA generation. For 12-HETE (NLR units), a significant increase in concentration (from about 7 ng / ml to about 269 ng / ml) was recorded from day 28 to day 42 of storage. However, this increase was not observed in LR (Figure 8B). The likely reason for this observation is likely due to the 12-LOX activity of WBCs in NLR units. Similar spikes in concentration were not consistent for 5-HETE and 15-HETE in stored human RBC units, and the trend was similar for both LR and NLR units (Figures 8A and 8B). The kinetic data provided a comprehensive insight into DAMP generation in stored RBC units over time.

[0110] The supernatant (supt) of 42D stored RBCs is a mixture of various charged species ranging from negatively charged extracellular DNA, bioactive lipids including arachidonic acid and eicosanoids, to positively charged free hemoglobin and histones. The predominance of charged species was the main factor leading to the design of charged biomaterials that can scavenge the charged species. The pAcr scaffold was specifically designed to bind DNA via intercalation, whereas pTau seeks out positively charged histones. The extracellular DNA in the supernatant of 42D stored NLR blood is concentrated within the surface area of ​​the scaffold (8 cm). 2 ~24cm 2 ) and incubation time (5 min to 30 min) were optimized to remove maximum DAMPs at 4°C (without breaking the cold chain). 2 suggested that pAcr could reduce extracellular DNA from 13ng / ul to 5ng / ul within 5 minutes from 1ml of supernatant (Figure 9). A similar trend was reported for the pTau scaffold.

[0111] Furthermore, these polymers did not leach out of the scaffolds (Figure 10). 2 of nanofiber pAcr was weighed (6.75 mg) and incubated with 1 ml of distilled water at room temperature for 30 min. The incubated water was collected after 30 min and freeze-dried. The freeze-dried product was processed by fluorescence spectroscopy. The pAcr was further incubated with distilled water for 5 min consecutively. The results clearly show that no significant amount of (pAcr) was leached out during the process.

[0112] Figure 2a shows the surface area (>8 cm 2 ) and stepwise increase in incubation time (>5 min) revealed no significant effect on the removal of extracellular DNA by the group of pTau and pAcr scaffolds. The idea of ​​using a combination of cationic (pAcr) and anionic (pTau) scaffolds in the subsequent removal experiments was based on the presence of negatively and positively charged lesions on pRBCs.

[0113] To confirm the effectiveness of the scaffold against positively and negatively charged DAMPs, a 12 cm2 scaffold with 12 cm2 area was used. 2 pTau scaffold and pAcr scaffold (total surface area 24 cm 2 ) was combined to process both NER 42D RBCs and supernatants. Washing of DAMPs from both groups by the scaffold was performed for 5 min at 4 °C. The overall idea in designing this experiment was to mimic a clinical scenario where the RBC units could be washed once by the scaffold during the storage timeline.

[0114] First, extracellular nucleosomes (DNA-histone complexes) were quantified by EEISA with or without treatment. Findings were plotted relative to nucleosome concentration of 100% for 42D untreated NLR supernatants. Treatment of 42D NLR supernatants with both scaffolds reduced extracellular nucleosomes by up to 50%. Similar removal efficiencies were reported for the RBC population (Figure 2). The high percentage of extracellular nucleosomes in the NLR set represents storage-associated extracellular DNA-histone complexes released from WBCs undergoing apoptosis, necrosis or neutrophil extracellular trap NETosis (Zimrin, AB and Hess, JR Current issues relating to the transfusion of stored red blood cells. Vox Sang. 96, 93-103 (2009)).

[0115] Furthermore, extracellular DNA in NLR units was reduced by up to about 75% in the supernatant group and by up to about 40% in the RBC group (Figure 2). The degree of reduction was significantly greater in the supernatant group compared to the RBC-treated group. The difference in efficiency was due to the presence of RBCs protecting the scaffold, which could prevent direct interaction between the scaffold and DNA.

[0116] Further quantification of free hemoglobin (Hb) was performed on NLR supernatants, and the data revealed that the concentration of free Hb was reduced from approximately 0.966 g / dl to 0.5 g / dl. This reduction in free Hb (g / dl) explains the 50% removal ability of the scaffold compared to the untreated group (Figure 2). Significant removal was also observed when RBC units were treated with the scaffold (Figure 2). Interestingly, in the LR units, the reduction in free Hb was very evident in the supernatant group (from approximately 0.466 g / dl to approximately 0.226 g / dl), but no significant reduction was observed in the RBC-treated group (LR) (Figure 11).

[0117] The universal role of scaffolds in binding and removing DAMPs was further substantiated by LCMS findings. AA and 12-HETE were removed in the blood supernatant primarily by the charged scaffolds. AA and 12-HETE were significantly reduced from 234ng / ml to 69.9ng / ml and from 270ng / ml to 47ng / ml, respectively (Figure 2). Similar reductions were recorded for 5-HETE and 15-HETE. RBC units also showed a significant reduction, with up to 70% removal observed for all lipids. Compared to NLR findings, LR blood revealed lower amounts of AA and other lipids. However, upon treatment with the scaffolds, AA values ​​were significantly reduced from 115ng / ml to 50ng / ml in the LR supernatant, whereas they dropped to 35ng / ml in the RBC units. Thus, the charged scaffolds also provide a method to purify LR blood containing soluble bioactive lipids generated from RBCs. These findings also demonstrate that -COOH groups and hydrophobic chains in bioactive lipids specifically bind to the charged hydrophobic scaffold aiding in their removal. The combined treatment with both scaffolds (pTau and pAcr) showed significant removal of extracellular DNA, nucleosomes and Hb, which could prove to be a game changer in transfusion research.

[0118] Example 3: Intermittent Removal of DAMPs: Improving the Quality of Older Stored RBCs by Treatment on Days 21 and 28 of Storage Storage damage is a general term that encompasses all the changes that occur as RBCs age in storage solutions. RBC units can be classified into "young" (<14-21 days) and "old" (>21D) RBC units based on the concentration of DAMPs produced (Hanna, VS and Hafez, EAA Synopsis of arachidonic acid metabolism: A review. J. Adv. Res. 11, 23-32 (2018)). Kinetic data (Figure 8) showed that DAMPs increased significantly from days 21 to 42 of storage. This observation led us to expect that DAMP production may accelerate the deterioration of healthy RBCs remaining in storage units. It was hypothesized that by decreasing DAMPs from blood units at specific points in the storage timeline (0-42D), we could increase the number of healthy RBCs at day 42 of storage.

