Method for producing extracellular vesicles, extracellular vesicles, and their use

Abstract

The present invention provides a method for producing extracellular vesicles (EVs), comprising incubating or culturing EV-producing cells in a culture medium. The present invention also provides compositions comprising populations and vesicles of EVs, as well as methods and uses thereof.

Description

[Technical Field]

[0001] The present invention provides a method for producing extracellular vesicles (EVs), comprising incubating or culturing EV-producing cells in a culture medium. The present invention also provides compositions comprising populations and vesicles of EVs, as well as methods and uses thereof. [Background technology]

[0002] There is a lack of treatment options for neurodegenerative diseases. The treatments available for neurodegenerative diseases primarily treat the symptoms, but not the underlying causes. Furthermore, there are currently no treatments for the majority of neurodegenerative diseases.

[0003] Neurodegeneration is a term that covers diseases or disorders involving the loss of neurons. Neurodegenerative diseases can occur in the brain or in other tissues or organs that contain neurons, such as the retina. In the retina, progressive neurological loss is typically referred to as retinal degenerative disease. A common form of retinal degeneration is age-related macular degeneration (AMD). AMD is a leading cause of blindness and affects more than 200 million people worldwide. Dry or atrophic AMD is a common condition affecting a significant portion of the elderly population worldwide. Causes of dry AMD include familial genetics and environmental factors, but there is growing recognition that oxidative stress and inflammation may play a role in the development and acceleration of eye diseases such as AMD, diabetic retinopathy, and retinal vein occlusion. With the exception of SYFOVRE by Apellis and IZERVAY by Astellas, recently approved by the FDA, there are no treatments for dry AMD, leaving patients with limited options for managing their condition. Existing treatment options for ocular diseases, including AMD, are limited to monthly, expensive, and invasive intravitreal injections that must be administered by an ophthalmologist. Adverse immune responses have been reported with such treatments.

[0004] The treatment of neurodegenerative diseases in the brain faces similar challenges to those faced in the treatment of retinal degeneration. These include the immune privileged environment and the blood-brain barrier (BBB) ​​(similar to the blood-retinal barrier (BRB)), which prevent systemic uptake of larger therapeutic molecules. Extracellular vesicles (EVs) show promise for the treatment of degenerative diseases in both organs, given that their small nanosize allows them to permeate the BBB and BRB and potentially be used to deliver therapeutic drugs to the brain and eyes. Furthermore, unlike standard synthetic encapsulation agents that induce immune responses, naturally supplied EVs can be utilized as low-immunogenic delivery vehicles. The contribution of EVs to disease onset and progression is well established in many neurodegenerative diseases, often involving alterations in cellular communication pathways or EV cargo.

[0005] Therefore, improved treatments for neurodegenerative diseases and / or ocular diseases or ocular disorders are needed. [Overview of the Initiative]

[0006] The inventors have developed a method for producing extracellular vesicles (EVs), comprising incubating or culturing EV-producing cells in a culture medium, wherein the extracellular vesicles possess advantageous properties. The inventors have also developed populations of EVs and compositions containing EVs, as well as methods and uses thereof.

[0007] In one embodiment, the present invention provides a method for producing extracellular vesicles (EVs), comprising incubating or culturing EV-producing cells in a medium containing an antioxidant and / or N1 medium component or equivalent thereof.

[0008] In one embodiment, the present invention provides a population of cell-derived EVs containing increased levels of one or both endogenous and exogenous antioxidants.

[0009] In one embodiment, the present invention provides a population of EVs produced by the method described herein.

[0010] In one embodiment, the present invention provides a population of EVs produced by the method described herein, the EVs having increased levels of one or both of endogenous and exogenous antioxidants.

[0011] In one embodiment, the present invention provides a composition comprising EVs produced by the method described herein, or a population of EVs described herein, for use in treating and / or preventing a disease or condition in a subject.

[0012] In one embodiment, the present invention provides a composition comprising EVs produced by the method described herein, or a population of EVs described herein, for use in reducing oxidative stress and / or inflammation in a subject.

[0013] In one embodiment, the present invention provides a composition comprising EVs produced by the method described herein, or a population of EVs described herein, for use in treating and / or preventing neurodegeneration in a subject.

[0014] In one embodiment, the present invention provides a method for treating and / or preventing neurodegeneration in a subject, comprising administering an EV produced by the method described herein, or a population of EVs described herein.

[0015] In some embodiments, the present invention provides the use of EVs produced by the methods described herein, or populations of EVs described herein, for the treatment and / or prevention of neurodegeneration in a subject.

[0016] In one embodiment, the present invention provides the use of EVs produced by the method described herein, or a population of EVs described herein, in the manufacture of a medicament for treating and / or preventing neurodegeneration in a subject.

[0017] In one aspect, the present invention provides a composition comprising extracellular vesicles (EVs) produced by incubating or culturing EV-producing cells in a medium comprising an antioxidant and / or an N1 medium component or an equivalent thereof for use in reducing oxidative stress and / or inflammation in the eye of a subject.

[0018] In one aspect, the present invention provides a composition comprising EVs produced by the methods described herein or a population of EVs described herein for use in reducing oxidative stress and / or inflammation in the eye of a subject.

[0019] In one aspect, the present invention provides a method of reducing oxidative stress and / or inflammation in the eye of a subject, the method comprising administering to the subject EVs produced by the methods described herein or a population of EVs described herein.

[0020] In one aspect, the present invention provides the use of EVs produced by the methods described herein or a population of EVs described herein for reducing oxidative stress and / or inflammation in the eye of a subject.

[0021] In one aspect, the present invention provides the use of EVs produced by the methods described herein or a population of EVs described herein in the manufacture of a medicament for reducing oxidative stress and / or inflammation in the eye of a subject.

[0022] In one aspect, the present invention provides a composition comprising EVs produced by incubating or culturing EV-producing cells in a medium comprising an antioxidant and / or an N1 medium component or an equivalent thereof for use in treating and / or preventing neurodegeneration in a subject.

[0023] In one embodiment, the present invention provides a composition comprising EVs produced by incubating or culturing EV-producing cells in a medium containing antioxidants and / or N1 medium components or equivalents, for use in treating and / or preventing retinal degeneration in a subject.

[0024] In one embodiment, the present invention provides a composition comprising EVs produced by the method described herein, or a population of EVs described herein, for use in treating and / or preventing retinal degeneration in a subject.

[0025] In one embodiment, the present invention provides a method for treating and / or preventing retinal degeneration in a subject, comprising administering an EV produced by the method described herein, or a population of EVs described herein.

[0026] In some embodiments, the present invention provides the use of EVs produced by the methods described herein, or populations of EVs described herein, for the treatment and / or prevention of retinal degeneration in a subject.

[0027] In one embodiment, the present invention provides the use of EVs produced by the method described herein, or a population of EVs described herein, in the manufacture of a medicament for treating and / or preventing retinal degeneration in a subject.

[0028] In one embodiment, the present invention provides a method for producing EVs, comprising incubating or culturing EV-producing cells in a medium containing an antioxidant and / or N1 medium component or equivalent thereof, or incubating or culturing EV-producing cells and EV-producing cell fragments.

[0029] Any embodiment described herein shall be construed as applicable with modifications to any other embodiment unless otherwise specifically stated. For example, as those skilled in the art will understand, the examples of antioxidants outlined for the methods of the present invention apply equally to the uses and compositions of the present invention.

[0030] The present invention is intended for illustrative purposes only and its scope should not be limited by the specific embodiments described herein. Functionally equivalent products, compositions, and methods, as described herein, are clearly within the scope of the present invention.

[0031] Throughout this specification, unless otherwise specifically stated or required by the context, any reference to a single step, composition of a substance, group of steps, or group of compositions of a substance shall be construed as encompassing one or more (i.e., one or more) of those steps, compositions of a substance, groups of steps, or groups of compositions of a substance.

[0032] The present invention is described below by the following non-limiting embodiments and with reference to the accompanying drawings. [Brief explanation of the drawing]

[0033] [Figure 1] Typical ERG waveforms. ERG waveforms are labeled with a-wave amplitude and latency, and b-wave amplitude and latency. [Figure 2-1]Isolation and characterization of RBC-EVs. (A) Experimental paradigm for isolation and incubation conditions of RBC-EVs. (B) Cryo-EM imaging of RBC-EVs. (C) Size distribution of RBC-EVs from cryo-EM images (N=72). (D-E) Western blots for the cell marker calnexin (CNX), the EV marker tumor susceptibility gene 101 (TSG101), and ALIX, as well as the reference protein GAPDH. (D) The CNX (90kDa) band was present in retinal lysates but not in RBC-EV lysates. (D-E) TSG101 (50kDa) was present in retinal, RBC, and RBC-EV lysates, and (E) ALIX (100kDa) and GAPDH (37kDa) were present in RBC and RBC-EV lysates. Scale = 200nm. [Figure 2-2] Same as above. [Figure 3-1] RBC-EV supplementation comparison regarding RBC health and RBC-EV quality. (A) RBC health (N=2-4) in incubation with or without N1 and SOD supplementation, with a 10% abnormality cutoff. (B) RBC-EV size distribution profiles measured using nanotracking analysis (Nanosight NS300). (C) Summary data showing EV count, abnormality %, and size per 1 mL of blood under different supplementation methods. (D) Mean EV count (N=13), (E) average mean and modal size (N-13), and (F) size distribution profile (N=3) were consistent across multiple batches of RBC cultures using combined N1 and SOD supplementation. [Figure 3-2] Same as above. [Figure 4-1]Therapeutic potential of topical RBC-EV delivery with or without N1 and SOD supplementation. (A) Experimental paradigm for intravitreal injection of RBC-EV and control. (B) Retinal function, as measured by ERG, shows conserved a-wave function for PBS(N1 / SOD) and RBC-EV(N1 / SOD) and (C) significantly conserved b-wave function compared to control without supplement (P<0.05, N=5). (D) TUNEL+ cells in ONL were significantly reduced in the supplement group compared to control, but (E) photoreceptor conservation was found to be significantly increased in RBC-EV(N1 / SOD) compared to the PBS(N1 / SOD) group. (F) IBA-1+ cells were significantly downregulated in RBC-EV(N1 / SOD) mice compared to PBS control (P<0.05, N=5). Compared to the PBS control, no change in the number of IBA-1+ cells was observed in the PBS(N1 / SOD) or RBC-EV groups. Representative confocal images show (G) reduced TUNEL+ cells in ONL in the supplement group, as well as photoreceptor conservation in the RBC-EV(N1 / SOD group), and (H) reduced IBA-1+ cells (arrow) in RBC-EV(N1 / SOD) injected mice (P<0.05, N=5). Scale = 50 μM. [Figure 4-2] Same as above. [Figure 5-1]SOD, as well as combined supplementation with N1 and SOD, provides protection against photooxidative damage in a retinal neurodegeneration model. (A) Retinal function, as measured by ERG, showed no changes in wave a and (B) wave function (P>0.05, N=5). (C) TUNEL+ cells in ONL were significantly reduced in the RBC-EV(SOD) and RBC-EV(N1 / SOD) groups compared to RBC-EV and RBC-EV(N1) injected mice, while (D) photoreceptor conservation was significantly increased in RBC-EV(N1 / SOD) injected mice. (E) IBA-1+ cells were significantly downregulated in RBC-EV(SOD) mice (P<0.05, N=5), with no differences observed among the other groups (P>0.05, N=5). (F) Overall retinal thickness was significantly increased between RBC-EV and RBC-EV(N1 / SOD) injected mice (P<0.05, N=5). Representative (G) fundus and (H) OCT images show improved retinal health in the RBC-EV(SOD) and RBC-EV(N1 / SOD) groups. [Figure 5-2] Same as above. [Figure 6-1] Safety of local RBC-EV(N1 / SOD) delivery. (A) Experimental paradigm. (B) Retinal function for both wave a response and (C) wave b response was unchanged compared to the PBS-injected control. Furthermore, there were no significant differences in (D) TUNEL+ cells in the ONL, (E) the number of photoreceptor rows, or (F) the presence of IBA-1+ microglia in the lateral retina. (G-H) Representative confocal images showed no difference between groups in TUNEL+ cells and IBA-1+ cells in the lateral retina. (N=5, P>0.05). Scale = 50 μM. [Figure 6-2] Same as above. [Figure 7-1]Therapeutic potential of local RBC-EV delivery. (A) Experimental paradigm for intravitreal injection of RBC-EV(N1 / SOD). Retinal function, measured by ERG, shows (B) significantly conserved a-wave function and (C) significantly conserved b-wave function compared to PBS control. (D) TUNEL+ cells in ONL were significantly reduced in RBC-EV(N1 / SOD) compared to control, while (E) photoreceptor conservation was found to be significantly increased in RBC-EV(N1 / SOD) compared to PBS. (F) IBA-1+ microglia were found to be significantly reduced in RBC-EV(N1 / SOD) mice compared to PBS control. (G-H) Representative confocal images show reduced photoreceptor cell death and the presence of inflammatory cells in the lateral retina of the RBC-EV(N1 / SOD) group. (N=5, P<0.05). Scale=50μM. [Figure 7-2] Same as above. [Figure 8-1] Safety of whole-body RBC-EV(N1 / SOD) delivery. (A) Experimental paradigm. (B) Retinal function for both wave a response and (C) wave b response was unchanged compared to PBS-injected controls. Furthermore, there were no significant differences in (D) TUNEL+ cells in the ONL, (E) the number of photoreceptor rows, or (F) the presence of IBA-1+ cells in the outer retina between groups. (G~H) Representative confocal images showed no differences between groups in TUNEL+ cells and IBA-1+ cells. (N=5, P>0.05). ONL = outer granular layer. Scale = 50 μM. [Figure 8-2] Same as above. [Figure 9-1]Therapeutic potential of systemic RBC-EV delivery. (A) Experimental paradigm for intraperitoneal injection of RBC-EV(N1 / SOD). Retinal function, as measured by ERG, shows (B) significantly conserved a-wave function and (C) significantly conserved b-wave function compared to PBS control (N=5, P<0.05). (D) TUNEL+ cells in the ONL were significantly reduced in RBC-EV(N1 / SOD) compared to control, while (E) photoreceptor conservation was found to be significantly increased in RBC-EV(N1 / SOD) compared to PBS. (F) IBA-1+ microglia were found to be significantly reduced in RBC-EV(N1 / SOD) mice compared to PBS control. (G) Representative confocal images show reduced TUNEL+ cells in the ONL and conserved photoreceptor thickness in RBC-EV(N1 / SOD) injected mice compared to PBS, as well as (H) reduced IBA-1+ cells in the lateral retina. (N=15, P<0.05). ONL = outer granular layer. Scale = 50 μM. [Figure 9-2] Same as above. [Figure 10-1] RBC-EV(N1 / SOD) uptake. (A) Experimental paradigms for in vitro and in vivo RBC-EV(N1 / SOD) uptake in retinal cells. (B) SYTO-labeled RBC-EV(N1 / SOD) uptake in retinal cell lines 661W, BV2, MIOM1, and aRPE19 after 24-hour incubation compared to SYTO-PBS control. (C) Transfection efficiency of SYTO-labeled RBC-EV(N1 / SOD) over 24 hours, measured using IncuCyte® ZOOM. Technical reproducibility N=3. (D) In ​​vivo SYTO-labeled RBC-EV(N1 / SOD) uptake after intravitreous delivery over 7 days shows progressive radial uptake from the superior retinal delivery site and penetration through all retinal layers over 7 days, and into the superior and inferior retina. (N=1) ONL = outer granular layer, INL = inner granular layer, GCL = ganglion cell layer. Scale bars = 100 μm and 50 μm. [Figure 10-2] Same as above. [Figure 11-1]Loading capacity of RBC-EV(N1 / SOD). (A) Experimental paradigm for miRNA loading setup using electroporation. (B) Paradigm for miRNA quantification using output measurement and Qubit miRNA assay. (C~F) Optimized electroporation settings showed that (C) miRNA retention was optimal with 350V, 125μF, 1 pulse (Gene Pulser), and (D) post-electroporation incubation at 37C in BioRad Gene Pulser buffer. (E) miRNA retention did not increase with increased miRNA input loading. (F) Cryo-EM shows that EV stability was maintained after electroporation. Scale = 200nm. (N=3). [Figure 11-2] Same as above. [Figure 12] N1 / SOD supplementation enhances EV production in vitro in 661w photoreceptor cells. (A) Experimental paradigms for 661w supplement incubation, in vitro photooxidative damage, and EV collection. (B) Nanoparticle tracking analysis (ZetaView x30) shows similar size distribution profiles for 661w-EV and 661w-EV(N1 / SOD) (N=6). (C) The concentration of 661w-EV(N1 / SOD) after 18 hours of incubation was significantly higher in the 661w-EV(N1 / SOD) group compared to the control (P<0.05, N=6). (D) No significant difference in mode size was observed between the groups (P>0.05, N=6). (E) 661w incubated with N1 / SOD preserved more morphology and reduced cell death than the control after 2 hours of photooxidative damage (N=3). [Figure 13] The antioxidant capacity of RBCs is increased by N1 / SOD supplementation. (A) Assay design in which increased SOD activity directly correlates with the presence of increased ROS. (B) RBC(N1), RBC(SOD), and RBC(N1 / SOD) had significantly reduced SOD activity compared to RBC(PBS) control (P<0.05, N=3). [Figure 14]Assessment of RBC health and quality after N1 / SOD incubation. RBCs were evaluated using the ImageJ plugin for morphological changes (A) before culture and (B) after overnight incubation at 37C with N1 / SOD supplementation. (C-F) The number of abnormalities (tear droplet shape, size / shape variation, and presence of burr cells) was counted and calculated as a percentage of normal RBCs. [Figure 15] Fundus health after intravitreal injection. (A) Representative fundus images taken using MICRON IV show fundus health after intravitreal injection of PBS or RBC+ / - combination supplementation, compared to PBS. (B) PBS(N1 / SOD) fundus images show localized hemorrhage near the optic nerve in all mice. [Figure 16-1] RBC-EV(N1 / SOD) provides protection against sodium iodate-induced retinal degeneration. (A) Experimental paradigm. (B)(C) Representative ERG traces for the control and treatment groups, showing that wave a protection in retinal function was observed in RBC-EV(N1 / SOD) injected mice (P<0.05), but (D) there was no change in wave b response between groups (P>0.05). (E) No change in photoreceptor cell death was observed when measured by TUNEL (P>0.05); however, (F) a significant reduction in the total number of IBA-1+ immune cells in the retina was measured (P<0.05). N=4~6. [Figure 16-2] Same as above. [Figure 17-1] RBC-EV(N1 / SOD) as a safe and effective therapeutic agent for the neuroinflammatory features of Parkinson's disease. (A) Experimental paradigm. (B) Western blotting for TH and IBA-1 showed (Bii) no change in TH expression in the control and degenerated state groups (P>0.05), but (Biii) a significant reduction in IBA-1 levels in 6-OHDA / RBC-EV(N1 / SOD) injected mice compared to 6-OHDA / PBS controls (P<0.05). (Ci and ii) No changes were observed in behavioral measures of rotarod or grip strength between either group (P>0.05). N=3~4. [Figure 17-2] Same as above. [Figure 17-3] Same as above. [Figure 18] RBC-EV(N1 / SOD) performs better than commercially available competitor RBC-EV products in protecting against retinal degeneration in vivo. After 5 days of photooxidative damage, retinal function was significantly improved in mice injected with RBC-EV(Comp) and vehicle (V CVR) for both (A) a-wave and (B) b-wave measurements (P<0.05). Retinal thickness measurements on OCT images showed significant ONL and overall retinal thickness in RBC-EV(CVR)-treated mice compared to the V(CVR) and RBC-EV(Comp) groups (C-E) (P<0.05). Photoreceptor survival was demonstrated by (F) a significantly increased number of photoreceptor cell rows in V(CVR) and RBC-EV(CVR)-treated mice compared to the other groups (P<0.05). No significant differences were observed in IBA-1+ cells or TUNEL+ cells in the lateral retina, respectively, as measures of inflammation and cell death (P>0.05). (N=4~5). V=vehicle, CVR=Clear Vision Research, Comp=competitor, ONL=outer granular layer, INL=inner granular layer, GCL+IPL=ganglion cell layer and inner plexiform layer, WR=whole retina. [Figure 19] Antioxidant doses used to test the health of RBCs. Compared to RBCs before treatment, RBC(N1 / SOD) had the lowest percentage of abnormalities among all antioxidants and doses tested. RBCs treated with resveratrol 100 μM, kaempferol (2 μM), and glutathione (0.1 mM) had the lowest percentage of abnormalities among the respective dose ranges tested (dashed lines). N=1~4. [Figure 20]Antioxidant doses were used to test the health and characterization of RBCs and RBC-EVs. (A) RBC health was assessed by the measured percentage of membrane abnormalities, showing a significantly reduced percentage of abnormalities in the RBC(N1), RBC(SOD), RBC(N1 / SOD), and RBC(R 100μM) groups compared to the control before treatment (P<0.05). (B) RBC counts were also measured after 18 hours of incubation with supplementation, with the highest counts observed in the RBC(SOD) and RBC(N1 / SOD) groups (N=1~4). (C) Nanoparticle tracking analysis (Nanosight NS300) was performed to characterize the RBC-EV size distribution, (D) RBC-EV concentration, and (E) RBC-EV mean and mode size, but no significant differences were observed between groups for these measurements. N=3. [Figure 21] Effects of supplementation on the endogenous characteristics of RBCs and RBC-EVs. (A(i)) SOD activity in the RBC(N1 / SOD) and RBC(K 2uM) groups was significantly higher than in the RBC(PBS) control (P<0.05), while (A(ii)) SOD activity in RBC-EV(R 100μM) and RBC-EV(K 2μM) was significantly higher than in the RBC-EV(PBS) control (P<0.05). (B(i)) For RBCs, there was no difference in hemoglobin levels between groups (P>0.05), but (B(ii)) RBC-EV(N1) and RBC-EV(R 100μM) had significantly reduced hemoglobin levels compared to the RBC-EV(PBS) control (P<0.05). N=6. [Figure 22] In vivo RBC-EV (Resv) did not provide protection against retinal degeneration. (A) Experimental paradigm. No changes were observed in retinal function between groups for (B) a-wave or (C) b-wave measurements. (D-E) The ONL was significantly thicker in RBC-EV (Resv) treated mice compared to the PBS (Resv) or PBS groups (P<0.05). No differences were observed in other retinal layers (P>0.05). ONL = outer granular layer, INL = inner granular layer, GCL + IPL = ganglion cell layer and inner plexiform layer, WR = entire retina. N=5. [Figure 23]Human RBC health. (A) Baseline abnormality scores showed approximately 30% RBC membrane abnormalities in mouse RBCs and approximately 40% in human RBCs (N=1-4). (B) Mouse RBC survival rates, assessed by ImageStream analysis, showed a significant decrease in survival rates, measured by the percentage of calcein+RBCs, in the RBC(PBS), RBC(N1), and RBC(N1 / SOD) groups compared to the pre-treatment control (P<0.05, N=3). No changes were observed in the percentage of annexin+ cells (cell death marker) between groups (P>0.05). (C) No significant differences were observed in RBC count or (D) RBC abnormalities among the human RBC supplementation groups, however, the RBC(SOD) and RBC(N1 / SOD) groups had the lowest percentage of abnormalities compared to the pre-treatment control (P>0.05, N=1-3). (E) RBC survival rates, assessed using FACS and ImageStream analysis, showed high survival rates measured by calcein+ cells and low cell death rates measured by annexin V+ cells. No differences were detected between groups compared to pre-treatment controls (P>0.05, N=3). [Figure 24] The health and quality of human RBC-EVs were comparable among the supplementation groups. (A) RBC-EV size distribution profiles using nanoparticle tracking analysis (ZetaView x30). (B) Mean, mode, and concentration did not differ significantly between groups. (N=3, p>0.05). [Figure 25] In vitro photoreceptor EV response to photooxidative damage. (A) Experimental paradigm of in vitro photooxidative damage to 661w photoreceptor and EV response. (B) EV distribution profile using nanotracking analysis (ZetaView x30) shows similar size distribution profiles for 661w-EV under dim (5k lux) and PD (25k lux) conditions, but (C) after PD, 661w-EV concentration increased significantly (P<0.050), but (D) there was no change in mode size (P>0.05). N=3. [Figure 26-1]SOD and N1 / SOD supplementation confers protection to 661w photoreceptor cells in vitro. (A) Experimental paradigm. (B(i)-(iv)) Cytotoxicity assays were performed on 661w cells after 18-hour incubation with supplementation (N1, 1:100; SOD, 1:500; N1 / SOD, R 100 μM, K 2 μM, and G 0.01 mM), before photooxidative damage (PD) (0 hours), as well as at 2, 3, and 4 hours. (B(v)-(vi)) Comparative analysis across all time points showed the lowest toxicity in the PBS, SOD, and N1 / SOD treatment groups compared to the untreated control. N=6. [Figure 26-2] Same as above. [Figure 27] SOD and N1 / SOD supplementation dose response to photooxidative damage in vitro. (A) Experimental paradigm. (B(i)-(iii)) Cytotoxicity assays were performed on 661w before (0 hours) and 2 hours after photooxidative damage (PD) following an 18-hour incubation with supplementation (N1, 1:200; SOD, 1:1000; N1 / SOD, R 50 μM, K 1 μM, and G 0.005 mM). Recovery time was also investigated 24 hours after 2 hours of PD. (B(iv)) Comparative analysis across all time points showed the lowest toxicity in the SOD-treated group compared to untreated controls. N=6. [Figure 28] Effect of photoreceptor EV with supplementation on photooxidative damage. (A) Experimental paradigm. (B) Retinal function measured by ERG showed no significant difference between groups in (B) wave a or (C) wave responses (P>0.05). (D~E) Retinal thickness measurements showed a significant increase in ONL thickness between the 661w-EV (N1 / SOD) group and the 661w-EV group, indicating protection after N1 / SOD supplementation (P<0.05). N=5. [Figure 29]Effect of photoreceptor EV with supplementation on photooxidative damage. (A) Experimental paradigm. (B) Retinal function measured by ERG showed no significant difference between groups in (B) wave a or (C) wave responses (P>0.05). (D~F) Retinal thickness measurements showed no significant increase in ONL thickness, TUNEL, or photoreceptor count between the 661w-EV(N1 / SOD) group and the PBS group (P>0.05). N=5. [Figure 30-1] RBC-EV has a different proteomics profile compared to RBC. (A) PCA of RBC vs. RBC-EV. (B) Cellular compartment of RBC-EV shows terms related to extracellular vesicles. Heatmaps show the enrichment of (C) EV cargo and (D) EV membrane proteins in RBC-EV compared to RBC. Pathway analysis of enriched proteins in RBC-EV compared to (E(i)) RBC-EV, (E(ii)) RBC, and (E(iii)) RBC, with downregulated proteins. N=3, P<0.05. [Figure 30-2] Same as above. [Figure 30-3] Same as above. [Figure 30-4] Same as above. [Figure 30-5] Same as above. [Figure 30-6] Same as above. [Figure 31-1] N1 / SOD supplementation confers a unique immunomodulatory signature to RBCs. (A) Number of proteins per sample. (B) PCA showing different clustering between groups. (C) Heatmap of differentially expressed proteins between the Pre-Tx and PBS groups. (D) Pathway analysis of differentially expressed proteins shows enrichment of hemostatic and immune pathways in the RBC(PBS) group compared to the Pre-Tx control. (E) Uniquely expressed proteins in each supplement group show that RBC(N1) samples have the most unique proteins compared to the other groups. (F) Pathway analysis of proteins within each supplement group compared to the PBS and Pre-Tx groups. N=3, P<0.05. [Figure 31-2] Same as above. [Figure 31-3] Same as above. [Figure 32-1] N1 / SOD supplementation confers immunomodulatory properties to RBC-EV. (A) Number of proteins in each RBC-EV sample. (B) UMAP showing different clusterings between sample groups. (C) Venn diagram showing uniquely expressed proteins within each group, highlighting the most unique proteins in RBC-EV(N1 / SOD). (D) Heatmap showing numerous downregulated proteins in RBC-EV(N1 / SOD) samples compared to other groups; pathway analysis indicates these proteins were associated with immune processes and transcriptional regulation. N=3, P<0.05. [Figure 32-2] Same as above. [Figure 32-3] Same as above. [Figure 32-4] Same as above. [Figure 33-1] Supplementation did not alter the microRNA signature of RBC-EV. (A) MicroRNA abundance in the sample. (B) Abundance of the top 11 microRNAs. (C) Pathway analysis of enriched microRNAs. (D) Venn diagram showing no unique microRNAs between supplement groups. N=3, P<0.05. [Figure 33-2] Same as above. [Figure 34] RBC-EV promoted a dose-dependent increase in PBMC survival. The dose-dependent response of increased PBMC survival was observed after RBC-EV incubation in all groups under both (A) control and (B) LPS-stimulated conditions. N=1, performed as biological double replicates. The red dotted line indicates baseline (culture medium). [Figure 35-1] RBC-EV(N1 / SOD) did not induce an inflammatory response under control conditions. (A-H) Cytokine output after 48 hours of incubation of PBMCs with RBC-EV. N=1, performed in biological double replicates. The red dotted line shows baseline (culture medium). The dotted line indicates values ​​below LLOQ. The gold dotted line indicates values ​​above ULOQ. [Figure 35-2] Same as above. [Figure 35-3] Same as above. [Figure 35-4] Same as above. [Figure 36-1]RBC-EV dose-dependently reduces inflammatory cytokine production in PBMCs. (A-F) Cytokine output after 48 hours of incubation of LPS-stimulated PBMCs with RBC-EV. N=1, performed in biological double replicates. The red dotted line represents baseline (culture medium). The dotted line indicates values ​​below LLOQ. The gold dotted line indicates values ​​above ULOQ. [Figure 36-2] Same as above. [Figure 37] A gating strategy was applied to isolate populations of TER119+, CD41+, CD71+, and CD45+ cells, as shown in Figure 37A. This analysis showed that mature RBCs and reticulocytes together constituted approximately 83% of the total cell population. Additionally, as illustrated in Figure 37B, CD41+ platelets represented 15–19%, and leukocytes accounted for less than 0.04% of the cells. This suggests that the EV population induced after incubation consisted of both RBC and platelet-derived vesicles. [Modes for carrying out the invention]