[0119] To identify distinct time points in the storage timeline, the kinetic data of DAMP generation in stored RBCs was reviewed (Figure 8). Findings revealed that DAMP generation was rapid during the storage period from days 21 to 42. A stepwise decline in RBC quality was also observed within the same period. Hence, days 21 and 28 of stored blood units (NLR and LR) were strategically selected for processing. 2 of charged nanofiber scaffolds (pTau and pAcr were 12 cm each). 2 Blood units were treated with 500 mM NaCl (NaCl) for 5 min at 4°C and stored until day 42 after collection. DAMPs (extracellular DNA, nucleosomes, free Hb, bioactive lipids) were quantified at day 42 of storage after intermittent treatments (days 21 and 28).

[0120] Extracellular DNA production was significantly reduced from 12.7ng / μl to 8.32ng / μl on day 42 after 21 days of treatment, and further reduced to -3.26ng / μl when treated on day 28 (Figure 3). This reduction was relative to untreated 42D NLR blood units. Extracellular DNA production in LR blood units was low as expected by the end of day 42. Intermittent treatment on day 21 significantly reduced concentrations, but treatment on day 28 showed a maximum reduction of 40% (from 0.3ng / μl to 0.186ng / μl) (Figure 12).

[0121] Nucleosome data revealed that treatment on day 21 was not sufficient to limit the amount produced on day 42. However, treatment on day 28 significantly reduced the amount of free nucleosome produced by about 60% (Figure 3). Thus, the data suggest that washing on day 21 may be a little too early to remove DAMPs, as the change in the amount of free nucleosome produced is higher for the remaining storage period, i.e., the next 21D. Intermediate treatment on day 28 in the LR unit was also effective. Compared to the NLR, the amount of free nucleosome produced was significantly reduced by 30%, despite the much lower amount of free nucleosomes (Figure 12). Following the quantification of free nucleosomes, the level of free hemoglobin was monitored. Intermediate treatment on days 21 and 28 in the NLR unit revealed a similar decrease in the level of free Hb, i.e., a decrease of almost 50% (from about 0.992 g / dl to 0.6 g / dl and 0.5 g / dl, respectively). However, treatment on day 21 did not affect the rate of RBC lysis in LR units, whereas treatment on day 28 limited the production of free Hb by 50% (from about 0.4 g / dl to about 0.2 g / dl) compared to the untreated group. The effectiveness of intermediate treatment was demonstrated by quantification of free nucleosomes and free Hb in NLR and LR units.

[0122] However, this was further strengthened by the evaluation of bioactive lipids. Compared to the untreated group (approximately 258 ng / ml), the abundance of AA in NLR units was reduced to approximately 32 ng / ml and approximately 34 ng / ml when treated on days 21 and 28. The effect of intermediate treatment was more evident in the case of the formation of 12-HETE in pRBC units. It was found that the level of 12-HETE was reduced 100-fold (from approximately 200 ng / ml to approximately 2 ng / ml) after treatment on days 21 / 28 (Figure 3). This indicates that the lower formation of the oxidized AA product, 12-HETE, is largely dependent on the low amount of AA formation in the stored RBCs due to the intermittent treatment. A similar trend was observed for 5-HETE and 15-HETE. In the LR storage units, the formation of AA was prominent, but the intermittent treatment on days 21 / 28 significantly reduced its formation by 10-fold (Figure 12).

[0123] This experiment suggested that interim treatment on days 21 and 28 significantly reduced the amount of low DAMPs produced on day 42. However, treatment on day 28 was more desirable as it outweighed the reduction in DAMPs after treatment on day 21. This strategy is expected to improve the overall quality of stored blood. Intermittent treatment can provide significant progress against transfusion-related complications. Stored blood with interim treatment on day 28 is expected to improve the quality of stored blood and significantly reduce transfusion-related complications.

[0124] Example 4: Intermittent treatment with scaffolds maintains structural integrity of RBCs at 42D Maintenance of structural integrity of pRBCs after treatment: Assessed by a conventional osmotic fragility assay. Stored RBC units (treated and untreated) were added to nine tubes containing saline (NaCl, pH 7.4). The NaCl concentration in each tube was increased stepwise by 0.1%, from 0% (distilled water) (tube 1) to 0.9% (tube 9). The NaCl in the tubes was gently mixed and incubated at room temperature for 30 min. After incubation, the tubes were centrifuged at 500G for 20 min and the supernatant was collected. The absorbance density of the supernatant was measured at 540 nm using a multimode microplate reader (Thermo fisher, MA, USA). Hemolysis in each tube was expressed as a percentage. The maximum absorbance value of hemolyzed RBCs was taken as 100% in distilled water. RBCs treated with saline were used as a negative control (0% hemolysis).

[0125] Primary and secondary fixation of RBCs for SEM: Glass coverslips were sonicated and washed in acetone. Further coating of APTMS (3-(aminopropyl)trimethoxysilane) was applied by immersion in 2% APTMS and diluting it with acetone. RBCs were primarily fixed with 2% glutaraldehyde (GTA), incubated for 30 min in the dark, and washed with PBS. Primary fixed RBCs were incubated on APTMS coated coverslips for 1 h. After 1 h, coverslips were rinsed twice with PBS and incubated for 10 min in 0.1 M cacodylate buffer. Secondary fixation was achieved with 1% (w / v) osmium tetroxide (OsO4) for 10 min. Secondary fixed coverslips were then rinsed with Milli-Q water for 10 min, followed by a dehydration step including graded ethanol washes ranging from 40% to 100%. The dehydration step was followed by HMDS (hexamethyldisilane) drying. Finally, the dried samples were mounted on aluminum stubs, sputter coated with gold, and imaged with a FESEM (Carl Zeiss MERLIN VP compact).