[0034] General techniques and definitions Unless otherwise specifically defined, all technical and scientific terms used herein shall be construed to have the same meaning as those generally understood by those skilled in the art (e.g., extracellular vesicles, proteins, neurodegeneration, and / or ocular diseases).

[0035] The term "and / or," for example, "X and / or Y," shall be understood to mean either "X and Y" or "X or Y," and shall be interpreted as providing explicit support for both meanings or either meaning.

[0036] Throughout this specification, the word “comprise,” or variations such as “comprises,” or “comprises,” implies the inclusion of the specified component, element, or step, or group of components, elements, or steps, but it should be understood that this does not exclude any other component, element, or step, or group of components, elements, or steps.

[0037] As used herein, the term “approximately” means + / - 10%, more preferably + / - 5%, and even more preferably + / - 1% of the specified value, unless otherwise specified.

[0038] As used herein, the term “subject” refers to any animal. In one example, an animal is a vertebrate. For example, an animal is a mammal, a bird, a chordate, an amphibian, or a reptile. In one embodiment, the subject is a mammal. In a preferred embodiment, the mammal is a human. In one embodiment, the subject is a model animal such as a mouse, rat, or guinea pig. In one embodiment, the subject is a mouse. In one embodiment, the mammal may be a companion animal such as a cat, dog, or horse. In one embodiment, the mammal may be a domestic animal such as a pig, cattle, horse, goat, sheep, and deer.

[0039] As used herein, the terms “increase” or “increases” or “increased” or “to increase” refer to having a given parameter at a higher or greater level compared to a control or baseline.

[0040] As used herein, the terms “reduce,” “reduces,” or “to reduce” refer to having a given parameter at a lower level compared to a control or baseline.

[0041] As used herein, the terms “to treat,” “to cure,” or “to treat” mean to obtain, at least partially, a desired therapeutic outcome. In one embodiment, treating includes preventing or delaying the onset of one or more symptoms of a disease or condition. In one embodiment, treating includes preventing or reducing the onset of one or more symptoms of a disease or condition.

[0042] As used herein, the terms “prevent,” “prevent,” or “prevention” mean reducing the likelihood of developing a disease or condition. Prevention does not have to be complete and does not imply that the subject will never develop a disease or condition.

[0043] As used herein, the term “population of extracellular vesicles” refers to any number, group, or collection of extracellular vesicles described herein, for example, the number or group of extracellular vesicles in a given solution.

[0044] As used herein, the term “endogenous” means originating within a cell or extracellular matrix (EV), for example, being produced within a cell or EV.

[0045] As used herein, the term “exogenous” means external to or originating from the outside of a cell or EV, for example, produced externally to a cell or EV.

[0046] Extracellular vesicles As used herein, the term “extracellular vesicle” or “EV” refers to a lipid-bound vesicle (lipid bilayer) secreted by a cell into the extracellular environment. EVs can carry cargo such as proteins, nucleic acids, lipids, and metabolites. In some embodiments, the EVs of the present invention are selected from microvesicles, exosomes, and apoptotic bodies. Microvesicles, exosomes, and apoptotic bodies are defined, for example, by Nederveen et al (2021) and Dellar et al (2022).

[0047] In one embodiment, the EV of the present invention has an average diameter of about 1,000 nm or less. In another embodiment, the EV of the present invention has an average diameter of about 800 nm or less. In yet another embodiment, the EV of the present invention has an average diameter of about 600 nm or less. In yet another embodiment, the EV of the present invention has an average diameter of about 500 nm or less. In yet another embodiment, the EV of the present invention has an average diameter of about 400 nm or less.

[0048] In one embodiment, the EV of the present invention has an average diameter of about 20 nm to about 1000 nm, or about 20 nm to about 800 nm, or about 20 nm to about 600 nm, or about 30 nm to about 600 nm, or about 40 nm to about 400 nm, or about 30 nm to about 400 nm, or about 30 nm to about 350 nm, or about 40 nm to about 200 nm, or about 30 nm to about 200 nm. In one embodiment, the EV has an average diameter of about 20 nm to about 600 nm. In one embodiment, the EV has an average diameter of about 40 nm to about 600 nm. In one embodiment, the EV has an average diameter of about 40 nm to about 400 nm. In one embodiment, the EV has an average diameter of about 40 nm to about 200 nm. In one embodiment, the EV has an average diameter of about 200 nm.

[0049] In one embodiment, approximately 85% of the EVs have a diameter of approximately 200 nm or less. In another embodiment, approximately 80% of the EVs have a diameter of approximately 200 nm or less. In yet another embodiment, approximately 75% of the EVs have a diameter of approximately 200 nm or less.

[0050] In one embodiment, about 60% to about 70% of the EVs have a diameter of about 100 to about 200 nm. In another embodiment, about 65% of the EVs have a diameter of about 100 to about 200 nm. In yet another embodiment, about 75% to about 80% of the EVs have a diameter of about 90 to about 220 nm.

[0051] In some embodiments, the EV of the present invention has advantageous properties for treating or preventing a disease or condition selected from the group including neurodegeneration, oxidative stress, and inflammation. In some embodiments, neurodegeneration is retinal degeneration. In some embodiments, the EV of the present invention has advantageous properties for treating oxidative stress and / or inflammation in the eye. In some embodiments, the advantageous properties include one or more of the following: a favorable safety profile, biocompatibility, non-immunogenicity, ease of manufacture on a clinically relevant scale, and systemic delivery. As used herein, “biocompatibility” means that the EV is autologous or from an object having the same blood type (O, A, B, AB) as the EV recipient.

[0052] In some embodiments, the EVs of the present invention are autologous. As used herein, “autologous” means obtained from the cells of the subject itself (for example, the EVs are for administration to a subject from which the EV-producing cells originate).

[0053] In some embodiments, the EVs of the present invention are heterogeneous. As used herein, “heterogeneous” means obtained from different subjects of the same species (for example, the EVs are intended for administration to subjects from which the EV-producing cells did not originate).

[0054] In some embodiments, the extracellular viable cells (EVs) of the present invention are allogeneic. As used herein, “allogeneic” means obtained from donor-matched subjects of the same species. In some embodiments, donor-matched subjects are subjects that match on one or more blood or immunomarkers. In some embodiments, donor-matched subjects are subjects that match or partially match on human leukocyte antigen (HLA).

[0055] In some embodiments, the EVs described herein have anti-inflammatory properties. As used herein, “anti-inflammatory” means reducing the expression of one or more cytokines, chemokines, and / or inflammatory mediators in target cells. In some embodiments, the cells are peripheral bone mononuclear cells. In some embodiments, the cells are target cells described herein or in target tissue.

[0056] In some embodiments, the EV of the present invention reduces the expression of one or more cytokines, chemokines, and / or inflammatory mediators. In some embodiments, the cytokine is selected from MIP-1α(Ccl3), IL-1β, IL-6, IL-8, IL-10, TNFα, MCP-1(Ccl2), and IL-1α. In some embodiments, the cytokine is MIP-1α. In some embodiments, the cytokine is IL-1β. In some embodiments, the cytokine is IL-6. In some embodiments, the cytokine is IL-8. In some embodiments, the cytokine is IL-10. In some embodiments, the cytokine is TNFα. In some embodiments, the cytokine is MCP-1. In some embodiments, the cytokine is IL-1α.

[0057] In some embodiments, the chemokine is MIP-1a or MCP-1. In some embodiments, the chemokine is MIP-1a. In some embodiments, the chemokine is MIP-1a.

[0058] In some embodiments, the EV of the present invention reduces the expression of one or more of MIP-1α(Ccl3), IL-6, IL-8, and MCP-1(Ccl2).

[0059] In some embodiments, the EV of the present invention reduces microglial activation in the subject.

[0060] In some embodiments, the EV of the present invention modulates the expression of one or more proteins in Table 1 compared to a control. In some embodiments, the EV of the present invention increases or decreases the expression of one or more proteins in Table 1 compared to a control.

[0061] As used herein, the term “control” refers to a reference point for quantification. The reference point may be an internal reference (i.e., from the same source) or from an established dataset (i.e., matched by source, cell type, EV type, supplementation type, or activation state). Suitable controls will be obvious to those skilled in the art and / or will be described herein. In some embodiments, the control is a sample of EVs obtained from EV-producing cells not cultured in N1, SOD, or N1 / SOD. In some embodiments, the control is a sample of EVs obtained from EV-producing cells not cultured in N1. In some embodiments, the control is a sample of EVs obtained from EV-producing cells not cultured in SOD. In some embodiments, the control is a sample of EVs obtained from EV-producing cells not cultured in N1 / SOD. In some embodiments, the control is a sample of EVs obtained from EV-producing cells cultured in PBS.

[0062] In some embodiments, the EV of the present invention comprises increased expression of one or more proteins compared to a control, where one or more proteins are Arih1, Rpl22, Cfi, Lnpk, Rps17, Rpl23a, Rps21, Cpox, Metap2, Farsb, Rpl18, Myg1, Rps3, Rps15a, Hsp90b1, Phb1, Eef1d, Ddx1, Rps4x, Eif2s3y, Ahsg, Rpl12, Ndufa4, Sacm1l, Rps5, Rpl18a, Rpl14 Selected from Calr, Rpl26, Rplp0, Hyou1, Eno3, Ctse, Aldh1a7, Pdia6, Prkcsh, Clns1a, Rps6, Hace1, Rpl17, Rpl27a, Eprs1, Prxl2a, Trim56, Canx, Npepl1, Pdia3, Ppib, Eef2, Spr, Ngp, Hspa5, Bag2, Snd1, Rangap1, Eno1, Rps14, Tfrc, Thg1l, Pfas, Ppp2r1a, Glo1, and Scamp3.

[0063] In some embodiments, the EV of the present invention comprises increased expression of one or more proteins compared to a control, one or more proteins selected from ARIH1, RPL22, CFI, LNPK, RPS17, RPL23A, RPS21, CPOX, METAP2, and FARSB. In some embodiments, the protein is ARIH1. In some embodiments, the protein is RPL22. In some embodiments, the protein is CFI. In some embodiments, the protein is LNPK. In some embodiments, the protein is RPS17. In some embodiments, the protein is RPL23A. In some embodiments, the protein is RPS21. In some embodiments, the protein is CPOX. In some embodiments, the protein is METAP2. In some embodiments, the protein is FARSB.

[0064] In some embodiments, the EV of the present invention comprises reduced expression of one or more proteins compared to a control, including Fcho2, Crlf3, Psma5, Gspt1, Psmb4, Acp1, Ccdc6, Uros, Psmd3, Tgm2, Psmc2, Psma3, Glrx3, Rnh1, Ppid, Usp25, Gmpr, Usp5, Pgls, Ostf1, Psmb6, Eif5, Tbcb, Oxsr1, Stip1, Usp14, Psmd5, Tollip, Psmd8, Gpi, Otu One or more proteins selected from b1, Sri, Agfg1, Cfap157, Psmd6, Chordc1, Phpt1, Psmd7, Psmd11, Psmd12, Psmd13, Gstm5, Synj1, Sh3glb1, Swap70, H4c1, Pzp, Psmd14, Wnk1, Gdi1, Pf4, Snx15, Coro1c, Ptgr2, Aldoart2, Ighg3, Lzic, Epn1, Pacs1, Skic2, Kyat3, Rnf213A, and Anxa6.

[0065] In some embodiments, the EV of the present invention comprises reduced expression of one or more proteins compared to a control, one or more proteins selected from ANXA6, RNF213, KYAT3, SKIC2, PACS1, EPN1, LZIC, IGGH3, ALDOART2, and PTGR2. In some embodiments, the protein is ANXA6. In some embodiments, the protein is RNF213. In some embodiments, the protein is KYAT3. In some embodiments, the protein is SKIC2. In some embodiments, the protein is PACS1. In some embodiments, the protein is EPN1. In some embodiments, the protein is LZIC. In some embodiments, the protein is IGGH3. In some embodiments, the protein is ALDOART2. In some embodiments, the protein is PTGR2.

[0066] In some embodiments, the EV of the present invention includes downward or upward control of one or more paths in Table 2, compared to a control.

[0067] In some embodiments, the EV of the present invention includes the expression of one or more pathways increased or decreased in Table 2.

[0068] In some embodiments, the EV of the present invention comprises increased or decreased expression of one or more proteins compared to a control, and the one or more proteins mediate one or more pathways shown in Table 2.

[0069] In some embodiments, the EV of the present invention comprises the increased or decreased expression of one or more proteins compared to a control, one or more of which are Rab regulation of transport, membrane-targeting Srp-dependent cotranslating proteins, major pathways of rRNA processing in the nucleolus and cytoplasm, exon junction-independent nonsense mutation-dependent degradation mechanisms, nonsense mutation-dependent degradation mechanisms nmd, raf gefs exchange of gtp to gdp on rab, cytoplasmic ribosomal proteins, trans-Golgi network vesicle budding, formation of a pool of free 40s subunits, Golgi-associated vesicle biosynthesis, MHC class II antigen presentation, Rab geranylgeranylation, ntrk1 Trka-mediated signaling, NTRK-mediated signaling, vesicle-mediated transport, eukaryotic translation initiation, receptor tyrosine kinase-mediated signaling, neurotransmitter receptor and postsynaptic signaling, membrane transport, cratrin-mediated endocytosis, interleukin-1 family signaling, RunX1-mediated transcriptional regulation, hedgehog ligand biosynthesis, hedgehog-on state, ubiquitin-mediated degradation of cdc25a, RunX3-mediated transcriptional regulation, p53 stabilization, RunX1 regulates gene transcription in HSCS differentiation, regulation of RunX3 expression and activity, axin degradation, cross-presentation of soluble exogenous antigen endosomes, asymmetric localization of PCP proteins, Apc C cdh1-mediated degradation of cdc20 target proteins in late mitotic G1, GAPS-mediated regulation of RAS, DVL degradation, KEEP1 nef212 pathway, activation of Apc C and Apc C cdc20-mediated degradation of mitotic proteins, PCP It mediates one or more pathways selected from the following: the ce pathway, transcriptional regulation by runx2, regulation of runx2 expression and activity, regulation of pten stability and activity, processing of gli3 to gli3r by the proteasome, the G2 m checkpoint, Auf1 hnrnp d0 binding to mRNA and destabilizing it, switching of origin to the post-replication state, removal of Orc1 from chromatin, APC c-mediated degradation of cell cycle proteins, the Tnfr2 non-canonical NF KB pathway, Ub-specific processing proteases, and beta-catenin-independent WNT signaling.

[0070] In some embodiments, the EV of the present invention comprises increased expression of one or more proteins compared to a control, one or more proteins mediating one or more pathways selected from Rab regulation of transport, membrane-targeted Srp-dependent cotranslating proteins, major pathways of rRNA processing in the nucleolus and cytoplasm, exon junction-independent nonsense mutation-dependent degradation mechanisms, nonsense mutation-dependent degradation mechanisms nmd, Raf GEFs exchange of GTP to GDP on Rab, cytoplasmic ribosomal proteins, trans-Golgi network vesicle budding, formation of a pool of free 40s subunits, Golgi-associated vesicle biosynthesis, MHC class II antigen presentation, Rab geranylgeranylation, signaling by ntrk1 trka, signaling by ntrk, vesicle-mediated transport, eukaryotic translation initiation, signaling by receptor tyrosine kinases, neurotransmitter receptor and postsynaptic signaling, membrane transport, and cratrin-mediated endocytosis. In some embodiments, the EV of the present invention comprises increased expression of one or more proteins compared to a control, one or more proteins mediating a pathway of Rab regulation of transport. In some embodiments, the EV of the present invention comprises increased expression of one or more proteins compared to a control, wherein one or more proteins mediate a pathway of membrane-targeting Srp-dependent cotranslating proteins. In some embodiments, the EV of the present invention comprises increased expression of one or more proteins compared to a control, wherein one or more proteins mediate a major pathway of rRNA processing in the nucleolus and cytoplasm. In some embodiments, the EV of the present invention comprises increased expression of one or more proteins compared to a control, wherein one or more proteins mediate a pathway of a nonsense mutation-dependent degradation mechanism that is independent of the exon junction complex. In some embodiments, the EV of the present invention comprises increased expression of one or more proteins compared to a control, wherein one or more proteins mediate a pathway of the nonsense mutation-dependent degradation mechanism nmd.

[0071] In some embodiments, the EV of the present invention comprises reduced expression of one or more proteins compared to a control, where one or more proteins are involved in interleukin-1 family signaling, runx1-mediated transcriptional regulation, hedgehog ligand biosynthesis, hedgehog-on state, ubiquitin-mediated degradation of cdc25a, runx3-mediated transcriptional regulation, p53 stabilization, runx1-mediated regulation of gene transcription in HSCS differentiation, regulation of runx3 expression and activity, axin degradation, cross-presentation of soluble exogenous antigen endosomes, asymmetric localization of pcp proteins, Apc c cdh1-mediated degradation of cdc20 target proteins in late mitotic G1, GAPS-mediated regulation of ras, dvl degradation, keap1 nef212 pathway, activation of Apc c and Apc c cdc20-mediated degradation of mitotic proteins, and pcp The EVs mediate one or more pathways selected from the following: the ce pathway, transcriptional regulation by runx2, regulation of runx2 expression and activity, regulation of pten stability and activity, processing of gli3 to gli3r by the proteasome, the G2 m checkpoint, the binding of auf1 hnrnp d0 to mRNA and destabilizing it, the switching of origin to the post-replication state, removal of Orc1 from chromatin, apc c-mediated degradation of cell cycle proteins, the Tnfr2 non-canonical nf kb pathway, Ub-specific processing proteases, and beta-catenin-independent wnt signaling. In some embodiments, the EVs of the present invention include the reduced expression of one or more proteins compared to a control, one or more of which mediate a pathway of interleukin-1 family signaling. In some embodiments, the EVs of the present invention include the reduced expression of one or more proteins compared to a control, one or more of which mediate a pathway of transcriptional regulation by runx1. In one embodiment, the EV of the present invention comprises the reduced expression of one or more proteins compared to a control, the one or more proteins mediating the hedgehog ligand biosynthesis pathway. In another embodiment, the EV of the present invention comprises the reduced expression of one or more proteins compared to a control, the one or more proteins mediating the hedgehog-on state pathway.In some embodiments, the EV of the present invention includes the reduced expression of one or more proteins compared to a control, one or more of which mediates the ubiquitin-mediated degradation pathway of cdc25a. In some embodiments, the EV of the present invention includes the reduced expression of one or more proteins compared to a control, one or more of which mediates the transcriptional regulation pathway by runx3. In some embodiments, the EV of the present invention includes the reduced expression of one or more proteins compared to a control, one or more of which mediates the stabilization pathway of p53. In some embodiments, the EV of the present invention includes the reduced expression of one or more proteins compared to a control, one or more of which mediates the pathway in which runx1 regulates gene transcription in HSCS differentiation. In some embodiments, the EV of the present invention includes the reduced expression of one or more proteins compared to a control, one or more of which mediates the regulation pathway of runx3 expression and activity. In some embodiments, the EV of the present invention includes the reduced expression of one or more proteins compared to a control, one or more of which mediates the degradation pathway of axin. In some embodiments, the EV of the present invention includes the reduced expression of one or more proteins compared to a control, one or more of which mediate the cross-presentation pathway of soluble exogenous antigen endosomes. In some embodiments, the EV of the present invention includes the reduced expression of one or more proteins compared to a control, one or more of which mediate the asymmetric localization pathway of pcp proteins. In some embodiments, the EV of the present invention includes the reduced expression of one or more proteins compared to a control, one or more of which mediate the Apc c cdh1-mediated degradation pathway of cdc20 target proteins in late mitotic G1. In some embodiments, the EV of the present invention includes the reduced expression of one or more proteins compared to a control, one or more of which mediate the RAs regulation pathway by gaps. In some embodiments, the EV of the present invention includes the reduced expression of one or more proteins compared to a control, one or more of which mediate the degradation pathway of dvl. In some embodiments, the EV of the present invention includes the reduced expression of one or more proteins compared to a control, one or more of which mediate the keap1 nef212 pathway.

[0072] In some embodiments, the EV of the present invention comprises exosomes. In some embodiments, the exosomes have a diameter of about 40 to about 200 nm. In some embodiments, the EV of the present invention comprises microvesicles.

[0073] In some embodiments, the microvesicles have a diameter of approximately 40 to 1,000 nm.

[0074] In some embodiments, the EV of the present invention comprises apoptotic bodies. In some embodiments, the apoptotic bodies have a diameter of about 50 to about 5000 nm.

[0075] In some embodiments, EV is not an exosome.

[0076] In some embodiments, the EV of the present invention can be delivered systemically (without the need for an ophthalmologist or anesthesia).

[0077] In some embodiments, the EV of the present invention can be delivered locally (for example, into the vitreous humor).

[0078] In some embodiments, the EV of the present invention is suitable for treating and / or preventing ocular diseases / conditions.

[0079] In some embodiments, the EV of the present invention is suitable for treating and / or preventing neurodegeneration.

[0080] In some embodiments, the neurodegeneration is retinal neurodegeneration.

[0081] In some embodiments, the EV of the present invention is suitable for treating and / or preventing oxidative stress and / or inflammation in the eye of a subject.

[0082] In some embodiments, the EV of the present invention is suitable for treating and / or preventing retinal degeneration in the eye of interest.

[0083] In some embodiments, protective effects (prevention / treatment) are achieved (without loading the therapeutic agent) through both systemic and local delivery routes.

[0084] In one embodiment, the EV described herein has an increased level of one or both of endogenous and exogenous antioxidants.

[0085] In one embodiment, the EV described herein comprises one or more N1 culture medium components or their equivalents.

[0086] In one embodiment, the present invention provides a population of EVs derived from cells containing increased levels of one or both endogenous and exogenous antioxidants, as described herein. In another embodiment, the present invention provides a population of EVs produced by the method described herein, the EVs having increased levels of one or both endogenous and exogenous antioxidants, as described herein.

[0087] In one embodiment, the SOD activity of the EV of the present invention is 1 × 10 11 The SOD activity per EV is approximately 0.3 to 0.4 U / ml. In one embodiment, the SOD activity of EV is 1 × 10⁻⁶ 11 The EV is approximately 0.33 to 0.37 U / ml. EV SOD activity can be measured by any method known to those skilled in the art, for example, the superoxide dismutase colorimetric activity kit ThermoFisher Scientific catalog number EIASODC.

[0088] In some embodiments, when administered systemically or topically, a portion of the EV localizes to the eye. In some embodiments, when administered systemically or topically, a portion of the EV localizes to the retina.

[0089] In some embodiments, when administered systemically or locally, a portion of the EV localizes to the brain. In some embodiments, when administered systemically or locally, a portion of the EV localizes to microglia cells. In some embodiments, when administered systemically or locally, a portion of the EV localizes to glial cells. In some embodiments, when administered systemically or locally, a portion of the EV localizes to immune cells. In some embodiments, when administered systemically or locally, a portion of the EV localizes to neurons.

[0090] As used herein, “a portion” refers to a therapeutically effective number of extravasation doses (EVs).

[0091] In one embodiment, the EV further includes an exogenous cargo.

[0092] In one embodiment, the method for producing EVs described herein increases the quality and / or quantity and / or therapeutic efficacy of the produced EVs.

[0093] As used herein, the phrase “increase the quality of the produced EVs” includes increasing the uniformity of the size of the produced EVs and increasing the levels of one or both of the endogenous and exogenous antioxidants in the EVs. In some embodiments, the quality is increased compared to EVs produced by the methods described in Gangadaran et al (2018), Usman et al (2018), or Chiangjong et al (2021). In some embodiments, the quality is increased compared to EVs produced by a calcium ionophore EV induction method (e.g., described in Chiangjong et al, 2021).

[0094] As used herein, the phrase “uniformity of the size of the produced EVs” refers to the uniformity of the diameter of the produced EVs. The mean and modal diameters of the EVs can be evaluated by methods known in the art, for example, by visual evaluation including microscopy and size distribution analysis. In some embodiments, about 85% of the EVs have a diameter of about 200 nm or less. In some embodiments, about 80% of the EVs have a diameter of about 200 nm or less. In some embodiments, about 75% of the EVs have a diameter of about 200 nm or less. In some embodiments, about 60% to about 70% of the EVs have a diameter of about 100 to about 200 nm. In some embodiments, about 65% of the EVs have a diameter of about 100 to about 200 nm. In some embodiments, about 75% to about 80% of the EVs have a diameter of about 90 to about 220 nm.

[0095] As used herein, the phrase "increase the amount of EVs produced" means increasing the total number of EVs produced for any given population of EV-producing cells of any size compared to the total number of EVs produced at baseline, including a population of equal-sized EV-producing cells produced without the application of the methods and media described herein.

[0096] As used herein, the phrase "increase the effectiveness of the produced EV" means increasing the therapeutic effectiveness of the EV for treating or preventing any disease or condition described herein.

[0097] Extracellular vesicle-producing cells As used herein, the terms “extracellular vesicle-producing cell” or “EV-producing cell” refer to a cell capable of producing one or more extracellular vesicles (EVs) when incubated or cultured in vitro. In some embodiments, the cells are derived from the subject described herein.

[0098] In some embodiments, the cells are animal cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human cells. In some embodiments, the cells are rat cells.

[0099] In some embodiments, the cells are anucleated cells. In some embodiments, the cells are nucleated cells. In some embodiments, the cells are from a continuous cell line. In some examples, the cells are from a primary cell line. In some embodiments, the cells are from an immortalized cell line.

[0100] In some embodiments, the cell line is selected from photoreceptor cell lines, microglia cell lines, Müller glial cell lines, and retinal pigment epithelial cell lines. In some embodiments, the cells are derived from humans. In some embodiments, the cells are derived from mice. In some embodiments, the cells are non-adherent cells (suspended cells). In some embodiments, the cells are adherent cells.

[0101] In one embodiment, EV-producing cells are selected from erythrocytes, reticulocytes, mesenchymal stem cells, epithelial cells, endothelial progenitor cells, umbilical cord cells, ophthalmic cell lines, neural cell lines, dental pulp stem cells, dendritic cells, leukocytes, cancer cells, microglia, glial cells, neurons, astrocytes, photoreceptor cells, embryonic fibroblasts, and megakaryocytes.

[0102] In some embodiments, EV-producing cells are erythrocytes. In some embodiments, EV-producing cells are erythrocytes and reticulocytes. In some embodiments, EV-producing cells are reticulocytes. In some embodiments, EV-producing cells are mesenchymal stem cells. In some embodiments, EV-producing cells are epithelial cells. In some embodiments, EV-producing cells are endothelial progenitor cells. In some embodiments, EV-producing cells are umbilical cord cells. In some embodiments, EV-producing cells are ocular cell lines. In some embodiments, EV-producing cells are neural cell lines. In some embodiments, EV-producing cells are dental pulp stem cells. In some embodiments, EV-producing cells are dendritic cells. In some embodiments, EV-producing cells are leukocytes. In some embodiments, EV-producing cells are cancer cells. In some embodiments, EV-producing cells are microglia. In some embodiments, EV-producing cells are glial cells. In some embodiments, EV-producing cells are astrocytes. In some embodiments, EV-producing cells are photoreceptor cells. In some embodiments, EV-producing cells are embryonic fibroblasts. In one embodiment, the cells are megakaryocytes.

[0103] In some embodiments, EV-producing cells are red blood cells, also known as RBCs or erythrocytes. RBCs can be isolated from whole blood using any method known to those skilled in the art, such as centrifugation or apheresis. In some embodiments, the RBCs are leukocyte-depleted. Leukocyte depletion can be achieved by any method known to those skilled in the art, such as centrifugation, washing, freezing, buffy coat removal, and filtration. In some embodiments, the RBCs are passed through a leukocyte-depletion filter (e.g., a Sterile Acrodisc® WBC syringe filter with a Leukosorb membrane, 25 mm; Pall) to remove leukocytes.

[0104] In one embodiment, if the EV-producing cells are leukocytes, the leukocytes are selected from monocytes, lymphocytes, neutrophils, eosinophils, basophils, and macrophages.

[0105] In one embodiment, the ophthalmic cell line is a cell line described by Lieto et al (2022), Al-Ubaidi et al (1992), Limb et al (2002), or Dunn et al (1996).

[0106] In some embodiments, the ophthalmic cell line is selected from photoreceptor cell lines, microglia cell lines, Müller glial cell lines, retinal cell lines, and retinal pigment epithelial cell lines. In some embodiments, the cells are derived from humans or mice. In some embodiments, the ophthalmic cell line is a retinal cell line. In some embodiments, the retinal cell line is selected from aRPE19 endothelial cells, BV2 microglia cell line, MIO-M1 Müller cells, D407 retinal pigment epithelial cells, iMG human glial cells, or primary retinal cells (photoreceptor, bipolar, microglia or recruited macrophages, Müller or RPE). In some embodiments, the retinal cell line is aRPE19. In some embodiments, the retinal cell line is BV2. In some embodiments, the retinal cell line is MIO-M1. In some embodiments, the retinal cell line is D407. In some embodiments, the retinal cell line is iMG.

[0107] In some embodiments, EV-producing cells are not stem cells. In some embodiments, EV-producing cells are not pluripotent stem cells. In some embodiments, EV-producing cells are not differentiated neural stem cells. In some embodiments, the cells are not induced pluripotent stem cells (iPSCs). In some embodiments, EV-producing cells are not mesenchymal stem cells. In some embodiments, EV-producing cells are not amniotic epithelial stem cells.

[0108] In some embodiments, EV-producing stem cells are unmodified. For example, they are not modified to confer additional properties to the cell or EV, or to contain the exogenous cargo described herein.

[0109] In one embodiment, the method for producing EVs described herein increases the levels of one or both of endogenous and exogenous antioxidants in EV-producing cells.

[0110] In some embodiments, the method for producing EVs described herein reduces the percentage of EV-producing cells having abnormal morphology compared to EV-producing cells not cultured in the culture medium described herein. As used herein, the term “abnormal morphology” refers to abnormal morphology of EV-producing cells compared to EV-producing cells not incubated or cultured in the culture medium described herein. In some embodiments, abnormal morphology is selected from one or more of the following: urchin-like cells, teardrop shape, abnormal size (larger or smaller), torn, or deformed. In some embodiments, if the cells are RBCs, the morphology of the cells is compared to the morphology of RBCs in healthy blood. In some embodiments, less than about 8% of EV-producing cells have abnormal morphology. In some embodiments, less than about 10% of EV-producing cells have abnormal morphology. In some embodiments, less than about 12% of EV-producing cells have abnormal morphology.

[0111] In some embodiments, the method for producing EVs described herein increases the percentage of EV-producing cells having abnormal morphology compared to the level of abnormal morphology of RBCs in healthy blood. In some embodiments, the abnormal morphology increases by about 5% compared to the level of abnormal morphology of RBCs in healthy blood. In some embodiments, the abnormal morphology increases by about 4% to about 6% compared to the level of abnormal morphology of RBCs in healthy blood. In some embodiments, the abnormal morphology increases by about 4% to about 7% compared to the level of abnormal morphology of RBCs in healthy blood.

[0112] In one embodiment, the present invention provides a method for producing extracellular viable cells (EVs), comprising incubating or culturing EV-producing cells in a medium containing an antioxidant and / or an N1 medium component or equivalent thereof, or incubating or culturing EV-producing cells and EV-producing cell fragments. In one embodiment, the EV-producing cell fragment is a platelet.

[0113] Platelets, or thrombocytocytes, are components of blood that play a role in blood coagulation, hemostasis, and vascular function. In humans, platelets lack a nucleus and are fragments of megakaryocytes. Those skilled in the art will understand that platelets can produce extracellular viable cells (EVs).

[0114] In one embodiment, EV-producing cell fragments comprise 0 to about 30% of the population of EV-producing cell material (EV-producing cells and EV-producing cell fragments). In another embodiment, EV-producing cell fragments comprise 0 to about 25% of the population of EV-producing cell material. In yet another embodiment, EV-producing cell fragments comprise 0 to about 20% of the population of EV-producing cell material. In yet another embodiment, EV-producing cell fragments comprise 0 to about 15% of the population of EV-producing cell material. In yet another embodiment, EV-producing cell fragments comprise 0 to about 10% of the population of EV-producing cell material.

[0115] In one embodiment, platelets comprise 0 to about 30% of the population of EV-producing cell material (red blood cells and platelets). In another embodiment, platelets comprise 0 to about 25% of the population of EV-producing cell material. In yet another embodiment, platelets comprise 0 to about 20% of the population of EV-producing cell material. In yet another embodiment, platelets comprise 0 to about 15% of the population of EV-producing cell material. In yet another embodiment, platelets comprise 0 to about 10% of the population of EV-producing cell material.

[0116] Incubation and culture In one embodiment, the present invention provides a method for producing EVs, comprising incubating or culturing EV-producing cells in a medium containing an antioxidant and / or N1 medium components or equivalents thereof. In another embodiment, the present invention provides a method for producing EVs, comprising incubating or culturing EV-producing cells in a medium containing an antioxidant and / or N1 medium components or equivalents thereof, or incubating or culturing EV-producing cells and EV-producing cell fragments. In one embodiment, the EV-producing cell fragment is a platelet.

[0117] As used herein, “incubate” means to keep cells (or cells and cell fragments) taken from or derived from their natural environment viable in an artificial environment for a period of time in which the cells have not undergone mitosis.

[0118] As used herein, “viable” means that they are isolated and function successfully outside of an environment in which they can produce EVs.

[0119] As used herein, the terms “culture” or “cell culture” refer to cells (or cells and cell fragments) that have been taken out of their natural environment or derived from cells taken out of their natural environment and that are made possible to grow (grow larger and / or divide) for a period of time in an artificial environment.

[0120] The conditions of the artificial environment that can be controlled are well known in the art and include pH, temperature, humidity, available nutrients, atmospheric conditions, light conditions, culture substrate, etc.

[0121] In some embodiments, the incubated cells are RBCs. In some embodiments, if the cells are RBCs, they are diluted before being added to the culture medium. In some embodiments, the cells are diluted with phosphate-buffered saline (PBS). As used herein, “PBS” refers to PBS (phosphate-buffered saline), which is a pH-adjusted blend of ultra-high purity grade phosphate buffer and saline, which, when diluted to a 1:1 working concentration, contains, for example, 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4. In some embodiments, the cells are diluted in PBS at a ratio of about 1:20 to about 1:5. In some embodiments, the cells are diluted in PBS at a ratio of about 1:15 to about 1:7. In some embodiments, the cells are diluted in PBS at a ratio of about 1:10. In some embodiments, the cells are RBC cells, and the cells are diluted in PBS at a ratio of about 1:10. In some embodiments, the cells are diluted in the culture medium.