[0126] Discussion: Human red blood cell (RBC) morphology gradually deteriorates during hypothermic storage. Acanthocytosis is a common pathway leading to these morphological deformations. As a result, each unit of stored blood contains a heterogeneous mixture of cells in various stages of acanthocytosis (disciform RBCs, spheroacanthocytocytes, and spherocytes). Discoid RBCs are considered healthy RBCs (Fu, X., Felcyn, JR, Odem-Davis, K. and Zimring, JC Bioactive lipids accumulate in stored red blood cells despite leukoreduction: a targeted metabolomics study. Transfusion 56, 2560-2570(2016)). Morphology determines the health status of RBCs or the fate of RBCs in circulation after transfusion. Fresh RBCs stored without processing (0D) and 42D NLR blood were imaged by FESEM to estimate the number of discoid RBCs present at the end of the storage period. Images revealed that the majority of RBCs in 42D NLR-pRBCs were spherocytic, spherocytes. Healthy discocytes were very few (Figure 3). Observable discocytes from multiple 42D samples were counted and considered as 100%. Discocytes counted in the 42D untreated set were used as a reference to analyze the intermediate treatment groups (days 21 and 28 of treatment). Image analysis revealed that the number of discocytes was 2-fold higher in the group that received pAcr and pTau treatment on day 21, whereas the number of discocytes was 3.5-fold higher in the group that received treatment on day 28 (Figure 4). The higher number of discocytes in the treatment group adds further evidence supporting the benefit of intermittent treatment, especially at day 28 of storage with artificial scaffolds, which helps maintain the excellent quality of RBCs.

[0127] Looking further into the quality of stored RBCs, the osmotic fragility test (OFT) is also an important parameter in characterizing the health of the red blood cell membrane. It depends on the degree of resistance of red blood cells (RBCs) to lysis when introduced in a hypotonic environment. OFT is also determined by 50% hemolysis at a specific NaCl concentration (%), known as MCF (mean cell fragility). The value of MCF increases with increasing storage time of pRBC units. In NLR pRBC units, a sharp increase in MCF value was observed from about 0.35 at 0D to 0.6 at 42D. With the increase in value, the hemolysis curve shifts to the right (Figure 4). This is also consistent with the FESEM data where the 42D RBC population is mainly in the form of spherocytes, which become more fragile and have a lower osmotic resistance. pRBC units with higher MCF values ​​may cause intravascular hemolysis after transfusion, which shortens the lifespan of RBCs in circulation. Surprisingly, the LR units also showed an increase in the MCF value, which indicates the degree of RBC health damage, from approximately 0.35 to 0.5 (Figure 13). However, this suggests that LR RBCs also become fragile during storage, although this is not as significant as the NLR data.

[0128] On the 21st / 28th day of storage at 4°C, the area was 24 cm for the NLR and LR units. 2 The data reveal that intermittent treatment performed for 5 min by both scaffolds (pTau and pAcr) of LR units. The data suggest that intermittent treatment on day 21 significantly reduced the MCF value to about 0.52, whereas intermediate treatment on day 28 brought the MCF of 42D RBCs to match that of the storage units on day 28 (Figure 4). Intermittent treatment on days 21 and 28 with LR units reduced the MCF value and shifted the hemolysis curve to the left, i.e., the healthy side, compared to the untreated group. The reduction in MCF values ​​suggests that the RBCs became healthier and more resistant to lysis after intermediate treatment. Of both time frames selected for washing, treatment on day 28 significantly reduced DAMPs on day 42. This clearly indicates that washing on day 28 is the optimal time to maintain healthier RBCs.

[0129] Example 5: Delayed aging by intermittent removal of DAMPs from stored mouse blood, demonstrating a strategy to extend shelf life In vivo experimental design: mice Male and female mice aged 8–12 weeks were used for the experiments. Animals were bred and housed in the animal facility at the National Centre for Biological Sciences, Bangalore. Animals were caged (maximum four per cage) during the experiments and were provided with food and water ad libitum. All mouse studies strictly adhered to institutional and national guidelines for humane use of animals. The experimental protocol was approved by the Animal Ethics Committee (IAEC) of the Institute of Stem Cell and Regenerative Medicine.

[0130] Packaging and storage of mouse RBCs and treatment with pTau and pAcr to remove DAMPs Blood collected at CPD was immediately transferred and centrifuged at 500g for 20 minutes at 4°C. Plasma was carefully aspirated and pooled with the pRBCs. Optisol, also known as AS-5, was added to pRBCs with a hematocrit of 65%. Finally, 550uL and 250uL blood units were aliquoted from these pRBCs and stored in sterile Eppendorf tubes (0.6ml and 0.3ml). On days 5 and 10 of storage, pTau and pAcr scaffolds (24cm 2 ) and mouse pRBCs were treated twice for 5 min at 40° C. After the depletion procedure, the RBCs were transferred again to heat-sterilized 2 ml Eppendorf tubes leaving a small residual space and stored until day 17, when the supernatants were harvested for estimation of extracellular Hb and nucleosomes.

[0131] Reduced exposure of annexin V (AV)-labeled phosphatidylserine in treated pRBCs The number of erythrocytes expressing phosphatidylserine (PS) on the outer membrane was determined as described elsewhere (Han, V., Serrano, K. and Devine, DV A comparative study of common techniques used to measure haemolysis in stored red cell concentrates. Vox Sang. 98, 116-123 (2010)). Briefly, AV labeling was performed by injecting 5 μL of AV-Alexa Fluor 106 erythrocytes in 100 μL of Annexin binding buffer. TM 568 conjugate (Invitrogen, USA). Binding buffer was prepared by adding 140 mM and 2.5 mM CaCl2 (all from Sigma-Aldrich) to 10 mM HEPES buffer. After 15 min incubation on ice, cells were diluted 4-fold with binding buffer and analyzed on an LSR Fortessa cytometer. Data analysis was performed using computer software (FACSDiva). AV-Alexa Fluor TM The erythrocyte population containing exposed PS labeled with the 568 conjugate was measured as the percentage of Annexin V positive RBCs.

[0132] Experimental design for biotinylation and RBC transfusion Biotinylated RBCs were obtained using EZ-Link Sulfo-NHS Biotin (Thermo Fisher Scientific, USA) via IV injection, which results in the formation of a covalent amide bond between biotin and lysine residues of outward-facing RBC membrane proteins (Singer, VL, Jones, LJ, Yue, ST and Haugland, RP Characterization of PicoGreen reagent and development of a fluorescence- based solution assay for double-stranded DNA quantitation. Anal. Biochem. 249, 228-238 (1997)). Briefly, sulfo-NHS-biotin powder was dissolved in sterile PBS, vortexed, and passed through a 0.22 μm filter. It was diluted to a final concentration of 1 mg biotin per 300 μL of IX PBS. Mice were restrained and the biotin reagent solution was administered via intravenous injection. Two days after biotin administration, under light anesthesia, approximately 1 ml of biotinylated blood was collected by cardiac puncture into a CPD-containing vial and immediately centrifuged at 500 g for 20 min at 4° C. Plasma was carefully aspirated and pooled with the pRBCs. Biotinylated blood was processed and stored in sterile Eppendorf tubes (0.5 ml and 1 ml) using Optisol as a preservative.

[0133] Treatment of biotin-labeled RBCs with pTau and pAcr scaffolds: Functionality test: Biotin-labeled RBCs were divided into treated and untreated groups. The pRBC units in the treated group (n=3) were washed with scaffolds on days 5 and 10 of storage and then packaged until day 18. The pRBC units in the untreated group (n=3) were not treated with scaffolds during the storage period. Both groups were analyzed for labeling efficiency and phosphatidylserine exposure during storage.

[0134] Evaluation of the efficiency of biotin-labeled RBCs and their extended shelf life by recovery experiments: At different time points during storage (0D, 7D, 14D, 15D, 17D), 6 μl of RBCs from both groups (treated and untreated, n=3) were conjugated with streptavidin APC-efluor® 780 conjugate (BD Biosciences, USA). The positive RBC population labeled with streptavidin APC-efluor was processed and analyzed by an LSR Fortessa cytometer (BD Bioscience, San Jose, CA). At different days of storage (0D, 7D, 14D, 15-19D), biotinylated pRBC units were diluted (1:1) in PBS with or without treatment. 200 μl of diluted biotinylated pRBCs were administered via intravenous injection to restrained mice (Burger, P., De Korte, D., Van Den Berg, TK and Van Bruggen, R. CD47 in erythrocyte ageing and clearance-the Dutch point of view. Transfus. Med. Hemotherapy 39, 348-352 (2012)). At specific time points, 6 μl of blood was collected by tail snip and stored in a microtube containing 250 μL of sterile PBS.

[0135] To detect biotinylated RBCs by flow cytometry, approximately 10 6 ~10 8 RBCs (100 μL of diluted sample) were diluted with 0.125 pg of streptavidin-APC-efluor® 780 conjugate (1:20 at 0.2 mg / mL, eBioscience TM) after transfusion. Samples were incubated for 5 min in the dark, washed with 400 μL PBS and centrifuged at 1000 g for 4 min. RBC pellets were resuspended in 200 μL PBS. Samples were analyzed using a BD LSR Fortessa cytometer (BD Biosciences, USA). A red laser was used to excite the dye (λ emission wavelength = 780 nm) through a 780 / 60 bandpass filter. Calculate post-transfusion survival rate (%) based on the following formula:

number

[0136] statistical analysis All data are presented as mean ± SD (standard deviation). Data were plotted and analyzed using Prism software. For comparison between multiple groups, one-way analysis of variance and Tukey's multiple comparison test ( * P < 0.05, ** P < 0.01, **** P<0.0001).

[0137] Discussion: Designing functional experiments of human RBCs after transfusion was not ethically feasible, so we performed a mouse model for scrutiny. A reported shelf life of 14 days for C57 BL / 6 mouse RBCs is comparable to that of human RBCs (42 days) (Piety, NZ, Reinhart, WH, Pourreau, PH, Abidi, R. and Shevkoplyas, SS Shape matters: the effect of red blood cell shape on perfusion of an artificial microvascular network. Transfusion 56, 844-851 (2016)). To identify potential periods of intermittent washing, we estimated the generation of DAMPs in mouse RBC units. Mouse blood was collected by retro-orbital puncture and processed as described in the previous section. We then assessed the generation of DAMPs at 14 days for stored RBC units. The data suggested that a large amount of free Hb was generated between 10 and 14 days of storage. The level of free Hb increased from approximately 0.2 g / dl to approximately 0.4 g / dl. A two-fold increase in the production of free nucleosomes was also observed. These observations suggested that DAMP formation was observed in RBCs stored in mice, similar to that in human blood.

[0138] Because DAMP generation was significant between days 10 and 14, intermediate treatments on days 5 and 10 were selected to evaluate the effects on stored mouse blood. 2The treatment was carried out at 4°C for 5 min on days 5 or 10 using scaffolds (pTau and pAcr). To further understand the intermediate treatment, another experimental group was added to this study. The blood of mice in this group was processed on days 5 and 10, i.e., blood was processed twice (double treatment). Evaluation of DAMPs in blood tests at 14D after single / double treatment revealed that double treatment outperformed single treatment. Single treatment significantly reduced the formation of free Hb, while double treatment reduced the free Hb level to about 0.15 g / dl (Figures 5 and 14). Double treatment also produced lower amounts of nucleosomes (about 30%) compared to the untreated group. These findings laid the foundation for future experiments in which increasing the number of intermittent treatments may improve the quality of RBCs over single treatment. The shelf life of RBCs is inversely proportional to the amount of DAMPs produced. The generation of DAMPs due to biochemical changes in RBCs accelerates RBC deterioration. Therefore, removing DAMPs may improve the quality of stored blood and extend its shelf life. Intermediate treatment with scaffolds opens a new avenue to extend the shelf life of mouse RBCs.