[0122] In one embodiment, EV-producing cells are incubated or cultured for approximately 10 to 24 hours, or approximately 12 to 20 hours, or approximately 12 to 18 hours, or approximately 14 to 18 hours, or approximately 16 to 18 hours, or approximately 18 hours. In one embodiment, EV-producing cells are incubated or cultured for approximately 10 to 24 hours. In one embodiment, EV-producing cells are incubated or cultured for approximately 12 to 20 hours. In one embodiment, EV-producing cells are incubated or cultured for approximately 12 to 18 hours. In one embodiment, EV-producing cells are incubated or cultured for approximately 14 to 18 hours. In one embodiment, EV-producing cells are incubated or cultured for approximately 16 to 18 hours. In one embodiment, EV-producing cells are incubated or cultured for approximately 18 hours. In one embodiment, EV-producing cells are incubated or cultured for approximately 16 hours.

[0123] In one embodiment, EV-producing cells are incubated or cultured at approximately 20°C to 42°C, or approximately 22°C to 40°C, or approximately 24°C to 39°C, or approximately 27°C to 39°C, or approximately 30°C to 39°C, or approximately 33°C to 39°C, or approximately 36°C to 38°C, or approximately 37°C. In one embodiment, EV-producing cells are incubated or cultured at approximately 20°C to 42°C. In one embodiment, EV-producing cells are incubated or cultured at approximately 22°C to 40°C. In one embodiment, EV-producing cells are incubated or cultured at approximately 24°C to 39°C. In one embodiment, EV-producing cells are incubated or cultured at approximately 27°C to 39°C. In one embodiment, EV-producing cells are incubated or cultured at approximately 30°C to 39°C. In one embodiment, EV-producing cells are incubated or cultured at approximately 33°C to 39°C. In one embodiment, EV-producing cells are incubated or cultured at approximately 36°C to 38°C. In another embodiment, EV-producing cells are incubated or cultured at approximately 37°C.

[0124] In one embodiment, EV-producing cells do not differentiate during incubation or culture.

[0125] In addition to EV-producing cells, EV-producing cell fragments may be present with EV-producing cells during the steps of the methods described herein.

[0126] Culture medium The EV-producing cells described herein are incubated or cultured in a medium containing antioxidants and / or N1 medium components or equivalents thereof. As used herein, “medium” refers to any gel or liquid capable of supporting cell viability and often growth in an artificial environment. In some embodiments, the medium comprises antioxidants. In some embodiments, the medium comprises at least one N1 medium component. In some embodiments, the medium comprises antioxidants and N1 medium.

[0127] Antioxidants Oxidation occurs when a substance loses electrons, for example, by contact with oxygen or other oxidizing agents. Free radicals are produced when oxidation occurs. Free radicals are involved in chain reactions that can disrupt normal cellular activity, cause cell damage, and lead to severe harm to organisms. Free radicals, including hydroxyl radicals, superoxide anion radicals, and hydrogen peroxide, are known as reactive oxygen species (ROS).

[0128] As used herein, “antioxidant” is an inhibitor of the process of oxidation. In some embodiments, the antioxidant is an enzymatic antioxidant or a non-enzymatic antioxidant.

[0129] In some embodiments, the enzymatic antioxidant is selected from one or more of superoxide dismutase (SOD), superoxide dismutase 2 (SOD2), catalase, peroxiredoxin, glutathione peroxidase, and glutathione reductase. In some embodiments, the enzymatic antioxidant is SOD. In some embodiments, the enzymatic antioxidant is SOD2. In some embodiments, the enzymatic antioxidant is catalase. In some embodiments, the enzymatic antioxidant is peroxiredoxin. In some embodiments, the enzymatic antioxidant is glutathione peroxidase. In some embodiments, the enzymatic antioxidant is glutathione reductase.

[0130] In some embodiments, the non-enzymatic antioxidant is selected from one or more of hemoglobin, kaempferol, glutathione, vitamin E, vitamin A, vitamin C, tocopherol, carotenoids, glutathione, and curcumin. In some embodiments, the non-enzymatic antioxidant is hemoglobin. In some embodiments, the non-enzymatic antioxidant is kaempferol. In some embodiments, the non-enzymatic antioxidant is glutathione. In some embodiments, the non-enzymatic antioxidant is vitamin E. In some embodiments, the non-enzymatic antioxidant is vitamin A. In some embodiments, the non-enzymatic antioxidant is vitamin C. In some embodiments, the non-enzymatic antioxidant is tocopherol. In some embodiments, the non-enzymatic antioxidant is a carotenoid. In some embodiments, the non-enzymatic antioxidant is glutathione. In some embodiments, the non-enzymatic antioxidant is curcumin.

[0131] In some embodiments, the antioxidant is an endogenous antioxidant. As used herein, “endogenous antioxidant” is an antioxidant produced in a host from which EV-producing cells are obtained, or produced within EV-producing cells. In some embodiments, the endogenous antioxidant is an enzymatic antioxidant. In some embodiments, the endogenous antioxidant is a non-enzymatic antioxidant.

[0132] In some embodiments, the endogenous antioxidant is selected from one or more of hemoglobin, SOD, glutathione, vitamin C, vitamin E, catalase, and glutathione peroxidase. In some embodiments, the endogenous antioxidant is hemoglobin. In some embodiments, the endogenous antioxidant is SOD. In some embodiments, the endogenous antioxidant is glutathione. In some embodiments, the endogenous antioxidant is vitamin C. In some embodiments, the endogenous antioxidant is vitamin E. In some embodiments, the endogenous antioxidant is catalase. In some embodiments, the endogenous antioxidant is glutathione peroxidase.

[0133] In some embodiments, the antioxidant is an exogenous antioxidant. For example, the exogenous antioxidant is supplied to the cell or organism by an external source, such as through feeding or supplementation during incubation or culture. In some embodiments, the exogenous antioxidant is selected from SOD and kaempferol. In some embodiments, the exogenous antioxidant is SOD. In one embodiment, the antioxidant comprises one or both of an enzymatic antioxidant and a non-enzymatic antioxidant, and examples of such antioxidants are described herein.

[0134] N1 culture medium components or their equivalents "N1 medium," also referred to as "N1 medium supplement" as used herein, is a commercially available medium containing 0.5 mg / ml human insulin, 0.5 mg / ml human transferrin (partially iron-saturated), 0.5 μg / ml sodium selenite, 1.6 mg / ml putrescine, and 0.73 μg / ml progesterone. The medium is prepared in Earl equilibrium salt solution (EBSS) without phenol red.

[0135] As used herein, “N1 medium component” refers to one or more of the following: transferrin, insulin, sodium selenite, putrescine, and progesterone.

[0136] As used herein, “its equivalent” refers to the functional equivalent of the N1 medium component.

[0137] It will be apparent to those skilled in the art that N1 medium components can be modified (derivatives) or replaced with their equivalents. In some embodiments, the equivalent has at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% of the functionality of the selected N1 medium component. In some embodiments, the equivalent has at least 80% of the functionality of the selected N1 medium component. In some embodiments, the equivalent has at least 85% of the functionality of the selected N1 medium component. In some embodiments, the equivalent has at least 90% of the functionality of the selected N1 medium component. In some embodiments, the equivalent has at least 95% of the functionality of the selected N1 medium component. In some embodiments, the equivalent has at least 98% of the functionality of the selected N1 medium component.

[0138] In one embodiment, the equivalent of human insulin is insulin derived from another organism, such as cattle or pigs. In another embodiment, the equivalent of human insulin is a derivative of human insulin (synthetic insulin), or a derivative of insulin derived from another organism.

[0139] In one embodiment, the transferrin equivalent is transferrin derived from another organism, for example, a bovine or a pig. In another embodiment, the transferrin equivalent is another protein that binds to and transports iron in the blood, for example, lactoferrin or ceruloplasmin.

[0140] Selenium is an essential trace element that can be found in different forms, such as selenite, selenic acid, and selenomethionine. Sodium selenite is the most common form used for supplementation. In some embodiments, the equivalent of sodium selenite is selenium / selenite in other forms, such as selenite, selenic acid, and selenomethionine.

[0141] In some embodiments, the progesterone equivalent is progesterone derived from another organism, such as a cow or a pig. In some embodiments, the progesterone equivalent is a progesterone derivative.

[0142] In one embodiment, the culture medium contains at least two N1 culture medium components. In one embodiment, the culture medium contains at least three N1 culture medium components. In one embodiment, the culture medium contains at least four N1 culture medium components. In one embodiment, the culture medium contains five N1 culture medium components. In one embodiment, the culture medium contains N1 culture medium component equivalents. In one embodiment, the culture medium contains at least two N1 culture medium component equivalents. In one embodiment, the culture medium contains at least three N1 culture medium component equivalents. In one embodiment, the culture medium contains four N1 culture medium component equivalents. In one embodiment, the culture medium contains an antioxidant and at least one N1 culture medium component. In one embodiment, the culture medium contains an antioxidant and at least two N1 culture medium components. In one embodiment, the culture medium contains an antioxidant and at least three N1 culture medium components. In one embodiment, the culture medium contains an antioxidant and at least four N1 culture medium components. In one embodiment, the culture medium contains an antioxidant and five N1 culture medium components.

[0143] In one embodiment, the culture medium comprises an antioxidant and at least one N1 culture medium component equivalent. In another embodiment, the culture medium comprises an antioxidant and at least two N1 culture medium component equivalents. In yet another embodiment, the culture medium comprises an antioxidant and at least three N1 culture medium component equivalents. In yet another embodiment, the culture medium comprises an antioxidant and four N1 culture medium component equivalents.

[0144] In one embodiment, the culture medium contains all N1 medium components (including N1 medium). In another embodiment, the culture medium contains N1 medium and SOD.

[0145] In one embodiment, the culture medium contains N1 medium at a concentration of approximately 1 μg / ml to 10 μg / ml. In another embodiment, the culture medium contains N1 medium at a concentration of approximately 1 μg / ml to 5 μg / ml. In yet another embodiment, the culture medium contains N1 medium at a concentration of approximately 5 μg / ml to 10 μg / ml. In yet another embodiment, the culture medium contains N1 medium at a concentration of approximately 3 μg / ml to 7 μg / ml. In yet another embodiment, the culture medium contains N1 medium at a concentration of approximately 4 μg / ml to 6 μg / ml. In yet another embodiment, the culture medium contains N1 medium at a concentration of approximately 1 μg / ml. In yet another embodiment, the culture medium contains N1 medium at a concentration of approximately 2 μg / ml. In yet another embodiment, the culture medium contains N1 medium at a concentration of approximately 3 μg / ml. In yet another embodiment, the culture medium contains N1 medium at a concentration of approximately 4 μg / ml. In yet another embodiment, the culture medium contains N1 medium at a concentration of approximately 5 μg / ml. In one embodiment, the culture medium contains N1 medium at a concentration of approximately 6 μg / ml. In another embodiment, the culture medium contains N1 medium at a concentration of approximately 7 μg / ml. In yet another embodiment, the culture medium contains N1 medium at a concentration of approximately 8 μg / ml. In yet another embodiment, the culture medium contains N1 medium at a concentration of approximately 9 μg / ml. In yet another embodiment, the culture medium contains N1 medium at a concentration of approximately 10 μg / ml.

[0146] In one embodiment, the culture medium contains SOD at a concentration of approximately 1 μg / ml to 10 μg / ml. In another embodiment, the culture medium contains SOD at a concentration of approximately 1 μg / ml to 5 μg / ml. In yet another embodiment, the culture medium contains SOD at a concentration of approximately 5 μg / ml to 10 μg / ml. In yet another embodiment, the culture medium contains SOD at a concentration of approximately 3 μg / ml to 7 μg / ml. In yet another embodiment, the culture medium contains SOD at a concentration of approximately 4 μg / ml to 6 μg / ml. In yet another embodiment, the culture medium contains SOD at a concentration of approximately 1 μg / ml. In yet another embodiment, the culture medium contains SOD at a concentration of approximately 2 μg / ml. In yet another embodiment, the culture medium contains SOD at a concentration of approximately 3 μg / ml. In yet another embodiment, the culture medium contains SOD at a concentration of approximately 4 μg / ml. In yet another embodiment, the culture medium contains SOD at a concentration of approximately 5 μg / ml. In yet another embodiment, the culture medium contains SOD at a concentration of approximately 6 μg / ml. In one embodiment, the culture medium contains SOD at a concentration of approximately 7 μg / ml. In another embodiment, the culture medium contains SOD at a concentration of approximately 8 μg / ml. In yet another embodiment, the culture medium contains SOD at a concentration of approximately 9 μg / ml. In yet another embodiment, the culture medium contains SOD at a concentration of approximately 10 μg / ml.

[0147] In one embodiment, the culture medium contains N1 medium and SOD at a concentration of approximately 1 μg / ml. In another embodiment, the culture medium contains N1 medium and SOD at a concentration of approximately 2 μg / ml. In yet another embodiment, the culture medium contains N1 medium and SOD at a concentration of approximately 3 μg / ml. In yet another embodiment, the culture medium contains N1 medium and SOD at a concentration of approximately 4 μg / ml. In yet another embodiment, the culture medium contains N1 medium and SOD at a concentration of approximately 5 μg / ml. In yet another embodiment, the culture medium contains N1 medium and SOD at a concentration of approximately 6 μg / ml. In yet another embodiment, the culture medium contains N1 medium and SOD at a concentration of approximately 7 μg / ml. In yet another embodiment, the culture medium contains N1 medium and SOD at a concentration of approximately 8 μg / ml. In yet another embodiment, the culture medium contains N1 medium and SOD at a concentration of approximately 9 μg / ml. In yet another embodiment, the culture medium contains N1 medium and SOD at a concentration of approximately 10 μg / ml.

[0148] Additional culture medium / supplements The media described herein, comprising antioxidants and / or N1 medium components or equivalents thereof, may comprise one or more of the additional media and supplements. Such additional media include any medium known to those skilled in the art (see, for example, Josefsberg et al., 2012; Wolf et al., 2011) that can incubate or culture EV-producing cells without preventing EV production. Additional media for incubation or culture of EV-producing cells of the present invention include, but are not limited to, Iscove medium, Opti-MEM UltraCHO, CD hybridoma serum-free medium, episerf medium, MediV SF103 (serum-free medium), stem cell media or supplements (e.g., from Stem Cell Technologies), Dulbecco's Modified Eagle Medium (DMEM), Eagle Modified Eagle Medium (EMEM), Glasgow Modified Eagle Medium (GMEM), SMIP-8, Modified Eagle Medium (MEM), VP-SFM, DMEM-based SFM, DMEM / F12, DMEM / Ham F12, VPSFM / William Medium E, ExCell 525 (SFM), adenovirus expression medium (AEM), and Excell 65629. In some embodiments, the additional medium is not DMEM / F12. Additional supplements may include, for example, calcium ionophores to help stimulate EV production.

[0149] It will be understood by those skilled in the art that a culture medium containing antioxidants and / or N1 medium components or equivalents thereof may be supplemented with additional growth factors, such as, but not limited to, amino acids, vitamins, carbohydrates, inorganic salts, glucose, basic and trace elements, serum, growth factors, adhesion factors, hormones, buffer systems, supplements, antibiotics, and minerals. In some embodiments, the medium is not supplemented with serum. In some embodiments, the medium does not contain components produced in humans. In some embodiments, the medium does not contain components produced in animals.

[0150] In some embodiments, the culture medium does not contain one or more of the following: differentiation factors, neuronal differentiation factors, neurotrophic factors, and growth factors.

[0151] In some embodiments, the culture medium does not contain one or more of the following: corticosterone, calcium ionophores, triiodothyronine, levocarnitine, linoleic acid, alpha-lipoic acid, levocarnitine HCl, vitamin H, dorsomorphin, alkaline fibroblast growth factor, biotin, and pancreatic islet components. In some embodiments, the culture medium does not contain corticosterone. In some embodiments, the culture medium does not contain calcium ionophores. In some embodiments, the culture medium does not contain triiodothyronine. In some embodiments, the culture medium does not contain levocarnitine. In some embodiments, the culture medium does not contain linoleic acid. In some embodiments, the culture medium does not contain alpha-lipoic acid. In some embodiments, the culture medium does not contain levocarnitine HCl. In some embodiments, the culture medium does not contain vitamin H. In some embodiments, the culture medium does not contain dorsomorphin. In some embodiments, the culture medium does not contain alkaline fibroblast growth factor. In some embodiments, the culture medium does not contain biotin. In some embodiments, the culture medium does not contain pancreatic islet components.

[0152] In one embodiment, the cells are not cultured or incubated under hypoxic conditions.

[0153] Isolation of extracellular vesicles In some embodiments, extracellular viable cells (EVs) are isolated or separated from cells after incubation or culture. Isolation or separation of EVs can be performed by any method known to those skilled in the art, including, for example, centrifugation, filtration, tangential flow filtration, size exclusion chromatography, nanoflow / facs, magnetic beads, EV isolation kits (e.g., ExoQuick® ULTRA EV Isolation System, system biosciences catalog number EQULTRA-20A-1), and extrusion. In some embodiments, EVs are isolated from reductive cells (RBCs) after incubation. In some embodiments, isolation requires centrifugation and removal of the supernatant (EVs are present in the precipitate).

[0154] Endogenous and exogenous cargo In some embodiments, the EV of the present invention comprises endogenous or exogenous cargo. In some embodiments, the EV of the present invention comprises endogenous cargo (e.g., endogenous antioxidants, DNA, or RNA molecules). The cargo may be inside the vesicle, within the lipid membrane of the vesicle, and / or on the surface of the vesicle. In some embodiments, the cargo is inside the vesicle. In some embodiments, the cargo is within the lipid membrane of the vesicle. In some embodiments, the cargo is on the surface of the vesicle.

[0155] In some embodiments, the endogenous cargo is an antioxidant. In some embodiments, the antioxidant is selected from one or more of hemoglobin, kaempferol, glutathione, vitamin E, vitamin A, vitamin C, tocopherol, carotenoids, glutathione, and curcumin. In some embodiments, the antioxidant is hemoglobin.

[0156] In some embodiments, the endogenous cargo is a miRNA. In some embodiments, the miRNA is selected from one or more of the following: mmu-miR-142a-3p, mmu-miR-486b-3p, mmu-let-7c-5p, mmu-miR-16-5p, mmu-miR-25-3p, mmu-miR-486a-3p, mmu-miR-486b-5p, mmu-miR-486a-5p, mmu-let-7f-5p, mmu-let-7a-5p, and mmu-miR-451a. In some embodiments, the miRNA is mmu-miR-142a-3p. In some embodiments, the miRNA is mmu-miR-486b-3p. In some embodiments, the miRNA is mmu-let-7c-5p. In one embodiment, the miRNA is mmu-miR-16-5p. In another embodiment, the miRNA is mmu-miR-25-3p. In another embodiment, the miRNA is mmu-miR-486a-3p. In another embodiment, the miRNA is mmu-miR-486b-5p. In another embodiment, the miRNA is mmu-miR-486a-5p. In another embodiment, the miRNA is mmu-let-7f-5p. In another embodiment, the miRNA is mmu-let-7a-5p. In another embodiment, the miRNA is mmu-miR-451a.

[0157] In some embodiments, the EV of the present invention comprises an exogenous cargo. In some embodiments, the cargo is a therapeutic cargo. In some embodiments, the exogenous therapeutic cargo is selected from one or more of the following: drugs, antioxidants, chemotherapy agents, proteins, lipids, nucleic acids (such as DNA, mRNA, miRNA, siRNA, circular RNA, long non-coding RNA, and snoRNA), CRISPR / Cas9, nanoparticles, and exogenous targeted molecules. In some embodiments, the exogenous cargo is a drug. In some embodiments, the drug is a retinal therapeutic drug, or a drug for reducing inflammation and / or oxidative stress in the eye. In some embodiments, the drug is selected from corticosteroids, inflammasome-targeting drugs, complement protein-targeting drugs, anti-VEGF antibodies (e.g., Lucentis or bevacizumab), and Syfovre. In some embodiments, the exogenous cargo is an antioxidant. In some embodiments, the exogenous cargo is a chemotherapy agent. In some embodiments, the exogenous cargo is a protein. In some embodiments, the exogenous cargo is a lipid. In some embodiments, the exogenous cargo is a nucleic acid. In some embodiments, the exogenous cargo is DNA. In some embodiments, the exogenous cargo is mRNA. In some embodiments, the exogenous cargo is miRNA. In some embodiments, the exogenous cargo is siRNA. In some embodiments, the exogenous cargo is circular RNA. In some embodiments, the exogenous cargo is a long non-coding RNA. In some embodiments, the exogenous cargo is snoRNA. In some embodiments, the exogenous cargo is CRISPR / Cas9. In some embodiments, the exogenous cargo is a nanoparticle. In some embodiments, the cargo is an exogenous targeting molecule.

[0158] In some embodiments, the EV described herein does not include exogenous cargo.

[0159] As used herein, “exogenous targeting molecule” is a molecule that facilitates the targeting of EVs to specific sites within a subject after administration. In some embodiments, the exogenous targeting molecule facilitates targeting of ophthalmic cells or tissues. In some embodiments, the exogenous targeting molecule facilitates targeting of nerve cells or tissues. In some embodiments, the exogenous targeting molecule facilitates targeting of the retina. In some embodiments, the exogenous targeting molecule facilitates targeting of nerve cells or tissues susceptible to neurodegeneration. In some embodiments, the exogenous targeting molecule facilitates targeting of ophthalmic cells or tissues susceptible to neurodegeneration.