[0139] Furthermore, to understand the storage potential of RBCs, we studied the phospholipid structure of RBCs. The loss of phospholipid asymmetry associated with storage may result in the exposure of phosphatidylserine (PS) in the outer leaflet of the RBC plasma membrane. This serves as an effective signal contributing to the recognition and phagocytosis of apoptotic cells and cell fragments. PS exposure is involved in the removal of damaged RBCs from the circulation. It is well established that PS exposure increases with storage and is associated with the generation of DAMPs (Hod, EA et al. Transfusion of red blood cells after prolonged storage produces harmful effects that are mediated by iron and inflammation. Blood 115, 4284-4292 (2010)). Increased PS exposure during storage is also associated with hemolysis and microvesicle concentrations in red blood cell concentrates. This ultimately leads to a lower survival rate of transfused RBCs in the circulation. Based on this background information, the idea was to investigate the percentage of PS exposure in the outer leaflet of the RBC membrane during storage. The findings suggest that the percentage of PS exposure in untreated samples was low (approximately 5-6%) until day 5. However, it increased to approximately 20% by the end of day 14 (Figure 5) and then spiked to two-fold (approximately 40-50%) within 3 days, i.e., at day 17. The treated groups were in contact with the scaffold on days 5 and 10 (two treatments). The treated groups revealed a marked decrease in the percentage of PS exposure at 14D (approximately 10%). Interestingly, when compared to the untreated (NT) group, the percentage of PS exposure at 17D was significantly lower (approximately 18%) (Figure 5). The low percentage of PS exposure in the treated groups indicates that the phospholipid structure of RBCs was maintained until day 17. It also provides solid evidence that two treatments (days 5 and 10) delay RBC aging by removing DAMPs from stored blood.

[0140] To further support this assertion, male and female mice (8-12 weeks old) were transfused with biotin. Sulfo-NHS-biotin covalently binds and labels membrane proteins with biotin (Dinkla, S. et al. Phosphatidylserine exposure on stored red blood cells as a parameter for donor-dependent variation in product quality. Blood Transfus. 12, 204-209 (2014)). After 24 hours, RBCs were harvested from the same group of mice, processed, and stored as previously described. PS exposure data established that the membrane structure of the processed RBC units remained intact until day 17, 3 days longer than the reported shelf life (14D).

[0141] This understanding led us to store biotin-labeled RBC units up to day 19 with or without treatment. Here, two intermittent treatments were performed as described above. It seems to be the first time that labeled RBCs have been stored for more than 2 weeks. A few μl of stored blood of different storage durations were taken from treated and untreated groups, and the percentage of labeled RBCs was quantified using flow cytometry. Statistics revealed that the membrane integrity of RBCs (approximately 85%) was maintained up to day 14 in the untreated group. However, the membrane integrity significantly decreased from day 14 onwards; it was approximately (approximately 25-40%) in stored blood on day 17 (Figure 5). The decrease in the percentage of labeled mouse RBCs confirms the loss of their membrane structure. RBCs undergo morphological changes during storage that are evident when stored beyond the shelf-life, i.e., 14 days.

[0142] In contrast, in the treated group, membrane integrity remained intact (approximately 80-85%) until day 17, suggesting that intermediate treatment with the scaffold (days 5 and 10) could preserve RBC membrane structure. However, labeling efficiency decreased from day 17 onwards, indicating a preservation limit for treated RBCs.

[0143] Example 6: Dose-Responsive Reduction in Inflammatory Cytokine Responses of Twice-Treated 17D Mouse RBCs and Increased RBC Survival Following Transfusion Transfusion of processed pRBCs reduces inflammatory cytokine levels: Mice (8-12 weeks old (male), housed in the inStem / NCBS animal house) were injected with 14D and 17D stored RBCs with or without pTau and pAcr treatment, respectively. Treated and untreated groups were created as described in section 8a. Two hours after transfusion, mouse plasma was collected and diluted 1:10 in array buffer to analyze inflammatory markers. Cytokines / chemokines, including complement component 5A (C5A), intercellular adhesion molecule 1 (ICAM-1), monocyte chemotactic factor 1 (MCP-1), stromal cell-derived factor 1 (CXCL12), macrophage inhibitory protein 1 (MIP-1), keratinocyte-derived chemokine / CXCL1 (KC / CXCL1), interleukin-16 (IL-16), TIMP metallopeptidase inhibitor 1 (TIMP-1), interferon-γ-inducible protein 10 (CXCL-10), interleukin-1 receptor antagonist (IL-1F3), and granulocyte colony-stimulating factor (G-CSF), were quantified using a Mouse Cytokine Array Panel A (R&D Systems, MN, USA) according to the manufacturer's instructions.

[0144] Quantification of total iron in spleen: digestion and preparation of samples. Two hours after transfusion, mice were splenectomized. Spleens from all groups were collected in 2 ml Eppendorfs and the dry weight was recorded. Then, 1 ml of protein precipitation solution and 1 ml (8%) nitric acid were added to the spleen tubes and homogenized for 2 minutes. Protein precipitation solution was prepared with 0.53 N HCl and 5.3% trichloroacetic acid in HPLC water. The solution containing the homogenized spleen with the cap closed was boiled at 2000C for 30 minutes. It was cooled at room temperature for 2 minutes and the cap was opened to remove air bubbles. Finally, the solution was centrifuged at full speed for 10 minutes. The supernatant from all groups (n=3) was collected for quantification of total iron.

[0145] ICP-MS method: The optimized operating conditions used for ICP-MS (Shimadzu-2030) are summarized in Supplementary Table 2. A peristaltic pump was used to deliver the sample to the nebulizer (0.1 mL / min) and argon (Ar) gas was used to convert the sample into a fine mist. Automatic adjustments of the torch positioning, detector voltage, and ion lens voltage were performed to optimize resolution, sensitivity, and stability over a wide range of atomic masses. The doubly charged ionic charge ratio (Ce2+ 69.95 vs. Ce 139.90) and oxide ratio (CeO+ 155.90 vs. Ce l39.90) were also monitored and maintained below 3% and 2.5% (intensity), respectively. The Prep Fast system interfaced with the ICP-MS automatically diluted the stock solutions to generate the calibration curve and QC samples. A 30 ppb indium (In) solution was also mixed in-line by the Prep-Fast during the analysis and used as an internal standard for all samples and standards analyzed. The method was evaluated for its selectivity, sensitivity, linearity, precision and accuracy. [Table 5]

[0146] Statistical analysis: For experiments with multiple groups, regular one-way ANOVA and Tukey's post-hoc test was used. For experiments with multiple groups that were time-course studies, one-way repeated measures ANOVA was used. To compare two experimental groups, a two-tailed Student's t-test with Welch's correction was used. Probability values ​​(P) < 0.05 were considered as statistically significant differences. Statistical analysis and graphing were performed using GraphPad PRISM9.