[0160] In some embodiments, the EV described herein does not include an exogenous targeting molecule. In some embodiments, the EV described herein does not include the exogenous targeting molecule PDFFA.

[0161] In one embodiment, the exogenous cargo is loaded into the EV by the EV-producing cell (the EV-producing cell produces an EV that contains (packages) at least a portion of the cargo present within the cell). In such an embodiment, the exogenous cargo may be provided to the EV-producing cell in a culture medium, or introduced into the EV-producing cell by means of electroporation, sonication, incubation, transfection, or mechanical permeation, for example.

[0162] In alternative embodiments, chemical substances or biomolecules can be directly loaded into isolated EVs using techniques known in the art, such as electroporation, sonication, incubation, transfection, or mechanical permeation.

[0163] Diseases and Conditions Exemplary diseases and conditions treated and / or prevented using the EVs, compositions, methods, and uses described herein include diseases and conditions that cause and / or result in oxidative stress and / or inflammation. In some embodiments, the disease or condition is a neurodegenerative disease or condition. In some embodiments, the disease or condition is oxidative stress and / or inflammation in the eye. In some embodiments, the disease or condition is retinal degeneration.

[0164] In one embodiment, the disease or condition is associated with elevated levels of one or more chemokines, cytokines, and / or inflammatory mediators.

[0165] As used herein, “oxidative stress” refers to an increase / accumulation of reactive oxygen species in cells, tissues, or organs. When there is an imbalance between reactive oxygen species, also known as free radicals, and the body’s innate antioxidant defenses, this results in oxidative stress in the body. The presence of oxidative stress and / or reactive oxygen species can be measured by any method known to those skilled in the art, for example, indirectly, by measuring DNA / RNA damage, lipid peroxidation, and protein oxidation / nitration.

[0166] As used herein, “inflammation” refers to both the normal bodily function of inducing inflammatory molecules in response to injury and pathogens or other substances, as well as an imbalance or excess of inflammatory molecules that can cause a variety of chronic diseases.

[0167] As used herein, “oxidative stress in the eye” refers to an increase / accumulation of reactive oxygen species in the eye.

[0168] As used herein, “inflammation of the eye” refers to inflammatory processes and increased levels of inflammatory molecules in the eye. Symptoms may include redness of the eye, eye pain, light sensitivity, blurred vision, and decreased visual acuity.

[0169] In one embodiment, a neurodegenerative disease or condition that causes and / or results from oxidative stress and / or inflammation.

[0170] In one embodiment, a neurodegenerative disease or condition that causes and / or results in the activation and / or proliferation of microglia.

[0171] In some embodiments, the neurodegenerative disease or condition includes neuroinflammation.

[0172] In one embodiment, the neurodegenerative disease or condition is retinal degeneration.

[0173] In one embodiment, diseases or conditions that cause and / or result in oxidative stress and / or inflammation in the eye are selected from macular degeneration, retinitis pigmentosa, diabetic retinopathy, Stargardt disease, Leber congenital amaurosis, Best's disease, cone-rod dystrophy, Usher syndrome, choroideremia, Valde-Wiedl syndrome, Refsum disease, macula telangana, macular telangiectasia, macular edema, retinal detachment, retinal ischemia, uveitis, scleritis, conjunctivitis, keratitis, corneal ulcer, glaucoma trachoma, choroidal melanoma, ocular melanoma, glaucoma retinal dystrophy, strabismus, and cataracts.

[0174] In one embodiment, the disease or condition that causes and / or results from oxidative stress and / or inflammation in the eye is selected from macular degeneration, retinitis pigmentosa, diabetic retinopathy, Stargardt disease, macular edema, retinal detachment, and retinal ischemia.

[0175] In one embodiment, the disease or condition that causes and / or results from oxidative stress and / or inflammation in the eye is selected from retinal degeneration, glaucoma, diabetic retinopathy, and retinal vein occlusion.

[0176] In one embodiment, the disease or condition that causes and / or results from oxidative stress and / or inflammation in the eye is retinal degeneration.

[0177] In some embodiments, retinal degeneration is selected from macular degeneration, retinitis pigmentosa, diabetic retinopathy, Stargardt disease, Leber congenital amaurosis, Best's disease, cone-rod dystrophy, Usher syndrome, colloideremia, Valde-Biedl syndrome, Refsum disease, macular terangana, macular edema (odema), retinal detachment, and retinal ischemia. In some embodiments, macular degeneration is selected from exudative macular degeneration and dry macular degeneration. In some embodiments, macular degeneration is geographic atrophy. Geographic atrophy refers to the later stage of dry AMD.

[0178] In one embodiment, the neurodegenerative disease or condition is Parkinson's disease (PD). PD is the second most common age-related neurodegenerative disorder. Chronic neuroinflammation is a key feature of the pathophysiology of PD (Araujo et al., 2022). Infiltration and accumulation of immune cells from the periphery are detected within and around the affected brain regions of PD patients.

[0179] In one embodiment, the neurodegenerative disease or condition is amyotrophic lateral sclerosis (ALS) (also known as motor neuron disease / Lou Gehrig). ALS is characterized by neuroinflammation. Neuroinflammation in ALS includes lymphocyte and macrophage infiltration, activation of microglia and reactive astrocytes, and complement involvement (Rojas et al., 2020; Mead et al., 2023; Benatar et al., 2022).

[0180] In one embodiment, the neurodegenerative disease or condition is Alzheimer's disease. Patients with AD have elevated levels of inflammatory markers (Leng et al., 2021; Sanchez-Sarasua et al., 2020).

[0181] In one embodiment, the neurodegenerative disease or condition is a tauopathy. Those skilled in the art will understand that tauopathy is a heterogeneous group of neurodegenerative disorders characterized by the aggregation of tau proteins into fibrous inclusions within neurons and glial cells. Tau pathology can arise independently and secondarily to a variety of triggers, each associated with an inflammatory process, including, for example, infection, recurrent mild traumatic brain injury, seizure activity, and autoimmune diseases (Cherry et al., 2022; Laurent et al., 2018).

[0182] In one embodiment, the neurodegenerative disease or condition is multiple sclerosis (MS). MS is a complex disease involving one or more of the following: neuroinflammation, demyelination, and continuous axonal loss. MS is the most common inflammatory, demyelinating, neurodegenerative disorder of the central nervous system (Naegele et al., 2014; Bjelobaba et al., 2017).

[0183] In one embodiment, the neurodegenerative disease or condition is Lewy body dementia (also known as dementia with Lewy bodies or DBL). DBL is the second most common cause of neurodegeneration in dementia (Amin et al., 2020).

[0184] In some embodiments, the neurodegenerative disease or condition is a stroke or transient ischemic attack. Stroke is the third leading cause of death and disability worldwide. Neuroinflammation is a major pathological event involved in the process of ischemic injury and repair (Jayaraj et al., 2019). In particular, microglia play a dual role in neuroinflammation.

[0185] In one embodiment, the neurodegenerative disease or condition is Huntington's disease (HD). HD is a devastating neurodegenerative genetic disorder that causes progressive motor impairment, emotional impairment, and cognitive impairment. Neuroinflammation is a component of HD, and the modulation of neuroinflammation has been suggested as a potential target for therapeutic intervention. Neuroinflammation in the HD brain includes reactive forms and chronic inflammatory states in these glial cells (Palpagama et al., 2019; Lee et al., 2021).

[0186] In one embodiment, the condition is not a demyelinating disease or condition.

[0187] In some embodiments, the EV of the present invention is administered during or after an eye surgery to aid in recovery after surgery in which inflammation and / or oxidative stress is present (e.g., retinal detachment, cataract surgery).

[0188] In some embodiments, the EV of the present invention is administered during or after a surgical procedure to aid in postoperative recovery in the presence of neuroinflammation and / or oxidative stress.

[0189] In some embodiments, the EV of the present invention reduces the expression of one or more cytokines, chemokines, and / or inflammatory mediators in the diseases or conditions described herein.

[0190] In some embodiments, one or more cytokines, chemokines, and / or inflammatory mediators are selected from the group including MIP-1α(Ccl3), IL-1β, IL-6, IL-8, IL-10, TNFα, MCP-1(Ccl2), and IL-1α. In some embodiments, the cytokine is MIP-1α. In some embodiments, the cytokine is IL-1β. In some embodiments, the cytokine is IL-6. In some embodiments, the chemokine is IL-8. In some embodiments, the cytokine is IL-10. In some embodiments, the cytokine is TNFα. In some embodiments, the chemokine is MCP-1. In some embodiments, the cytokine is IL-1α.

[0191] Administration The EVs or compositions described herein can be administered to a subject by an appropriate route, either alone or in combination with a therapeutic agent.

[0192] Various routes of administration are possible, but are not limited to, systemic and topical administration routes. In some embodiments, the delivery route is selected from intravenous, intra-arterial, intramuscular, intradermal, intravascular, oral, or subcutaneous injection routes. In some embodiments, systemic delivery is intravenous delivery. In some embodiments, systemic delivery is subcutaneous injection.

[0193] In some embodiments, the delivery route is selected from topical administration to the eye (e.g., eye drops), intraocular injection, subretinal injection, periocular injection, intravitreous injection, subconjunctival injection, intracranial injection, subarachnoid injection, intracerebral injection, and intracerebral implantation. In some embodiments, topical delivery is topical administration. In some embodiments, topical delivery is intravitreous injection. In some embodiments, topical delivery is subretinal injection.

[0194] In some embodiments, the EV or composition described herein is administered systemically or topically and localizes to ophthalmic cells or tissues. In some embodiments, the EV or composition described herein is administered systemically or topically and localizes to nerve cells or tissues. In some embodiments, nerve cells are selected from neurons, glia, microglia, and astrocytes.

[0195] In one embodiment, the EV or composition described herein is administered systemically or topically and localizes to the retina. In one embodiment, the retinal cells are selected from one or more of the following: photoreceptors, ganglion cells, bipolar cells, Müller cells, microglia / macrophages, retinal pigment epithelial cells, and endothelial cells. In one embodiment, the EV or composition described herein is administered systemically or topically and localizes to the retinal blood vessels.

[0196] The formulation of the administered composition will vary depending on the chosen route of administration (e.g., systemic or topical). In one embodiment, the formulation comprises PBS.

[0197] Compositions containing EVs as described herein may contain physiologically acceptable carriers. For solutions or emulsions, suitable carriers include aqueous or alcoholic / aqueous solutions, emulsions, or suspensions containing physiological saline and a buffer medium. Parenteral carriers include sodium chloride solution, ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixative oil. Intravenous carriers may include various additives, preservatives, or fluids, nutrients, or electrolyte replacement solutions. [Examples]

[0198] Example 1: Materials and Method Animal paradigm Animal handling Adult male and female C57BL / 6J wild-type (WT) mice (60-80 days old at the start of the experiment (P60-80)) were purchased from the Animal Resources Centre (ARC), Canning Vale, Western Australia (WA). The mice were bred, reared, housed under a 12-hour light / dark cycle (5 lux), and given free access to food and water.

[0199] photooxidative damage Mice were subjected to photooxidative damage (PD) for 5 days, as previously described by Natoli et al (2016). Briefly, the mice were placed in a Perspex box coated with a reflective inner surface and exposed to 100K lux white light from a light-emitting diode (LED). The animals were administered a pupillary dilator (Minims® atropine sulfate 1% w / v; Bausch and Lomb) to both eyes twice daily (9 a.m. and 4 p.m.) during the course of the damage paradigm. After the experiment, the mice were euthanized with CO2. Representative EGR waveforms are shown in Figure 1. The amplitude and latency of waves A and B were plotted and analyzed in GraphPad Prism 9, and the data were expressed as mean wave amplitude or latency ± SEM (mean standard error) (μV).

[0200] Induction of retinal degeneration by sodium iodate To induce chemical retinal degeneration, 50 mg / kg of sodium iodate in H2O was injected into the intraperitoneal fossa of C57BL6 / J mice (8-12 weeks old). This dosage was chosen as one that does not induce toxicity but still causes significant eye damage, as described by Koster et al (2022). The sodium iodate injection was administered on the morning of day 0. The mice were then treated according to the treatment regimen described below. On the evening of the last day of treatment, the mice were placed overnight in dark adaptation for functional and morphological evaluation the following morning. Throughout this paradigm, the animals were checked daily for signs of discomfort and to ensure there were no adverse effects from any injected compounds.

[0201] Induction of 6-OHDA lesions Mice received bilateral intracerebral injections into the striatum, with one side serving as a control and the other side being experimental according to Masini et al (2021). The experimental side either received vehicle injections or was lesioned with 6-OHDA; this was followed by therapeutic injections of therapeutic RBC-EV or vehicle. Injections were administered to the bregma at stereotactic coordinates of anterior / posterior +0.6 mm, medial / lateral ±2.2 mm, and dorsal / abdominal -3.2 mm.

[0202] The mice were mounted in a stereotactic frame, and once anesthetic stability was achieved, the top of the head was shaved and a midline incision was made to expose the skull. The injection sites were marked on the skull (anterior / posterior, medial / lateral coordinates), and small pilot holes were drilled to allow for needle insertion.

[0203] A borosilicate pipette (50 μm tip) was inserted into the brain through a hole guided by a stereotactic frame, and mice were injected with 1 μL of 6-OHDA hydrochloride (4 micrograms / microliter) in 0.9% sterile saline and 0.02 mg / mL% ascorbic acid, or an equal volume of sterile saline and ascorbic acid. On the experimental side, the mice then received 1 μL of RBC-EV (N1 / SOD) or an equal volume of PBS.

[0204] To reduce the historically high mortality rate during this procedure, an "enhanced care" regime was employed. This regime involved timely interventions to address the needs of the animals, following Masini et al (2021). Specifically, for 72 hours post-surgery, mice were provided with water-softened solid feed pellets and daily subcutaneous meloxicam injections (5 mg / kg).

[0205] This Parkinson's disease model was previously demonstrated by Masini et al (2021), inducing early molecular changes associated with neuroinflammation as early as 3–7 days after induction, and leading to significant behavioral deficits within 3–4 weeks. For this reason, and to focus on the potential of early treatment, we selected a 7-day time point to evaluate molecular and behavioral responses to treatment.

[0206] Rotor rod motion defect test The Rotarod test (Panlab Rotarod LE 8200, Harvard instruments) is a behavioral assessment routinely used for neuromuscular coordination or motor deficiencies (Deacon, 2013). Prior to the assessment, mice were familiarized with the Rotarod apparatus in a pre-training session (24 hours prior). Mice were then exposed to the apparatus and placed on the rod at a low rotation speed (4 rpm) for 5 minutes. Mice that fell off the rod were returned to the rod until the 5-minute duration was complete. On the day of the experiment, individual mice were placed on the rotating rod, and a timer was started upon contact. The rod spun from a base speed of 4 rpm up to a maximum of 40 rpm over a 1-minute period, ending the trial after the mouse fell off the rod. If a mouse spontaneously jumped off the rod, the trial was considered invalid and the trial was repeated. The latency (time) each mouse remained on the Rotarod was recorded. For each mouse, two trials with a minimum of 5 minutes between them were recorded. The final value was obtained as the average of the two trials.

[0207] Grip strength Grip strength was measured using a portable scale fitted with a grid to allow the mouse to grip. All force measurements were obtained as pressure in kilograms / gram. Briefly, the mouse was held by its tail and lifted to the force transducer device until it grasped the handle. Once the mouse consistently grasped the handle when placed near the device, the mouse was left undisturbed for at least 5 minutes before evaluation. Three evaluation trials were recorded, by which the mouse gripped the device and was gently pulled until it lost its grip. The maximum force recorded by the device from the three trials was recorded and used for analysis.

[0208] RBC-EV preparation incubation Whole mouse blood in EDTA was purchased from Applied Biological Products Management (MSBX 0005; 5 ml tube). Whole blood was spun at 1500 × g for 10 minutes to separate plasma, buffy coat, and erythrocytes. Plasma and buffy coat were removed by pipetting, and the remaining erythrocytes were diluted 1:10 in 1 × phosphate-buffered saline (PBS) (Gibco; pH 7.2). The erythrocyte (RBC) suspension was passed through a leukocyte depletion filter (Sterile Acrodisc® WBC syringe filter with Leukosorb membrane, 25 mm; Pall) to remove contaminating leukocytes. Next, the RBC suspension was incubated in a T25 flask supplemented with 1:100 dilution of 100×N1 medium supplement (Sigma; N6530) and 1:500 dilution of superoxide dismutase (SOD) (Worthington Biochemicals; LS003540) (5 μg / ml; final active concentration of SOD). The RBC suspension with the supplements was incubated on a shaker incubator for 18 hours at 37°C.

[0209] replenishment Trans-resveratrol (Sigma PHR2201-200MG), kaempferol (Sigma, 60010-25MG), and reduced L-glutathione (Sigma, G4251-1G) were added to RBCs at increasing doses for 18 hours at 37C according to the incubation paradigm described above, and compared with RBCs (PBS), RBCs (N1 / SOD), and pre-treatment (freshly isolated RBCs from whole mouse blood in EDTA, without supplementation or incubation).

[0210] RBC health checkup After incubation, the RBC suspension was transferred to a 15 mL Falcon tube and spun at 600 × g for 20 minutes to separate RBCs from extracellular vesicles (EVs) in the suspension (Usman et al, 2018). To determine the effect of incubation on RBC health, the 600 × g pellet was resuspended in 1 × PBS (Gibco; pH 7.2) at 1:1000 and 1:10000 dilutions for qualitative and quantitative evaluation. The total cell count was determined by applying 200 μL of pellet suspension to a glass hemocytometer (Westlab, product no. 071301-9877) and counting the number of RBCs in the large central square. Images of RBCs were taken at 4x and 20x magnification using a Zeiss Axiovert 200 microscope, and the images were processed using ImageJ software (version 2.1.0). The percentage of abnormal RBCs was determined by studying the RBC membrane and classifying them based on RBC size (normal, microcytes, and macrocytes) and membrane morphology variations (normal, urchin cells, tear droplets, sickle cells, elliptic cells, and mitotic cells), and all statistical analyses were performed on GraphPad Prism 9.

[0211] Cell viability of RBCs was evaluated before and after treatment / incubation by staining with calcein-AM (Thermo Fisher, catalog number C1430) and annexin V (BD Biosciences, catalog number 563973). Calcein-AM was prepared as a 10 mM stock solution in dimethyl sulfoxide, and a 100 μM working solution in PBS buffer, pH 7.4 was prepared as needed. RBCs, 2 × 10⁵ in 200 μl of PBS, were incubated with the calcein-AM working solution (final concentration of calcein-AM - 5 μM) at 37°C (in the dark) for 45 minutes. Next, the cells were isolated by centrifugation (1000×g for 5 minutes at 4°C), resuspended in 0.5 ml of Annexin V-binding buffer containing (145 mM NaCl, 7.5 mM KCl, 2 mM CaCl2, 10 mM glucose, 50 mM HEPES pH 7.4, 0.5% BSA), and incubated with 5 μl of PE-Annexin-V in the dark at room temperature for 15 minutes. The cells were analyzed for two-parameter histograms FL1 (calcein) vs. FL2 (PE-Annexin-V) using ImageStreamX Mk II (Amnis Corporation, Seattle, WA, USA) and BD LSRFortessa II (BD Biosciences, San Jose, California). RBCs were used as a control for dead cells, and permeabilization was easily performed using saponins according to Jacob et al, 1991. All experiments were performed in triple replication.

[0212] EV isolation After spinning at 600 × g, the RBC supernatant was transferred to a 15 mL Falcon tube and spun at 1600 × g for 15 minutes and then at 3200 × g for 10 minutes, removing the pellet at each spin step by transferring the supernatant to a new tube. The resulting RBC suspension S1 was transferred to an ultracentrifuge tube (Beckman Coulter Ultra-Clear Thinwall Tubes 13.2 ml; 50-pack) and spun at 10,000 × g for 35 minutes using an ultracentrifuge. The resulting pellet P2 was discarded, and the supernatant S2 was transferred to a new ultracentrifuge tube and spun again at 100,000 × g for 1 hour and 30 minutes. Then, P3 containing RBC-EV was collected, washed in PBS by pipetting, and spun again at 100,000 × g for 1 hour and 35 minutes (Usman et al, 2018). Finally, the P4 (RBC-EV pellet) was resuspended in 50-500 μL (depending on downstream experiments) and frozen at -80°C until use.

[0213] SOD and hemoglobin activity assays Rapidly frozen aliquots of RBC and RBC EV were thawed and dissolved in ice-cold ultrapure water and PBS + 0.1% Triton at dilutions of 1:150 and 1:4, respectively. The samples were left on ice for 20 minutes to ensure complete dissolution and spun down at 10,000 × g for 15 minutes. The supernatant was used for SOD and hemoglobin activity assays. SOD activity of RBC and RBC-EV, with or without N1, SOD, and N1+SOD, was measured using a superoxide dismutase (SOD) colorimetric activity kit according to the manufacturer's instructions (catalog number: EIASODC, Thermo Fisher Scientific) and compared to an unsupplemented control. Similarly, hemoglobin activity was evaluated using a hemoglobin colorimetric assay (Abcam, ab234046) according to the manufacturer's protocol.

[0214] Characterization of EVs The concentration and size distribution of human RBC-EVs were measured using nanoparticle tracking analysis on a ZetaView x30 QUATT (Particle Metrix GmbH) with a 488 nm excitation laser, after calibration with a 100 nm polystyrene latex reference standard (Applied Microspheres, Netherlands). The following parameters were used: (Sensitivity: 80, Shutter: 100, Minimum Brightness: 30, Minimum Size Region: 10, Maximum Size Region: 1000, Frame Rate: 30 frames / second). RBC-EVs were diluted 1:100,000 in sterile 1×PBS. Eleven positions in one cycle were captured across the flow cell and averaged. For plotting and statistical analysis, the average concentration values ​​for each sample in particles / mL, as well as the average and mode size (nm), were exported to Prism 9.