[0147] Discussion: To determine transfusion-associated inflammation in treated and untreated groups, RBCs from days 14 and 17 were transfused into C56B6 / J mice (male and female). Treatment conditions were as described above. Mice were sacrificed 2 hours after transfusion and plasma cytokine levels were measured. Elevated cytokine concentrations were recorded for stored RBCs from the 17D untreated group, i.e., twice as high as those in the 17D treated group. In the 17D NT group, increased expression of C5A, CXCL12, M-CSF, and KC / CXCL1 was observed (Figure 6). In the same group (17D untreated), increased IL-16, TIMP-1, CXCL-10, IL-1F3, and G-CSF were also observed. However, this cytokine group was not observed in the 14D untreated group, supporting the previous assertion that the rate of deterioration of RBCs increases rapidly after their shelf life. (Figure 6) In the 17D-treated group, cytokine levels were significantly lower, and inflammatory cytokines were barely detectable (Figure 15). Furthermore, spleens from both treated and untreated mice were removed and transfused with 17D untreated RBCs, and necropsy revealed blackening of the spleens.

[0148] The observation of darkened spleens is closely related to the deposition of free iron in the spleen after transfusion of poor quality blood containing lysed RBCs and free Hb. On the other hand, no darkening of the spleen was observed in the 17D treated blood, conclusively confirming that the RBCs were in good condition even after their shelf life (14D) had passed (Figure 6). The spleens of the groups were removed and the free Fe was detected by ICP spectroscopy. 2+ The quantification of the effect of the electrochemically charged scaffolds on the inflammatory response, improved RBC quality, and delayed RBC aging was performed to support our claims.

[0149] Next, the functionality of the stored red blood cells was investigated based on the viability of biotinylated RBCs in the circulation of male / female mice (8–12 weeks old) after transfusion. According to the FDA, successful transfusion is established based on RBC viability ≥75% 24 hours after transfusion (Mock, DM et al. Development, validation, and potential applications of biotinylated red blood cells for posttransfusion kinetics and other physiological studies: evidenced-based analysis and recommendations. Transfusion 58, 2068-2081 (2018)). To monitor the successful pRBC transfusion rate in our study, biotinylated RBCs stored up to 19D with or without processing were transfused into new groups of mice. For the labeled RBCs, viability was tracked 1, 24, 48, and 72 hours after transfusion by flow cytometry. First, we quantified the viability up to 72 hours of 0D and 7D old pRBCs transfused without processing. Since the transfused labeled RBCs were mixed with unlabeled RBCs from the recipient mice, the percentage of labeled population circulating in the recipient mice 1 h after transfusion was considered as 100%. The 0D and 7D RBC recovery data revealed that the viability of circulating RBCs was more than 75% at 24 and 72 h after transfusion (Figure 17).

[0150] As expected, 14D-stored NT RBCs transfused into recipient mice show a recovery rate of over 75% after 24 hours. If the time point was extended to 72 hours, a notable decrease in labeled RBCs was observed after 48 hours, with the survival rate dropping to 50% (Figure 6). Following FDA guidelines, we performed the survival study within 24 hours, which is the "gold standard" criteria. Therefore, in our study, we fine-tuned the current experimental setup and showed that labeled RBCs survive for up to 72 hours. The recovery data after 24 hours provides new insights into the outcome of RBC quality, which plays a pivotal role in function and survival during circulation. In contrast, 14D-stored RBCs, intermediately treated twice by the scaffold, showed a survival rate of over 75% RBCs over 3 days (Figure 6). The set of recovery rates (%) from 48 hours to 72 hours proves that the transfused RBCs were in good health. This also argues that intermittent treatment effectively delays aging of stored RBCs and avoids their removal from the circulation in mice by the spleen.

[0151] When 15D unprocessed RBCs were transfused into mice, the percent survival rate was significantly reduced to 50%, which was completely predictable (Figure 6). A similar trend was observed for 15D, 16D and 17D stored RBCs that were not processed by the scaffold, as they were immediately removed from the circulation. Their post-transfusion recovery rate was also less than 75% (Figure 4). Transfusion of 15D old processed RBCs results in 85% survival rate after 24, 48 and 72 hours (Figure 6). 16D old RBC units also circulated for 72 hours in recipient mice after transfusion in the treatment group (Figure 16). However, transfusion of 17D old processed RBCs reduced survival rate after 24 hours. Survival rate dropped to 50% after 48 and 72 hours (Figure 6).

[0152] Recovery data elicits equivalence of 17D processed RBCs with l4D NT RBCs. In both groups, the transfused population was cleared from the circulation after 24 hours. This equivalence indicates that the shelf life of 14D mouse RBCs can be extended to 17D with intermittent treatment during the storage period. The extended shelf life of 3 days in mice corresponds to 9 days for human RBCs. The shelf life could not be further extended because the recovery rate (%) of 18D processed RBCs fell below 75% (Figure 16). This exclusive study of RBC functionality adds new properties of treatment with scaffolds that can improve the quality of RBCs and extend their shelf life.

[0153] Transfusion of red blood cells (RBCs) is a life-saving process and their stored units are the most demanded transfusion product worldwide. They are essential in symptomatic anemia, acute sickle cell crisis, acute blood loss of more than 30% of blood volume, and in restoring tissue oxygenation. However, the storage period affects the RBC units at a qualitative level. RBCs age in the storage bag and storage damage (DAMPs) occurs. These DAMPs (nucleosomes, extracellular DNA, free Hb and bioactive lipids) were quantified in this study. The levels of DAMPs reached a maximum at the end of 42 days, i.e., the shelf life of the RBCs. Therefore, transfusion of RBC units with large amounts of DAMPs may lead to transfusion-related complications such as TRALI, deep vein thrombosis, and organ dysfunction (Garcia-Roa, M. et al. Red blood cell storage time and transfusion: Current practice, concerns and future perspectives. Blood Transfus. 15, 222-231 (2017) and Martinod, K. and Wagner, DD Thrombosis: tangled up in NETs. Blood 123, 2768-2776 (2014)).

[0154] Currently, leukoreduction (LR) reduces the generation of DAMPs by eliminating white blood cells (WBCs) as the only source of extracellular DNA and nucleosomes. However, it failed to reduce bioactive lipids, free Hb or iron ions generated from RBCs. There are also various filter units on the market, consisting of polymers as adsorbents and whose membranes are decorated with artificial proteins that bind pathogen toxins, i.e. PAMPs (pathogen-associated molecular patterns) (Roussel, C., Buffet, PA and Amireault, P. Measuring post-transfusion recovery and survival of red blood cells: Strengths and weaknesses of Chromium-51 labeling and alternative methods. Front. Med. 5, 1-8 (2018)). However, these filter units are unable to cover the full range of storage damage associated with RBCs.