[0215] RBC-EV administration To test the safety and therapeutic efficacy of RBC-EV, mice were administered RBC-EV topically and systemically. Mice were injected with 1 μL of RBC-EV(SOD / N1), RBC-EV(SOD), RBC-EV(N1), RBC-EV(PBS), or 1 μL of PBS(SOD / N1), PBS(SOD), PBS(N1), or PBS as a control. In some experiments, RBC-EV or PBS was labeled with SYTO RNASelect before administration to fluorescently label the EV RNA as described below. Mice from the same litter were used in each experiment for comparison and were randomly assigned across treatment groups.

[0216] Intravitreal injection Mice were first anesthetized by intraperitoneal injection of ketamine (100 mg / kg) and xylazil (10 mg / kg), and then the pupils were dilated by applying 1% w / v atropine to the ocular surface of each eye. The mice were kept on a heated mat while anesthetized to maintain their body temperature. String loops were first tied around the mice's eyes to lift them out of their sockets, allowing easier access to the injection site. The surface of the eyes was wiped with 10% w / v povidone-iodine disinfectant. A pilot hole was made at the injection site approximately 1 mm posterior to the temporal corneal margin using a 33G needle. 1 μL (2.0 × 10⁶) of each EV mixture or control solution was injected using a 10 μL NanoFil syringe with a 34G needle. 9 Individual EVs were injected into the vitreous humor of the eye through a pre-fabricated pilot hole, angled toward the optic nerve, with the help of a stereomicroscope. After injection, the eyes were wiped with 1% chlorsig to prevent bacterial infection and with GenTeal® gel to prevent eye dryness. To aid recovery, animals were intraperitoneally injected with ReversaMed (1 mg / kg). After allowing the animals to recover under dim light conditions for approximately 24 hours, they were subjected to photooxidative damage for 5 days (degenerative paradigm) or held under standard containment conditions for 7 days (safety assessment).

[0217] intraperitoneal injection Using the regimen outlined in the drawing, the mouse controls 2.0 × 10 11 The drug was administered daily via intraperitoneal injection at a dose of 1 EV / 100 μL.

[0218] Intracranial injection 2.0×10 9 RBC-EV or vehicle control (PBS) at doses of 1 EV / μL (1 μL bolus) is administered during intracranial delivery of 6-OHDA / vehicle using the regimen outlined in the drawings.

[0219] Characterization of extracellular vesicles Cryo-electron microscopy To visualize and image RBC-EVs, cryo-EM was used for sizing and characterization purposes. RBC-EV samples in PBS were applied to a 300-mesh glow-discharge EM grid with lace-like carbon (PELCO easiGlow® Glow Discharge Cleaning System) for vitrification at 80–90% humidity at room temperature. Any excess sample present was removed by blotting with filter paper. The loaded grid was placed in liquid ethane (held in equilibrium with solid ethane) using a Leica EMGP2 automated plunge freezer (Centre for Advanced Microscope facility, JCSMR). The grid was then stored in liquid nitrogen until further use. EVs were imaged using a JEOL JEM-F200 microscope at a voltage of 200kV and a magnification of 16,000x. Images were processed using ImageJ. All visible EVs across 10 images were measured for size (in nanometers) and exported to GraphPad Prism 9 for plotting and statistical analysis.

[0220] Characterization of NanoSight The concentration and size distribution of RBC-EVs were measured using nanoparticle tracking analysis on a NanoSight NS300. RBC-EVs were diluted 1:10,000 in sterile 1×PBS to achieve particle values ​​of 20–100 per frame. A constant flow rate of the sample was provided by a syringe pump set to a rate of 35 (equivalent to approximately 3.1 μL / min) (Wooff et al, 2020). Nine 30-second videos were captured for each sample. The detection threshold, set to 4–5, was not changed between measurements. The average concentration values ​​for each sample in particles / mL were exported to GraphPad Prism 9 for plotting and statistical analysis.

[0221] Western blot analysis Total lysates were extracted from cells and RBC-EVs by incubation with RIPA buffer supplemented with a 1:100 protease inhibitor cocktail (Sigma-Aldrich, MO, United States). An additional homogenization step was applied to retinal tissue samples. 10–20 μg of protein lysate / well was separated on a 4–12% polyacrylamide gel (Thermo Fisher Scientific, MA, United States) at 100 V for 60 minutes and then transferred to a nitrocellulose membrane (Bio-Rad, CA, United States) using a Power Blotter semi-drying system (Thermo Fisher Scientific, MA, United States) at 20 V for 15 minutes. The membranes were washed in PBS-Tween (0.01%; PBS-T), blocked in Pierce® Clear Milk Blocking Buffer (Thermo Fisher Scientific, MA, United States) for 1 hour, and then incubated overnight at 4°C with the primary antibody TSG101 (1:1000, ab30871, Abcam, Cambridge, United Kingdom), Alix (1:1000, EPR23653-32, ab275377, Abcam, Cambridge, United Kingdom), Calnexin (1:1000, ab22595, Abcam, Cambridge, United Kingdom), or GAPDH (1:2000, G9545-100UL, Sigma-Aldrich, United States).

[0222] After washing three times in PBS-T, the blots were incubated for 2 hours at room temperature with a suitable secondary antibody, either HRP-conjugated goat anti-rabbit IgG (H+L) (1:1000, 170-6515, Bio-Rad, CA, United States) or goat anti-mouse IgG (1:1000, 170-6516, Bio-Rad, CA, United States). The membranes were washed in PBS-T and developed for 2 minutes on Clarity® Western ECL Substrate (Bio-Rad, CA, United States). Imaging was performed using a ChemiDoc® MP Imaging System (Bio-Rad, CA, United States) with Image Lab® software.

[0223] retinal tissue analysis Collection and preparation of tissues The animals were ethically euthanized with CO2 after PD. The upper surface of the left eye was marked, excised, and then immersed in 4% paraformaldehyde for 3 hours. The eye was then cryopreserved overnight in 15% sucrose solution, embedded in OCT medium (Tissue Tek, Sakura, Japan), and frozen-sectioned at 12 μm in the lateral sagittal plane (upper to lower) using CM 1850 Cryostat (Leica Biosystems, Germany). To ensure accurate comparison for histological analysis, only sections containing the optic nerve head were used for analysis. The retina from the right eye was excised through a corneal incision, placed overnight in RNAlater solution (Thermo Fisher Scientific, MA, United States) at 4°C, and then stored at -80°C until further use.

[0224] immunolabeling Immunohistochemical analysis of frozen retinal sections was performed as previously described (Rutar et al, 2015). Fluorescence was visualized, and images were scanned with a laser at 20x and 40x magnification (A1). +Images were taken using a confocal microscope (Nikon, Tokyo, Japan). Image panels were analyzed using ImageJ V2.0 software and assembled using Illustrator software (Adobe Systems, CA, United States).

[0225] Immunohistochemistry and analysis Immunolabeling for IBA1, a marker for microglia and macrophage immune cells (1:500, 019 - 19741, Wako, Osaka, Japan) was performed as previously described (Rutar et al, 2015). Retinal frozen sections were stained with the DNA-specific dye bisbenzimide (BBZ; 1:10,000, Sigma-Aldrich, MO, United States) to visualize cell layers. IBA1 + The number and morphology (branched vs amoeboid) of cells were counted across the superior and inferior retinas using two retinal sections per mouse. Intensity analysis was performed 0.5 mm above the optic nerve using ImageJ V2.0 software and calculated as the relative intensity from the control.

[0226] TUNEL assay Terminal deoxynucleotidyl transferase (Tdt) dUTP nick end labeling (TUNEL) was used as a measure of photoreceptor cell death. TUNEL in situ labeling was performed on retinal frozen sections using a Tdt enzyme (catalog number 3333566001, Sigma-Aldrich, MO, United States) and biotinylated deoxyuridine triphosphate (dUTP) (catalog number 11093070910, Sigma-Aldrich, MO, United States) as previously described (Natoli et al, 2010). Images of TUNEL staining were captured at magnifications of 20x and 40x with an A1 + Nikon confocal microscope. TUNEL + The total number of cells was counted using two retinal sections per animal, including both the superior and inferior retinas.

[0227] To further quantify photoreceptor survival, the thickness of the outer nuclear layer (ONL) on retinal cryosections was determined by counting the number of nuclear rows (photoreceptor cell bodies) in the area of retinal lesion development (1 mm above the optic nerve head). Quantification of photoreceptor cell rows was performed five times per retina using two retinal cryosections at equivalent positions per mouse.

[0228] Brain tissue analysis Tissue collection and preparation After the behavioral tests, the mice were individually sacrificed by cervical dislocation, and the brains were immediately removed and placed in ice-cold PBS. Incisions were made through the entire left cortex of the brain to ensure that the hemispheres were not mixed throughout the slicing procedure. The brains were then sliced coronally in ice-cold PBS using a vibratome (Leica 1200s; 1 mm thickness, 0.75 mm amplitude, speed 1 mm / sec), and the striatum was carefully dissected from each slice in ice-cold PBS using sharp-pointed forceps. Control and experimental hemispheres were collected in separate tubes. Once the entire striatum was collected, the tissue was snap-frozen on dry ice and placed at -80 degrees until Western blotting was completed.

[0229] Western blot analysis The Bradford assay Rapid Gold BCA assay (ThermoFisher, A55860) was performed on each sample to confirm protein concentration. Samples were placed in 4× Laemmli sample buffer (BioRad, 1610747) with 5% 2-mercaptoethanol for reduction. Samples were denatured at 95 degrees for 5 minutes on a heating block and then loaded into wells of a Bolt™ Bis-Tris Plus Mini Protein Gel, 4–12% (Invitrogen, NW04122BOX). The gel was electrophoresed at 120 V for 1 hour in 1× MES SDS running buffer (Invitrogen, NP0002) at room temperature. The gel was then transferred onto a nitrocellulose membrane (BioRad, #1620112, 0.2 um pore size) and run at 100 V for 60 minutes at 4 degrees.

[0230] Next, the membrane was blocked for 1 hour in 5% non-fat dry milk in PBS-T (0.05% Tween) with constant rocking. Then, the primary antibodies diluted 1:1000 in PBS-T (2.5% non-fat dry milk, 0.05% Tween) were incubated overnight at 4°C (IBA1, Abcam, AB5076; beta-actin, Cell signalling, 8457S; tyrosine hydroxylase, ThermoFisher, OPA1-04050). Then, the membrane was washed in PBS-T for 30 minutes at room temperature, and then the secondary antibodies were added for 1 hour (anti-rabbit 41460; anti-goat AP163P; 1:10,000 dilution, PBS-T w / 2.5% non-fat dry milk, 0.05% Tween). Then, the membrane was washed in PBS-T.

[0231] The membrane was developed using Clarity Western ECL Substrate (BioRad #1705061) incubated for 5 minutes. Then, the membrane was imaged using a Chemidoc MP imaging system (BioRad).

[0232] All analyses were completed by measuring band intensity using the measurement function in ImageJ. All signals were normalized to the intensity of the housekeeping protein beta-actin, and the experimental side was normalized to the control side.

[0233] MiRNA Loading RBC-EV Electroporation RBC-EV electroporation was performed using a Gene Pulser II (BioRad) electroporation system with a fixed volume of 125 μF and an exponential program, in 0.2 cm cuvettes. For optimization, 1–2 × 10^11 RBC-EVs were mixed with 2–8 μg of microRNA and diluted to a total volume of 200 μl in either 1 × PBS (Gibco 7.2 pH), Opti-MEM® reduced serum medium, GlutaMAX® supplement (Thermo Fisher Scientific, MA, United States), or Gene Pulser electroporation buffer (BioRad). 200 μl of the EV mixture was added to each cuvette and incubated on ice for 10 minutes. Electroporation was tested at different voltages: 100–450 V. Aggregates of RBC-EVs formed during electroporation were dissolved by vigorous pipetting. To quantify the efficiency of electroporation, electroporated RBC-EVs were lysed and miRNA was quantified using the Qubit® microRNA assay kit.

[0234] Passive loading RBC-EV (1–2 × 10¹¹) was mixed with 2–8 μg of microRNA and diluted to a total volume of 200 μl in 1 × PBS (Gibco 7.2 pH), OptiMEM® reduced serum medium, GlutaMAX® supplement (Thermo Fisher Scientific, MA, United States), or Gene Pulser electroporation buffer (BioRad). The 200 μl EV mixture was incubated at 4 or 37°C for 1, 4, and 18 hours to determine the optimal conditions for passive loading. To quantify the passive loading efficiency and compare it to electroporation, RBC-EV was lysed and miRNA was quantified using the Qubit® microRNA assay kit.

[0235] miRNA quantification The actual concentration of miRNA encapsulated in RBC-EV was measured using the Qubit® MicroRNA Assay Kit. The RBC-EV-miRNA complex was first lysed by adding 5 μL of the complex to SDS buffer containing 90 μL of sterile 1×PBS and 3 μL of 0.5% SDS, releasing the incorporated miRNA. The protocol was modified from (Lamichhane and Jay, 2018). The miRNA-SDS solution was thoroughly mixed and incubated at 85°C for 15 minutes, with further thorough mixing midway. The miRNA was then quantified using the kit according to the manufacturer's instructions. 1 μL of miRNA-SDS solution was added in a Qubit® assay tube to 199 μL of Qubit® working solution, prepared by diluting the provided Qubit® MicroRNA reagent 1:200 in Qubit® MicroRNA buffer. The tube was vortexed for 5 seconds and then incubated at room temperature for 10 minutes. The sample concentrations were read and calculated using a Qubit® 3 fluorometer calibrated with the provided standards.

[0236] In vitro experiment Mouse photoreceptor-derived 661W cells (generous donation from Dr. Muayyad R. Al-Ubaidi, University of Houston (Al-Ubaidi et al, 1992)), human Müller-like MIO-M1 cells (Moorfield's Institute of Ophthalmology) (Limb et al, 2002), human RPE-like ARPE-19 cells (ATCC) (Dunn et al, 1996), and mouse microglia-like BV2 cells (Accegen) (Blasi et al, 1990) were used in in vitro experiments. The cells were cultured in growth medium in flasks in a humidified incubator at 37C with 5% CO2. The cells were subculturised every 3-5 days by trypsin treatment with 0.25% trypsin using a Nunc® Cell Scraper for MIO-M1 and ARPE-19 cells.

[0237] RBC-EV label SYTO(trademark) RNASelect™ green fluorescent cell staining To determine RBC-EV uptake in vivo and in vitro, RBC-EVs were labeled using SYTO® RNASelect® green fluorescent cell stain, a cell-permeable RNA-specific stain. The stain was first diluted 1:5 in DMSO (#ICN19141880, Thermo Fisher Scientific, MA, United States) to obtain a 1 mM DMSO stock. The RBC-EV stock was diluted to a total volume of 100 μL in sterile 1×PBS. 1×PBS was used as a control for background staining. Next, 1 μL of DMSO stock was added to the diluted RBC-EVs, thoroughly mixed, and incubated at 37C for 30 minutes. At approximately halfway through incubation, the solution was mixed to ensure that the dye did not settle at the bottom of the tube. Subsequently, the solution was added to an Amicon® 50kDa MWCO filter unit (UFC505024, Merck) pre-rinsed with UltraPure® distilled water, and spun at 5000×g for 20 minutes to remove excess unbound dye. The solution was further washed three times by adding 500 μL of sterile 1×PBS and spinning the column at 10,000×g for 10 minutes each time. The solution was then prepared to the same volume using sterile 1×PBS and immediately added to the cells. The cells were placed in an incubator set to 37C with 5% CO2 and fitted with an IncuCyte® ZOOM system. Phase images for visualization of cell morphology, and fluorescence images in the green channel (with excitation wavelengths of 440–480 nm and emission wavelengths of 504–544 nm) for visualization of labeled RBC-EVs were taken at 10x magnification every 2 hours for 48 hours. Control solutions containing only fluorescently labeled RBC-EV or PBS were also administered to mice via intravitreous injection (locally) or intraperitoneal injection (systemically), as described above.

[0238] In vitro experiment 661W cell culture Mouse photoreceptor-derived 661W cells (kindly donated by Dr. Muayyad R. Al-Ubaidi, Department of Biomedical Engineering, University of Houston, Houston, TX, United States) (Al-Ubaidi et al., 2013) were used in in vitro experiments with 1 to 5 passages. Cell authenticity was verified by short tandem repeat analysis (CellBank, Sydney, Australia). Cells were cultured in growth medium (Dulbecco's modified Eagle medium (DMEM; Sigma-Aldrich, MO, United States) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, MO, United States), 6 mM L-glutamine (Thermo Fisher Scientific, MA, United States), and antibiotic-antifungal agents (100 U / ml penicillin, 100 μg / ml streptomycin; Thermo Fisher Scientific, MA, United States)). Unless otherwise specified, cells were maintained, and all incubation steps were performed in the dark at 37°C in a humidified atmosphere of 5% CO2. Cells were subcultured every 3-4 days by trypsin treatment.

[0239] To deplete the FBS of extracellular life (EVs), serum was centrifuged using a Beckman Coulter Optima XE-100 ultracentrifuge (Beckman Coulter, CA, United States) equipped with an SW41Ti rotor (Beckman Coulter, CA, United States) (200,000 × g, 4°C for 18 hours), and the supernatant was used as FBS supplement in all EV collection experiments.

[0240] N1 medium supplement at a 1:100 dilution (Sigma; N6530) and superoxide dismutase (SOD) at a 1:500 dilution (Worthington Biochemicals; LS003540) were added to 661 watts for 18 hours before collecting the cell supernatant. EVs were isolated using fractional ultracentrifugation as previously published (Wooff et al., 2020).

[0241] In vitro photooxidative damage After 18 hours of N1 / SOD incubation, the cells were exposed to 15,000 lux (2.2 mW / cm²) of light from two white fluorescent lamps (2 x 10W T4 triphosphor 6500K daylight fluorescent tubes; Crompton, NSW, Australia). 2 The cells were exposed for 2 hours to illuminance measured using a PM100D optical power meter (Thorlabs, NJ, United States) (Lu et al., 2017; Fernando et al., 2018). Control cells were completely wrapped in aluminum foil with six small notches to allow air / gas exchange.

[0242] Quantitative proteomics Protein isolation Frozen aliquots of RBCs and RBC-EV+ / - supplements (N1, SOD, and N1 / SOD) were sent to the Australian Proteome Analysis Facility (APAF), Macquarie University, NSW, Australia, for protein isolation and digestion, as well as for LC-MS / MS tandem mass spectrometry using Orbitrap Exploris (Thermo Fisher Scientific) and NanoLC Vanquish Neo UHPLC (Thermo Fisher Scientific) systems.

[0243] Bioinformatics and Analysis Preprocessing: Initial processing of protein intensity data began with the application of base-2 logarithmic transformation. To ensure data integrity, proteins exhibiting more than two missing values ​​within any single experimental group were subsequently excluded from the expression matrix.

[0244] Missing Value Interpolation: For the remaining proteins showing missing values, an interpolation strategy was employed. This involved generating random values ​​from a Gaussian distribution anchored by the minimum value required for interpolation for each protein. This minimum value was equal to the 0.01 quantile of the observed data for each sample, representing a robust estimate of low expression levels. The standard deviation used in the Gaussian distribution was equal to the standard deviation of the samples requiring interpolation.

[0245] Data Normalization: After imputation, data normalization was performed using the Variance Stabilization Transform (VST). For this purpose, the `normalize_vst` function in the R DEP package was used. This function adjusted the count data by a normalization coefficient to produce a matrix with values ​​that are homoskedastic. Thus, this transformation ensured that the variance of the data remained constant across the range of the mean.

[0246] Differential Expression Analysis: The processed data was then subjected to differential expression analysis according to the limma pipeline. This involved using the lmFit function to fit a linear model to each protein, followed by the application of empirical Bayesian smoothing of the standard errors through the eBayes function. Finally, the topTable function was used to create a table of the most differentially expressed proteins. The limma pipeline is a well-established methodology in the field of bioinformatics for accurately and reliably identifying differentially expressed genes or proteins.

[0247] Enrichment analysis: Pathway analysis was performed through gene set enrichment analysis (GSEA) using the fgsea function. This method used the t-statistic as a ranking metric to prioritize genes, and the C2 canonical pathway and C5 gene ontology databases from the Molecular Signatures Database (MsigDB) functioned as reference gene sets. Additionally, the Enrichr platform was used to perform overrepresentation analysis (ORA) to identify significantly enriched pathways.

[0248] RNA sequencing RNA isolation and quality assessment Small RNA sequencing started with the lysis of RBC-EVs in 300 μL of lysis buffer, followed by the addition of 30 μL of miRNA homogenate and incubation on ice for 10 minutes. Then, 330 μL of acid-phenol:chloroform was added, and the mixture was vortexed for 60 seconds. Centrifugation at 10,000×g for 5 minutes facilitated phase separation. The aqueous phase was transferred and mixed with 1.25 volumes of ice-cold absolute ethanol for total RNA isolation or 1 / 3 volume of ice-cold absolute ethanol for small miRNA enrichment. The mixture was then passed through a purification column. The column was washed with 700 μL of miRNA wash solution #1, washed twice with 500 μL of wash solution #2, and then spin-dried. RNA was eluted with a heating solution (95 °C), collected in 30 μL, and stored at -80 °C. The quality and concentration of RNA were measured using a Nanodrop ND-1000 spectrophotometer and an Agilent 2100 Bioanalyzer.