[0155] Stored NLR units used for transfusion in clinical scenarios harbor the entire spectrum of charged insults. This includes free histones, Hb, Fe, 2+, DNA and bioactive lipids (Rimmele, T. and Kellum, JA Clinical review: Blood purification for sepsis. Crit. Care 15, 1-10 (2011)). The lack of reported approaches to eradicate these storage lesions prompted the inventors to design a technology that addresses transfusion-related complexities. Current approaches involve removing soluble DAMPs from old stored blood without disrupting the RBC membrane. Hydrophobic cationic (pAcr) and anionic (pTau) polymers were engineered into charged nanofiber scaffolds. These bind to DAMPs present in old stored pRBC units through non-covalent interactions (electrostatic, π-π stacking, hydrophobic). Experimental findings claim that the combination of pTau and pAcr scaffolds removes a wide range of charged lesions in 42D NLR and LR groups (supernatant and RBC units). In the 42D NLR-treated group (supernatant and RBCs), DAMPs such as extracellular DNA and nucleosomes were removed by up to 50%.

[0156] Furthermore, the scaffolds were able to remove 60-70% of non-polar bioactive lipids and 40-50% of free hemoglobin. These values ​​verified the extensive cleaning of lesions by cationic and anionic scaffolds. The charged scaffolds were hemocompatible and did not leach into the aqueous medium (RBC units). After incubation with RBC units, FESEM images showed that RBCs were not attached to the scaffolds. Thus, eliminating DAMPs from old blood units prior to transfusion is expected to address transfusion-related complications. The basis of this study is the intermittent treatment with the scaffolds on days 21 and 28 of RBC storage. The obtained data showed that intermittent treatment significantly reduced DAMPs on day 42 of storage when compared to untreated RBC units. The results suggest that treatment on day 28 reduced DAMPs to the greatest extent on day 42, resulting in healthier NLR RBCs. This may be because accelerated deterioration of RBCs occurred mainly between days 28 and 42 of storage, with significant formation observed after day 21 in a few cases.

[0157] This study was not limited to NLR RBC units, but investigated the kinetics of DAMP production in LR units. Although their production rates were not as significant as in NLR units, the release of free Hb and bioactive lipids was of interest. Thus, washing LR units before transfusion can significantly reduce the amount of RBCs associated with damage. In this case, the concept of intermittent treatment worked effectively, with a significant reduction in the concentrations of free Hb and bioactive lipids when treated on days 21, 28 or 42 of storage.

[0158] The structural integrity of RBCs plays an important role in extending their remaining circulation time. Osmotic fragility data confirms that scaffold-treated RBCs (interim treatment on day 28) were healthier at day 42 of storage. Their membrane integrity was significantly more intact compared to untreated RBCs.

[0159] It was predicted that maintaining membrane integrity would prolong circulation survival (enhanced functionality). To prove this hypothesis, a mouse model was chosen for functional experiments. Provided that the shelf life of mouse RBCs is 14 days, stored RBCs were treated intermittently twice with pTau and pAcr scaffolds on days 5 and 10 of storage. Free nucleosomes and Hb after intermediate treatments were quantified in blood supernatants on day 14. Compared to the untreated group, extracellular nucleosomes and DNA were reduced to 70% and 50%, respectively, reinforcing the appropriateness of increasing the number of treatments.

[0160] Biotin labeling experiments confirmed that treatment with the scaffolds maintained lipid symmetry of the plasma membrane. The data also demonstrate that the shelf life of RBCs can be extended by 3 days. Recovery experiments present a new standard for evaluating the quality and functionality of transfused RBCs. RBCs stored at 14D without treatment showed recovery of more than 75% 24 hours after transfusion, which was in line with FDA standards. However, recovery decreased after 24 hours. This was attributed to a deteriorated state due to the formation of DAMPs. This phenomenon was not observed in the 14D-treated group, as the labeled RBCs had been circulating for 72 hours. The functionality of 17D old treated RBCs was also more than 75% after transfusion, comparable to untreated 14D stored RBCs.

[0161] This suggests that treatment with the scaffold extended the shelf life of mouse RBCs by 3 days, which is comparable to 9 days for human RBCs. Furthermore, inflammatory cytokine levels in 17D old treated RBCs were very low after transfusion, and no significant blackening of the spleen was observed. Thus, our study reveals a link between accelerated deterioration of RBCs caused by generated DAMPs. Looking to a brighter future, treatment with the scaffold before transfusion can significantly reduce storage damage and prevent transfusion-related complications. Moreover, intermittent treatment adds a new dimension to maintain RBC quality.

[0162] The detailed investigations herein explain the concept of DAMP removal during storage, which ultimately leads to an extension of the shelf life of RBCs. This work not only has enormous potential to meet the huge demand for RBC units, but also addresses the transfusion-related complications associated with old stored blood. Although this disclosure provides experimental data for RBCs only, the invention is also applicable to other biomaterials and stored cells, such as platelets and stem cells.

[0163] Example 7: Synthesis of zwitterionic polymers: Synthesis of zwitterionic polymers from PMVEMA. PMVEMA-C14 (50%): PMVEMA (500 mg, 3.2 mmol) was added to tetrahydrofuran (10 ml) in a pressure tube and stirred at 50° until dissolved. After dissolution, a solution of tetradecylamine (341 mg, 1.6 mmol) in THF (10 ml) was added to the PMVEMA solution and the reaction mixture was kept at 50° for 3 h. The solvent was removed using a rotary evaporator to give the polymer as a dry powder (800 mg, approximately 90% yield).