[0249] Library preparation Library preparation for high-throughput sequencing of RNA from RBC EVs was performed at the Biomolecular Research Facility (JCSMR, ANU). Libraries were synthesized using Capture and Amplification by Tailing and Switching (Diagenode). The libraries were sequenced on an Illumina NovaSeq 6000, yielding at least 10 million 50 bp single-ended reads per sample.

[0250] Bioinformatics and Analysis Preprocessing: The index, adapter, and template switching oligonucleotides were removed using cutadapt with the following Unix shell clip, according to the product instructions.

[0251] Alignment: Use subread-align for reads into the mm10 mouse genome, specifying the following Unix shell script: subread-align -t 1 -i / mouse mm10 index -n 35 -m 4 -M 3 -T 10 -I 0 --multiMapping -B 10 -r miRNA_reads.fastq -o result.sam

[0252] Quantification: featureCounts was performed to quantify miRNA alignment using the following Unix shell script: featureCounts -t miRNA -g Name -O -s 1 -M -a / data / human / miRBase / mmu.gff3 -o miR_counts.txt result.sam

[0253] Differential expression analysis: The following command sequence, voom-->lmFit-->ebayes-->topTable, was used with the limma package to evaluate differential gene expression data in RStudio.

[0254] The Voom function, part of the limma package, is used to convert count data from RNA-seq into log2 counts per million (log-CPM), which are suitable for linear modeling. Voom estimates the mean-variance relationship of the log counts, assigns weights to each observation, and generates a "voom" object containing the weighted expression data. These weights are used in subsequent modeling steps to account for heterospersity (non-constant variance) inherent in the count data.

[0255] Once the lmFit:voom transformation is complete, the lmFit function fits a linear model to each gene (or transcript). This function corresponds to the experimental design matrix, which specifies the different conditions under which RBCEVs are provided. lmFit takes the experimental design into account and uses the weighted data from the voom step to fit the model.

[0256] ebayes: After fitting a linear model, empirical Bayesian relaxation is applied using the ebayes function. This step borrows information across all genes to obtain more accurate estimates of gene-specific variances, thereby obtaining more stable inferences about differential expression.

[0257] topTable: The final step involves summarizing the results using the topTable function. This function ranks genes based on ebayes' results using p-values ​​or adjusted p-values ​​and log-multiplier changes. Genes with p-values ​​< 0.05 and log-multiplier changes > 0.5 were considered biologically relevant.

[0258] Survival rate and inflammatory profiling of RBC-EV RBC-EV+ / - supplements (N1, SOD, N1 / SOD) in frozen -80C aliquots were sent to CruxBioLabs (VIC, AUS) for independent validation of their safety / toxicity and anti-inflammatory properties using control and LPS-stimulated human peripheral bone mononuclear cells (PBMCs).

[0259] Flow cytometry analysis of RBC-enriched fraction To evaluate the percentage of red blood cells (RBCs) after plasma removal and leukocyte depletion, RBCs were diluted 1:10 in PBS with 1×N1 medium supplement and SOD. Ten μl of this diluted sample was then combined with each of the following antibody preparations: TER119-PE-Cy7 (BD Biosciences, 557843), CD41-FITC (BD Biosciences, 553848), CD71-eFluor450 (LifeTech, 48-0711-82), CD45-APC-Cy7 (LifeTech, 47-0451-82), and a control without antibody, and incubated on ice for 30 minutes. Two hundred μl of FACS buffer (PBS, 0.5% BSA) was added to the sample, and cells were pelleted by centrifugation at 1500×g for 5 minutes. The pellet was resuspended in 200 μl of FACS buffer and analyzed on LSR-II, recording 1 million RBC events to identify populations of RBCs, platelets, leukocytes, and reticulocytes.

[0260] Example 2: Extracellular vesicles can be isolated from mouse erythrocytes. Extracellular vesicles (RBC-EVs) derived from erythrocytes were isolated from mouse whole blood using a novel experimental paradigm. After plasma removal and leukocyte depletion, RBCs were incubated overnight at 37°C in 1:10 dilution PBS with or without culture supplement N1 and / or SOD. Small EVs were isolated from the RBC suspension (pellet 4; P4) using serial fractionation ultracentrifugation (Figure 2A). RBC-EVs were characterized for morphology and size using cryo-electron microscopy (cryo-EM), exhibiting a rounded shape (Figure 2B) and a size distribution of 40 nm to 320 nm, peaking at 160 nm (Figure 2C). Western blotting was performed on retinal cell lysates and RBC-EV lysates to identify cellular contaminant markers and EV markers. A strong 90 kDa band was present in the retinal lysate for the cellular marker calnexin (CNX), but no expression was shown in RBC-EVs. Both retinal and RBC-EV lysates contained a 50 kDa band for the EV marker tumor susceptibility gene 101 (TSG101) (Figure 2D). To identify EV marker enrichment, the EV marker proteins TSG101 and ALIX were compared on RBC and RBC-EV lysates against the reference protein GAPDH (37 kDa). Both TSG101 (50 kDa) and ALIX (100 kDa) were found to be expressed at higher levels in RBC-EV compared to RBC (Figure 2E). In summary, these results suggest that EVs can be isolated from RBC and exhibit classical EV marker and undetectable contamination.

[0261] Example 3: Supplementation with N1 and SOD improves RBC health and RBC-EV quality. To improve the incubation and RBC-EV quality of RBCs, N1 medium supplement and the antioxidant SOD were added individually and in combination to the overnight incubation paradigm of RBCs and compared with RBC-PBS. RBC abnormalities (teardrop shape, urchin-like cells) were quantified before and after overnight incubation at 37°C (Figure 14). Compared to fresh blood (pre-treatment; Pre-Tx), RBCs incubated in PBS alone and N1 supplement had higher abnormalities, but RBCs with SOD, or a combination of N1 and SOD, had significantly lower abnormalities compared to the RBC-PBS and RBC-N1 groups (Figure 3A, P<0.05). The size distribution profiles of RBC-EVs were also measured for each preparation using nanotracking analysis (Nanosight NS300), showing a uniform size distribution with a single peak EV for the N1, SOD, and N1 / SOD groups, while RBC-PBS alone produced a dual-peak EV according to our measurements, further testing the reproducibility of this incubation. On average, the number of RBC-EVs from the N1 / SOD incubation (Figure 3D(i)), mean and modal sizes (Figure 3D(ii)), and size distribution profiles (Figure 3D(iii)) remained consistent across blood batches, demonstrating a reproducible and reliable incubation and isolation paradigm.

[0262] Example 4: Combined supplementation of N1 and SOD provided neuroprotective properties in a photooxidative damage-induced retinal neurodegeneration model. To investigate the potential protective effects of RBC-EV and N1 / SOD supplementation in slowing progressive retinal degeneration, RBC-EV(N1 / SOD) was added in a quantity of 2.0 × 10⁶ per 1 μl. 9Mice were injected with RBC-EV at doses prior to photooxidative damage and compared to RBC-EV, PBS(N1 / SOD), and PBS-injected controls (Figure 4A). Retinal function was measured after 5 days of photooxidative damage, and both the RBC-EV(N1 / SOD) and PBS(N1 / SOD) groups showed significantly higher retinal function than the unsupplemented control, for both wave a (Figure 4B, P<0.05) and wave b (Figure 4C, P<0.05) measurements.

[0263] Fundus imaging using MICRON IV did not show any degenerative areas in any group, but vascular hemorrhage near the optic nerve was observed in PBS(N1 / SOD) injected mice (Figure 15). Photoreceptor cell death was measured using the TUNEL assay and photoreceptor row counting.

[0264] The results showed that both PBS(N1 / SOD) and RBC-EV(N1 / SOD) injected mice had a significantly reduced number of TUNEL+ cells in the ONL compared to the unsupplemented control (Figure 4D, P<0.05), but RBC-EV(N1 / SOD) injected mice had a significantly higher number of photoreceptor rows than the PBS(N1 / SOD) control (Figure 4E, P<0.05). Retinal inflammation was further measured using IBA-1 immunohistochemistry as a marker of the presence of microglia / macrophage immune cells. RBC-EV(N1 / SOD) injected mice had a significantly reduced number of IBA-1+ cells in the lateral retina compared to the PBS control (Figure 4F, P<0.05), but no significant difference was observed between any of the other groups. Representative confocal images show reduced photoreceptor cell death in the supplement group, while RBC-EV(N1 / SOD) shows the lowest levels of photoreceptor cell death and the presence of inflammatory cells (Figure 4G-H).

[0265] These results support the use of N1 / SOD supplementation in RBC incubation, with the highest retinal protection observed in the RBC-EV(N1 / SOD) group compared to the control group without supplementation.

[0266] Example 5: SOD, and combined supplementation of N1 and SOD, provided neuroprotective properties in a photooxidative damage-induced retinal neurodegeneration model. To further investigate the potential protective effect of N1 and SOD supplementation in slowing progressive retinal degeneration, RBC-EV(N1), RBC-EV(SOD), and RBC-EV(N1 / SOD) were added in 2.0 × 10⁶ units per 1 μl. 9 Mice were injected with individual doses of EV prior to photooxidative injury and compared to RBC-EV controls. Retinal function was measured after 5 days of photooxidative injury, and no significant differences were observed in wave a (Figure 5A, P>0.05) or wave b (Figure 5B, P>0.05) measurements.

[0267] Photoreceptor cell death was measured using the TUNEL assay and photoreceptor row count. The results showed a significant reduction in the number of ONLs in RBC-EV(SOD) and RBC-EV(N1 / SOD) injected mice compared to unsupplemented controls. + Cells (Figure 5C, P<0.05), and RBC-EV(N1 / SOD) injected mice showed a significantly higher number of photoreceptor rows (Figure 5D, P<0.05). Retinal inflammation was further measured using IBA-1 immunohistochemistry as a marker of the presence of microglia / macrophage immune cells. No differences were observed among RBC-EV, RBC-EV(N1), and RBC-EV(N1 / SOD) injected mice, however, RBC-EV(SOD) injected mice showed a higher number of IBA-1 in the lateral retina. +The number of cells was significantly reduced (Figure 5E, P<0.05). OCT thickness measurements showed no changes in ONL, INL, or GCL+IPL between any group (Figure 5F, P>0.05), but RBC-EV(N1 / SOD) injected mice showed a significant increase in overall retinal thickness compared to controls. The preserved retinal health was reflected in representative fundus and optical coherence tomography (OCT) images (Figures 5G-H).

[0268] These results support the use of N1 / SOD supplementation in RBC incubation, as it reduced cell death compared to controls without supplementation.

[0269] Example 6: RBC-EV(N1 / SOD) is a safe delivery vehicle. Considering the protective effect of N1 / SOD supplementation, the safety of RBC-EV(N1 / SOD) as a delivery vehicle for topical treatment was evaluated. Mice were administered 2.0 × 10⁶ units per 1 μL using intravitreous injection. 9 Individual doses of RBC-EV(N1 / SOD) were injected and left for 7 days under standard containment conditions (Figure 6A). Retinal function was measured using ERG, and no significant differences were observed in either wave a (Figure 6B) or wave b (Figure 6C) responses compared to PBS-injected controls (P>0.05). No differences were observed in the measurement of cell death (Figure 6D-E; P>0.05) or inflammation (Figure 6F; P>0.05), as shown in representative confocal images (Figure 6G-H). Overall, RBC-EV(N1 / SOD) did not cause toxicity or damage to the retina in the measured parameters, suggesting that they can be used as a safe delivery vehicle via topical intravitreal injection.

[0270] Example 7: Local delivery of RBC-EV(N1 / SOD) provides neuroprotection in a retinal neurodegeneration model. The therapeutic efficacy of RBC-EV(N1 / SOD) was investigated in a rodent model of retinal neurodegeneration using intravitreous delivery (Figure 7A). Retinal function was measured using ERG after 5 days of photooxidative damage.

[0271] The results showed that RBC-EV(N1 / SOD)-injected mice significantly conserved function in both wave a (Figure 7B, P<0.05) and wave b (Figure 7C; P<0.05) responses compared to PBS-injected controls, along with significantly reduced levels of photoreceptor cell death as measured by the TUNEL assay (Figure 7F, P<0.05) and photoreceptor column counting (Figure 7E, P<0.05). Furthermore, RBC-EV(N1 / SOD)-injected mice significantly reduced the number of IBA-1+ cells in the lateral retina, indicating the presence of reduced inflammatory cells (Figure 7F; P<0.05). Representative confocal images show retinal protection against cell death and inflammation measurements after local administration of RBC-EV(N1 / SOD) (Figures 7G-H).

[0272] Example 8: Systemic delivery of RBC-EV(N1 / SOD) is safe. Following the robust safety and therapeutic effects observed in the local delivery paradigm, systemic administration of RBC-EV(N1 / SOD) was investigated. RBC-EV(N1 / SOD) was administered in a dose of 2.0 × 10⁶ units per 100 μL. 11 Individual doses of EV were administered daily via intraperitoneal injection for 7 days and compared to a PBS-injected control (Figure 8A). As in previous paradigms, retinal function and morphological assessments were performed after 7 days of administration under standard containment conditions. No significant differences in retinal function were found between the groups for a-wave (Figure 8B; P>0.05) or b-wave (Figure 8C; P<0.05) measurements. In addition, there were no differences in the levels of cell death (Figure 8D-E; P<0.05) or inflammation (Figure 8F; P<0.05), as shown in representative confocal images (Figure 8G-H). Systemic administration of RBC-EV(N1 / SOD) appeared not to cause any retinal toxicity after 7 days of daily administration.

[0273] Example 9: Systemic delivery of RBC-EV(N1 / SOD) provides retinal protection against photooxidative damage. The therapeutic efficacy of whole-body RBC-EV(N1 / SOD) was 2.0 × 10⁶ per 100 μL, after 5 days of photooxidative damage. 11This was investigated by using daily intraperitoneal injections of individual EV doses and compared with PBS (Figure 9A). Retinal function, measured using ERG, showed significantly conserved retinal function in RBC-EV(N1 / SOD)-injected mice for both a-wave (Figure 9B; P<0.05) and b-wave (Figure 9C; P<0.05) responses. Furthermore, compared to PBS-injected controls, RBC-EV(N1 / SOD)-injected mice had significantly reduced levels of TUNEL+ cells in the ONL (Figure 9D; P>0.05), significantly higher numbers of photoreceptor rows (Figure 9E; P<0.05), and significantly reduced numbers of IBA-1+ microglia / macrophages in the outer retina (Figure 9F; P<0.05). Representative confocal images show reduced levels of retinal cell death and inflammation in systemically injected RBC-EV(N1 / SOD) mice (Figure 9G-H), suggesting that potent therapeutic protection may be conferred to the retina via systemic administration.

[0274] Example 10: RBC-EV(N1 / SOD) demonstrates efficient uptake into retinal cells in vitro and in vivo. To confirm safety and therapeutic outcomes and determine the uptake efficiency of RBC-EV(N1 / SOD) in the retina, RBC-EV(N1 / SOD) was fluorescently labeled using SYTO RNASelect (green) and incubated on retinal cell lines or injected into mice using intravitreous injection. Uptake efficiency and localization output measurements were observed (Figure 10A). In vitro uptake in four retinal cell lines, 661w (photoreceptor cell line), BV2 (microglia cell line), MIOM1 (Müller cell line), and aRPE19 (RPE cell line), was measured over 24 hours using the IncuCyte® ZOOM system. Representative microscopic images show the fluorescent green labeling in all retinal cell lines after 24 hours of incubation (Figure 10B). Quantitative analysis of uptake efficiency shows that by 24 hours, both 661w and MIOM1 cell lines achieved nearly 100% uptake of RBC-EV(N1 / SOD), while BV2 and aRPE19 cell lines achieved approximately 50% uptake (Figure 10C). Subsequently, 2.0 × 10⁶ per 1 μl9 Following intravitreal injection of individual EV doses on day 0, in vivo retinal uptake was observed for 7 days. Uptake could be observed as early as 2 hours after injection at the injection site (superior retina), progressively spreading radially across the retina (superior-inferior) and through the retinal layers. Strong labeling was observed within the ONL by 6 hours, and labeling in the outer segments was observed from 48 hours. Labeling was still observed throughout the entire retina for 7 days (Figure 10D).

[0275] Example 11: RBC-EV(N1 / SOD) can be used as a miRNA delivery vehicle to the retina. To determine whether RBC-EV(N1 / SOD) could be used as a therapeutic delivery vehicle, we optimized the microRNA (miRNA) encapsulation method by comparing electroporation and passive loading techniques with modifications to voltage settings, incubation time, temperature, and buffer (Figure 11A). Following loading in each optimization experiment, the amount of encapsulated miRNA (μg) was quantified using the Qubit miRNA assay (Figure 11B). miRNA retention was found to increase with increasing voltage up to 350V, but no improvement in miRNA retention was observed at 450V (Figure 11C). Furthermore, Gene Pulser buffer (BioRad) was found to be the most optimal for miRNA retention, and post-electroporation incubation at 37C improved miRNA retention compared to incubation at 4°C for all electroporation media and buffers tested (Figure 11D). miRNA retention was not found to increase proportionally with the miRNA input; rather, the retention percentage decreased as the input increased (Figure 11E), suggesting that a maximum threshold should exist using electroporation. Finally, the integrity of RBC-EV(N1 / SOD) was evaluated before and after electroporation using cryoEM at optimal settings (350V, Gene Pulser buffer, 37C post-incubation). No significant morphological changes or aggregations were found in RBC-EV(N1 / SOD) after electroporation (Figure 11F).

[0276] In summary, these results support the capability of RBC-EV(N1 / SOD) for use in therapeutic miRNA encapsulation for delivery to the retina.

[0277] Example 12: N1 and SOD supplementation confer protection to 661w photoreceptor cells and enhance EV production in vitro. To validate the protective properties of N1 and SOD supplementation for cell health, retinal health, and EV health, 661w photoreceptor-like cells were incubated for 18 hours in or without N1 / SOD before 2 hours of photooxidative damage at 15,000 lux (Figure 12A). EV distribution profiles and sizes determined by nanoparticle tracking analysis showed a significant increase in EV number after N1 / SOD supplementation, but not in size (Figures 12B-D, P<0.05). Representative images taken before N1 / SOD administration, after 18 hours of incubation, and after 2 hours of photooxidative damage showed no changes in visible cell morphology and confluence measurements between the control and supplementation groups at 0 and 18 hours. However, after photooxidative damage, cells incubated with N1 / SOD showed better preserved morphology (processes) and less visible cell death (rounded and darker cells) than 661w control cells (Figure 12E).

[0278] In summary, these results suggest that 661w incubated with N1 / SOD supplementation exhibited increased protection against photooxidative damage and increased levels of EV production (N=3).

[0279] Example 13: The antioxidant capacity of RBCs is increased by N1 / SOD supplementation. Levels of SOD activity, measured by superoxide quenching ability, were assessed in RBCs with and without N1 or SOD supplementation using a SOD colorimetric activity assay. Increased SOD activity correlated with increased reactive oxygen species (ROS) accumulation (Figure 13A). The results showed that RBC(N1), RBC(SOD), and RBC(N1 / SOD) significantly reduced SOD activity compared to the control, supporting the reduction in intracellular ROS construction after 18 hours of incubation with supplementation (Figure 13B, P<0.05). No significant differences in SOD activity were observed between the supplementation groups (Figure 13B, P>0.05). These results suggest that N1, SOD, or N1 / SOD supplementation can improve the antioxidant capacity of RBCs.

[0280] Example 14: Health and quality evaluation of RBCs after N1 / SOD incubation RBCs were evaluated for morphological changes using the ImageJ plugin (Figure 14A) before culture and (Figure 14B) after incubation at 37°C overnight with N1 / SOD supplementation (Figures 14C-F). The number of abnormalities (tear droplet shape, size / shape variation, and presence of urchin-like cells) was counted and calculated as a percentage of normal RBCs.

[0281] Example 15: Systemic delivery of RBC-EV(N1 / SOD) protects against sodium iodate-induced panretinal neurodegeneration. Considering the observed local and systemic protection of RBC-EV(N1 / SOD) administration for photooxidative damage-induced retinal degeneration, the therapeutic efficacy of RBC-EV(N1 / SOD) was investigated in a secondary model of retinal degeneration—sodium iodate (NaIO3) (Figure 16A). Compared to PBS-injected controls, RBC-EV(N1 / SOD)-injected mice showed improved retinal function in terms of a-wave measurements (Figures 16B-C; P<0.05). No significant difference was observed between groups in b-wave response (Figure 16D, P>0.05), which reflects the characteristic chemical properties of the model and was not expected to improve regardless of treatment. We also measured photoreceptor cell death (TUNEL) and the presence of immune cells (IBA-1) as a marker of inflammation. RBC-EV(N1 / SOD)-treated mice showed no significant reduction in cell death (Figure 16E, P>0.05) and a significant reduction in total IBA-1+ immune cells (Figure 16F, P<0.05) compared to PBS-injected controls. H2O-injected mice were also included as baseline controls, and comparisons showed that NaIO3-injected mice treated with RBC-EV(N1 / SOD) were closer to control levels for all measurements compared to untreated mice (Figures 16A-F). Overall, these results demonstrate significant improvements in retinal function and inflammation in mice treated with RBC-EV(N1 / SOD) for NaIO3-induced retinal degeneration, further supporting the therapeutic potential of this treatment.