[0164] PMVEMA-C14-Zwitr: A solution of a zwitterionic compound, a sultone derivative of an amine (134.59 mg, 0.6 mmol) was prepared in 10 ml of DMF and added dropwise to a solution of PMVEMA-C14 (800 mg) in THF (20 ml) and left at 80 degrees for 12-16 hours with stirring. The reaction mixture was cooled and precipitated with chilled ethyl acetate. The polymer was washed with water to remove salts. The solid product was then freeze-dried and a white powder (510 mg) was observed. (Figure 21)

[0165] However, the foregoing description reveals such a general nature of the invention that others, by applying current knowledge, may easily modify and / or adapt it for various applications of such specific description without departing from the general concept. Thus, such adaptations and modifications should and are intended to be understood within the meaning and range of equivalents of the disclosed description. Moreover, precise and systematic details regarding all the above aspects are currently being worked out. Work on this invention is still in progress. It is understood that the expressions or terms used herein are for the purpose of description and not for the purpose of limitation. Thus, although the description herein is of the preferred embodiment, those skilled in the art will understand that the embodiments herein can be practiced with modifications within the spirit and scope of the embodiments.

Claims

1. (i) an anionic group; and (ii) a cationic group; and (iii) a combination of anionic and cationic groups; and (iv) a zwitterionic group; A nanofibrous scaffold for selectively removing storage damage from stored cells comprising a backbone polymer functionalized with a group selected from:

2. A system for selectively removing storage damage from stored cells, comprising: a nanofiber scaffold having a backbone polymer functionalized with anionic groups; A scaffold having a main chain polymer functionalized with a cationic group. Including, The system, wherein the preserved lesions are selectively removed when preserved cells are placed in contact with the scaffold.

3. A system for selectively removing storage damage from stored cells, comprising: a nanofiber scaffold having a backbone polymer functionalized with zwitterionic groups; The system, wherein the preserved lesions are selectively removed when preserved cells are placed in contact with the scaffold.

4. The scaffold of claim 1 and the system of claims 2 and 3, wherein the functionalized polymer is in the form of an electrospun fiber.

5. 4. The scaffold of claim 1 and the system of claims 2 and 3, wherein the backbone polymer is selected from the group consisting of poly(acrylic acid), poly(4-vinylpyridine), poly(4-vinylphenol), poly(allylamine), poly(vinyl alcohol), poly(methyl vinyl ether-alt-maleic anhydride) (PMVEMA), poly(vinyl chloride), poly(glycerin), poly(2-hydroxyethyl methacrylamide), poly(2-((allyloxy)methyl)oxirane), poly(vinylpyrrolidone), poly(4-vinylbenzyl chloride-alt-maleic anhydride), and poly(vinylimidazole).

6. The scaffold of claim 1 and the system of claims 2 and 3, wherein the backbone polymer is poly(methyl vinyl ether-alt-maleic anhydride) (PMVEMA).

7. Scaffold according to claim 1 and system according to claims 2 and 3, wherein the polymer is functionalized with an anionic group selected from 1,3-propane sultone, taurine, 1,3-dioxolan-2-one, 1,2-oxathiane 2,2-dioxide, phosphorochloric acid, (3-aminopropyl)phosphonic acid, valeric acid, 2-(methylamino)ethyl hydrogen phosphate, pentyl hydrogen phosphate and 4-(butylamino)butyl sulfate, preferably functionalized with taurine.

8. The scaffold of claim 1 and the system of claims 2 and 3, wherein the polymer is functionalized with taurine present in an amount of 8-50%.

9. Scaffold according to claim 1 and system according to claims 2 and 3, wherein the polymer is functionalized with cationic groups selected from ethidium bromide derivatives, cyanine dye YO-1, proflavine derivatives, acridine orange, ethylene blue derivatives, ellipticine derivatives, phenanthridine derivatives, quinolinium derivatives, thalidomide derivatives, naphthalimide derivatives and quinoline derivatives, preferably functionalized with acridine.

10. The scaffold of claim 1 and the system of claims 2 and 3, wherein the polymer is functionalized with acridine present in an amount of 3-35%.

11. The scaffold of claim 1 and the system of claims 2 and 3, wherein the cationic polymer has intercalation properties and positive charge properties.

12. The scaffold of claim 1 and the system of claims 2 and 3, wherein the scaffold is in the form of a sheet, bead, bag, insertion device, tube, etc., preferably in the form of a nanofiber sheet.

13. 4. The scaffold of claim 1 and the system of claims 2 and 3, wherein the stored cells are selected from red blood cells (RBC), white blood cells (WBC), platelets and stem cells, preferably red blood cells (RBC).

14. 1. A method for processing preserved cells, comprising: The method, wherein the preserved cells are placed in contact with the scaffold of any one of claims 1, 4 to 13, or in the system of claims 2 or 3, 4 to 13.

15. 1. A method for removing damage associated molecular patterns (DAMPs) from preserved cells, comprising: The method, wherein the preserved cells are treated by a scaffold according to any one of claims 1, 4-13, or in a system according to claims 2 or 3, 4-13.

16. 1. A method for improving the quality and shelf life of preserved cells by removing DAMPs from the preserved cells, comprising: The method, wherein the preserved cells are treated by a scaffold according to any one of claims 1, 4-13, or in a system according to claims 2 or 3, 4-13.

17. 1. A method for improving cell membrane integrity of preserved cells by removing DAMPs from the preserved cells, comprising: The method, wherein the preserved cells are treated by a scaffold according to any one of claims 1, 4-13, or in a system according to claims 2 or 3, 4-13.

18. The method according to any one of claims 15 to 17, wherein the DAMPs are extracellular DNA, histones, nucleosomes, cell-free hemoglobin, iron, proteins and polyunsaturated fatty acids.

19. Use of a scaffold according to any one of claims 1, 4 to 13 in the treatment of stored cells, comprising: Use comprising the step of passing said preserved cells through said scaffold.

20. A scaffold according to any one of claims 1, 4 to 13, instructions for passage of the preserved cells through the scaffold; Including the kit.

21. The method, use or kit according to claims 15 to 21, wherein said stored cells are red blood cells (RBC), white blood cells (WBC), platelets and stem cells, preferably red blood cells (RBC).

22. (a) preparing a solution of a polymer functionalized with groups selected from anionic, cationic or zwitterionic groups; (b) electrospinning the solution to obtain a nanofibrous scaffold; A method for preparing the nanofibrous scaffold of any one of claims 1, 4 to 13, comprising:

23. 24. The method of claim 23, wherein the polymer is rendered hydrophobic prior to being functionalized with groups selected from anionic, cationic or zwitterionic groups.

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