[0282] Example 16: RBC-EV(N1 / SOD) is safe and provides therapeutic protection against neuroinflammatory and motor deficit features in a neurodegenerative Parkinson's disease model. To test the broad applicability of RBC-EV(N1 / SOD) therapy, RBC-EV(N1 / SOD) was administered topically (striatal injection) to a neurotoxin model of Parkinson's disease (6-OHDA). The 6-OHDA model of Parkinson's disease induces selective destruction of dopaminergic and noradrenergic neurons through the generation of reactive oxygen species (Masini et al., 2021). Due to the structural similarity of 6-OHDA to dopamine, dopaminergic cells take up 6-OHDA via dopaminergic receptors and membrane transporters, which accumulate in these neurons. 6-OHDA is then oxidized by the enzyme monoamine oxidase, resulting in the release of reactive catecholamine quinones, hydrogen peroxide, and other reactive oxygen species. When injected directly into catecholamine-mediated regions such as the striatum, 6-OHDA rapidly depletes dopaminergic neurons and progressively induces dopaminergic denervation over a 3-5 week period, which can be observed through behavioral tests, molecular, and histochemical analyses.

[0283] Neuromotor responses and neuroinflammation were measured in vehicles, as well as in RBC-EV(N1 / SOD) injected controls (healthy; without 6-OHDA) and degenerated mice (6-OHDA), 7 days after degeneration-induction / treatment-administration (Figure 17A). Western blotting was performed for tyrosine hydroxylase (TH) and IBA-1 as measures of dopamine production in dopaminergic neurons (neuronal activity) and the presence of immune cells (inflammation), respectively (Figure 17B(i)). To test neuromotor deficits as a result of 6-OHDA, the strength of the mouse forelimbs was tested using a grip strength paradigm.

[0284] Mice injected with PBS / 6-OHDA showed significantly reduced TH levels compared to PBS-injected controls (Figure 17B(ii), P<0.05), supporting the use of 6-OHDA to induce dopamine loss. No significant difference was observed between the control and degenerated groups (Figure 17B(ii), P>0.05), indicating that while RBC-EV(N1 / SOD) is safe, it did not provide potent therapeutic protection against TH loss at this early disease stage investigated. Importantly, IBA-1 levels were found to be significantly increased in 6-OHDA / PBS-injected mice compared to PBS / PBS-injected controls and compared to 6-OHDA / RBC-EV(N1 / SOD)-injected mice (Figure 17B(iii), P<0.05). In mice injected with 6-OHDA / RBC-EV(N1 / SOD), no difference was measured in IBA-1 levels compared to controls, demonstrating both the safety and potent therapeutic efficacy of RBC-EV(N1 / SOD) treatment in reducing neuroinflammation.

[0285] Finally, no significant differences were observed between any group in either rotarod or grip strength behavioral measurements. However, mice treated with 6-OHDA / RBC-EV(N1 / SOD) showed increased latency to fall compared to mice injected with 6-OHDA / PBS (Figure 17C(i), P>0.05), and further evaluation of potential improvements should be made at later time points when behavioral effects become more evident in this model. Overall, these results support the use of RBC-EV(N1 / SOD) as a safe and effective treatment for the early neuroinflammatory features of Parkinson's disease, and further evaluation is needed at later time points in the model to see its efficacy against neuromotor deficits.

[0286] Example 17: RBC-EV(N1 / SOD) performs better than commercially available competing RBC-EV products in protecting against retinal degeneration in vivo. RBC-EV(N1 / SOD)(RBC-EV CVR) was compared to RBC-EV from a market competitor (RBC-EV Comp) along with its respective vehicle control. PBS-injected mice, healthy mice (housed in dim light; DR), and uninjected mice (photo-oxidative damage; PD) were also included as control groups.

[0287] Functional assessments showed that, for both a-wave (Figure 18A, P<0.05) and b-wave (Figure 18B, P<0.05) measurements, photooxidatively damaged mice injected with RBC-EV(Comp) or vehicle (CVR) 5 days later significantly preserved retinal function compared to PBS-injected controls. No significant functional differences were observed between RBC-EV(CVR) or RBC-EV(Comp), or between the RBC-EV(CVR) and vehicle (CVR) groups, but RBC-EV(Comp) and vehicle (CVR) injected mice significantly preserved retinal function compared to vehicle (Comp). No significant difference was observed between PBS-injected controls and vehicle (Comp), suggesting that this vehicle does not have a protective effect on the retina, while vehicle (CVR) was found to be protective against retinal degeneration (Figures 18A and B).

[0288] Retinal thickness measurements showed that mice injected with RBC-EV(CVR) had significantly thicker outer granular layer (ONL) and overall retinal thickness compared to the RBC-EV(Comp) and vehicle(CVR) groups (Figure 18C-E), indicating increased photoreceptor survival in these mice. Increased photoreceptor survival can also be indicated by increased photoreceptor column counts in both the vehicle(CVR) and RBC-EV(CVR) groups compared to the PD control, vehicle(Comp), and RBC-EV(Comp) groups, respectively (Figure 18F, P<0.05).

[0289] Inflammation (IBA-1 in the lateral retina) + Cells) or cell death (TUNEL during ONL) +No significant differences were observed between the groups (measured by cells) (Figure 18G and H, P>0.05). These results support the protective effect of RBC-EV(N1 / SOD) and, overall, demonstrate a more potent therapeutic efficacy in protecting against retinal degeneration than the leading commercially available RBC-EV(Comp) product tested.

[0290] Example 18: Antioxidant supplementation test for RBC / RBC-EV Resveratrol is a naturally occurring phytoestrogenic polyphenol / stilbene found in red grapes, berries, and peanuts, possessing high antioxidant properties. Resveratrol acts by inducing antioxidant enzymes such as SOD, glutathione peroxidase-1, and heme oxygenase, thereby inhibiting ROS production. Resveratrol has shown protective effects in cancer, cardiovascular disease, and neurodegeneration, with anti-apoptotic, anti-angiogenic, and anti-inflammatory effects (Salehi et al., 2018).

[0291] Kaempferol is a natural antioxidant found in fruits and vegetables, a phytoestrogenic polyphenol flavonol / flavonoid with known antioxidant and anti-inflammatory effects. Kaempferol acts through metalloproteinase inhibition, ROS inhibition, and SOD and glutathione regulation (Silva, 2012).

[0292] Glutathione is a potent non-protein / non-enzymatic thiol antioxidant, one of the most important in the body, and is produced by cells in the cytoplasm (not dietary). Glutathione exists in millimolar concentrations. Glutathione exists in reduced (GSH) and oxidized (GSSG) states, and the GSH:GSSG ratio indicates cellular stress (higher GSSG = more oxidative stress). Glutathione protects cells by scavenging / reducing ROS and as a cofactor for detoxification enzymes such as glutathione peroxidase (Averill-Bates, 2023). In the retina, GSH is the most potent and abundant antioxidant, and is reduced in patients with retinal degeneration (Sreekumar et al., 2021).

[0293] Compared to untreated RBCs, RBCs treated with N1 / SOD had the lowest membrane abnormality percentage compared to RBCs treated with PBS, as well as all doses of RBCs treated with resveratrol, kaempferol, and glutathione (Figure 19A). Within the dose range, resveratrol (R 100 μM), kaempferol (K 2 μM), and glutathione (G 0.1 mM) were found to produce RBCs with the lowest membrane abnormality percentage compared to the other doses tested (Figure 19A). These doses were selected for future studies and compared to the RBC(PBS), RBC(N1), RBC(SOD), and RBC(N1 / SOD) groups. The results showed that, compared to untreated RBCs, RBC(N1), RBC(SOD), RBC(N1 / SOD), and RBC(R 100μM) significantly reduced the membrane abnormality percentage (Figure 20A, P<0.05), while RBC(PBS), RBC(K 2uM), and RBC(G 0.1mM) did not exhibit any observed protection (Figure 20A, P>0.05). Of all the supplements tested, RBC(N1 / SOD) had the lowest membrane abnormality percentage. RBC counts were also measured to assess RBC loss compared to newly isolated RBCs (untreated), with the RBC(N1 / SOD) group showing the highest preservation of RBC counts and the RBC(PBS) and RBC(R 100uM) groups showing the lowest. (Figure 20B) The RBC-EV size distribution was also compared among the supplement groups. The most uniform distribution was observed in the RBC-EV(N1), RBC-EV(SOD), and RBC-EV(N1 / SOD) groups, while the distribution profiles of RBC-EV(PBS), RBC-EV(R 100μM), RBC-EV(K 2μM), and RBC-EV(G 0.1mM) were heterogeneous / uneven and biased (Figure 20C). The results showed a reduction in RBC-EV concentration for RBC-EV(N1) and RBC-EV(R 100μM) compared to the other groups, but this was not statistically significant (Figure 20D, P>0.05). No differences in mean (Figure 20E) or mode (Figure 20F) size were observed among any of the supplement groups (P>0.05). Overall, RBC-EV(K 2μM) and RBC-EV(G 0.1mM) showed the least protective effect on both the quality and quantity of RBC and RBC-EV.

[0294] Example 19: Effect of supplementation on the endogenous properties of RBCs and RBC-EVs To assess the endogenous levels of SOD and hemoglobin in RBCs and RBC-EVs, SOD activity assays and hemoglobin assays were performed on RBCs and RBC-EVs after 18 hours of incubation in response to supplementation. The results showed that RBCs incubated with (N1 / SOD) and (K 2 μM) had significantly higher SOD activity compared to RBC(PBS) controls (Figure 21A(i), P<0.05), while RBC-EVs (R 100 μM) and RBC-EVs (K 2 μM) had significantly increased SOD activity compared to RBC-EV(PBS) controls (Figure 21A(ii), P<0.05). No significant differences in hemoglobin levels were measured among any of the RBC groups (Figure 21B(i), P>0.05). However, RBC-EV(N1) and RBC-EV(R 100μM) had significantly lower levels of hemoglobin compared to the RBC-EV(PBS) control (Figure 21B(ii), P<0.05).

[0295] Example 20: Evaluation of in vivo protection from RBC-EV resveratrol supplementation Following in vitro testing and analysis, RBC health was found to be significantly preserved (reduced membrane abnormality %) when incubated with resveratrol (100 μM) compared to the untreated control. Therefore, RBC-EV (Resv) was tested in vivo for its therapeutic efficacy in a photooxidative damage model of retinal degeneration (1) compared with PBS (Resv 100 μM) and the PBS control (Figure 22A).

[0296] Retinal function and morphology were measured after 5 days of photooxidative damage. No significant differences in retinal function were observed for wave a (Figure 22B, P>0.05) or wave b (Figure 22C, P>0.05) measurements. Retinal thickness measurements were performed on OCT images, and the thickness of the outer granular layer (ONL) was significantly increased between the PBS (Resv) group and the RBC-EV (Resv) group (Figures 22D-E, P<0.05), but no significant differences were observed in any other group or retinal layer measured. Overall, RBC-EV (Resv) did not provide significant protection to the retina against photooxidative damage-induced retinal degeneration, but it did increase ONL thickness.

[0297] Example 21: Characterization of human RBCs and RBC-EVs To validate our RBC incubation pipeline in mice, human blood was collected from LifeBlood Australia and incubated in 1:10 PBS with N1, SOD, or N1 / SOD supplementation according to the methodology described above. Baseline abnormality measurements were obtained before incubation and compared to mouse RBC abnormality percentage. Mouse RBCs showed approximately 30% abnormality (N=4), while human RBCs had approximately 40% abnormality (N=1) at baseline (Figure 23A). Mouse RBC viability was analyzed using imaging flow cytometry (Amnis, ImageStream) analysis, and calcein + (Survival rate marker), Annexin V + (Apoptosis marker), and dual + Analysis was performed by the percentage of cells. The results showed that, compared to the pre-treatment control, the RBC(PBS), RBC(N1), and RBC(N1 / SOD) groups showed increased calcein levels. + A significant decrease in the percentage of RBCs was observed (Figure 23B, P<0.05). Annexin + or double + No significant differences were observed between the groups in terms of the percentage of cells (Figure 23B, P>0.05).

[0298] Human RBC counts were counted after an 18-hour incubation period for supplementation and compared to pre-treatment RBC counts, but no significant difference in counts was observed between groups (Figure 23C, P>0.05). The health of human RBCs was assessed, and the lowest percentage of abnormalities was observed in the RBC(SOD) and RBC(N1 / SOD) groups (Figure 23D). Human RBC survival rates were analyzed using flow cytometry, as was done for mouse RBCs. Results for both FACS Aria II and Amnis ImageStream analysis showed high RBC survival rates across all RBC groups, with no significant differences between groups (Figure 23E, P>0.05).

[0299] Collectively, these results indicate that human RBCs are of high quality and that SOD and N1 / SOD supplementation demonstrate the most potent preservation of RBC health. Characterization and quality of RBC-EVs were also performed on the human RBC group, showing no significant differences in size distribution profiles or mean or mode sizes (Figures 24A-B, P>0.05), and no significant differences in concentrations between groups (Figures 24B(i)-(iii), P>0.05). The distribution of human RBC-EVs, as well as their mean and mode sizes, for the supplement groups were very similar to those of mouse blood, indicating a robust and transportable pipeline.

[0300] Example 22: In vitro photo-oxidative damage induces photoreceptor EV release. To evaluate the effects of photooxidative damage on photoreceptors, 661w photoreceptor-like cells were exposed to bright white light at 25,000 lux for 4 hours, and extracellular volumes (EVs) were collected from the cell supernatant and compared to a dimly lit control (standard cell culture incubator environment; DR) (Figure 25A). EV distribution profiles and sizes determined by nanoparticle tracking analysis showed a significantly increased number of 661w-EVs after photooxidative exposure compared to control cells (Figures 25B-C, P<0.05), although no change was observed in their mode size (Figure 25D, P>0.05). Overall, these results support increased photoreceptor EV release after stress.

[0301] Example 23: Supplementary effects of 661w on health and protection against photo-oxidative damage To quantify the protective capacity of supplements against 661w cells in response to photooxidative damage, N1 and SOD were compared individually and in combination with resveratrol, kaempferol, and glutathione supplements, as well as PBS and untreated controls. Prior to photooxidative damage, supplements were incubated in 661w cells for 18 hours, followed by recovery under bright white light at 25,000 lux for 2, 3, and 4 hours, and then 24 hours under standard conditions after 2 hours. Supplements were used at either full dose (R 100 μM, K 2 μM, and G 0.01 mM) (Figure 26A) or half dose (R 50 μM, K 1 μM, and G 0.005 mM) (Figure 27A). Cell health was assessed using cytotoxicity assays.

[0302] The results showed that in the baseline (0h) group, compared to untreated cells, some toxicity was caused by supplementation, with the exception of SOD and K 2μM. However, after 2 and 3 hours of PD, R 100μM and N1 supplementation were found to confer significant toxicity to 661w cells, respectively. By 4 hours of PD, all cells had high levels of toxicity, reflecting high levels of cell death from photooxidative damage exposure at this point (Figure 26B(i)-(iv)). Comparative analysis across all time points showed that cells incubated with N1, R 100µM, K 2µM, or G 0.01mM had progressive levels of toxicity over photooxidative damage, whereas cells incubated with SOD or N1 / SOD did not. N1 / SOD-incubated cells had lower cytotoxicity measurements than untreated controls, which was most evident at 3 hours of photooxidative damage (Figure 26B(v) and (vi)). A similar trend was observed in half-dose measurements, with R 100uM exhibiting significant baseline toxicity to 661w cells, which was also seen at 2 hours of photooxidative damage (Figure 27B(i)-(ii), P<0.05). Other supplements did not cause toxicity at these time points. Given the high toxicity observed after 4 hours of photooxidative damage, the long-term effects of supplementation on cell health were then assessed by allowing photooxidatively damaged cells to recover for 24 hours after 2 hours. The results showed significant toxicity for cells incubated with N1, R 100uM, and G 0.005mM compared to untreated controls (Figure 27B(iii)-(iv), P<0.05), but there were no differences in toxicity for the other supplementation groups. Overall, these results suggest that SOD and N1 / SOD supplementation may provide some protection against photooxidative damage-induced degeneration when utilizing 661w and 661w-EV, although the protective effect was not as pronounced as with RBC and RBC-EV.

[0303] Example 24: In vivo therapeutic efficacy of 661w-EV and 661w-EV(N1 / SOD) does not demonstrate protection against retinal degeneration. To evaluate the therapeutic potential of 661w-EV for protection against retinal degeneration, mice were given 5.0 × 10⁶ 661w-EV and 661w-EV(N1 / SOD) prior to 5 days of photooxidative damage. 6 The EV / mL dose was injected intravitreously (Figure 28A). Retinal function, as measured by ERG, showed no significant protection in any group compared to the PBS-injected control group (Figures 28B and C, P<0.05). Retinal thickness measurements showed a significant increase in ONL thickness in 661w-EV(N1 / SOD)-treated mice compared to the 661w-EV-only group, but no significant improvement compared to the PBS-injected control group (Figures 28D and E, P<0.05). Overall, 661w-EV with or without supplementation did not provide protection against retinal degeneration, while N1 / SOD supplementation provided some protection compared to 661w-EV alone.

[0304] To evaluate whether an increased dose of 661w-EV(N1 / SOD) could confer protection against photooxidative damage-induced degeneration, mice were given 2.0 × 10⁶ units in 1 μL via intravitreous injection. 9 Individuals were injected with 661w-EV(N1 / SOD) and exposed to photooxidative damage for 5 days (Figure 29A). After 5 days of photooxidative damage, retinal function was measured using ERG and showed no significant difference in wave a (Figure 29B) or wave b (Figure 29C) responses compared to PBS-injected controls (P>0.05). No differences were observed in retinal thickness measurements (Figure 29D, P>0.05) investigated using optical coherence tomography, or in cell death (Figures 29E-F, P>0.05) and photoreceptor column counts measured using the TUNEL assay. Overall, 661w-EV(N1 / SOD) did not provide therapeutic efficacy.

[0305] Example 25: RBC-EV has a different proteomic profile compared to RBC and RBC-EV with N1 / SOD supplementation that confers immunomodulatory properties to RBC. To understand the effect of supplementation on the molecular signatures of RBC and RBC-EV, RBC and RBC-EV groups (PBS, N1, SOD, and N1 / SOD) were sent for LC-MS / MS tandem mass spectrometry. Computational analysis comparing the total protein signatures of RBC and RBC-EV revealed a clear proteomic shift between the RBC and RBC-EV groups, as represented by the PCA plot (Figure 30A). Pathway analysis of RBC-EV proteins was found to be strongly associated with terms such as “endosomes,” “endocytosis vesicles,” and “vesicular membranes,” supporting the presence of extracellular vesicles in these groups (Figure 30B). Furthermore, in the RBC-EV group, there was significant enrichment of both known EV cargo proteins (Figure 30C) and EV membrane proteins (Figure 30D) compared to RBC host cells. Pathway analyses of RBC-EV enriched protein (Figure 30E(i)) and RBC enriched protein (Figure 30E(ii)) further supported known vesicle and erythrocyte functions, respectively. Finally, pathway analysis of downregulated RBC-EV protein compared to RBC identified reduced enrichment of inflammatory terms such as "interleukin-1 family signaling" and transcriptional pathways (Figure 30E(iii)), suggesting that RBC-EVs are likely to be anti-inflammatory and possess reduced transcriptional regulatory proteins compared to their host cells.

[0306] Next, the proteomic signatures of RBCs were analyzed by comparing them between the supplementation group and the pre-treatment (Pre-Tx) control (before incubation or supplementation; 0h). The results showed that there were similar total protein counts within each group (Figure 31A), but there was significant variability between the treatment groups, particularly as represented by PCA, compared to the Pre-Tx sample (Figure 31B). Analysis of the top differentially expressed proteins between the Pre-Tx group and the RBC(PBS) group showed that proteins concentrated in the RBC(PBS) sample were associated with pathways controlling hemostasis, coagulation, and inflammation, while proteins with less concentration were involved in metabolic and translational processes (Figures 31C and D). Finally, there were independently expressed proteins among the supplementation groups (PBS, N1, SOD, and N1 / SOD), and RBC(N1) was shown to have the most independently expressed proteins when compared to the RBC(PBS) sample (Figure 31E). Protein pathway analysis in each supplement group compared to Pre-Tx and PBS controls identified that proteins enriched with RBC(N1 / SOD) are involved in hemostatic and immunomodulatory pathways (Figure 31F).

[0307] The proteomics composition of RBC-EV samples was also investigated, showing a similar number of proteins within each group (PBS, N1, SOD, and N1 / SOD) (Figure 32A). Significant variability in protein composition was observed between groups, with RBC-EV(N1 / SOD) exhibiting the most variability compared to the RBC-EV(PBS), RBC-EV(N1), and RBC-EV(SOD) groups (Figure 32B), which was also reflected in the numerous unique proteins found in this group (Figure 32C). Compared to the RBC-EV(PBS), RBC-EV(N1), and RBC-EV(SOD) groups, RBC-EV(N1 / SOD) also showed a significantly downregulated population of proteins (Figure 32D), which were found to be associated with inflammatory pathways including the "interleukin-1 family signaling," the "non-canonical NfκB pathway," and the "keap1 / nef2l2 pathway" (Figure 32E), as well as transcriptional regulation of inflammatory, apoptotic, mitotic, and hematopoietic pathways. A complete list of differentially expressed proteins is provided in Table 1 below. In Table 1, proteins with positive log-2 values ​​are upregulated compared to the control, and proteins with negative log-2 values ​​are downregulated compared to the control. A complete list of pathways to which, interact with, or mediate the differentially expressed proteins is provided in Table 2 below. In Table 2, paths with a positive magnification change value are paths that are controlled upward compared to the control, and paths with a negative magnification change value are paths that are controlled downward compared to the control. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0308] Small RNA sequencing was performed on RBC-EV treated groups (PBS, N1, SOD, and N1 / SOD) to identify any arbitrary molecular changes in the microRNA signature in response to supplementation. MicroRNA content, expressed as a percentage of total reads, was similar across samples and groups, accounting for approximately 5–15% of the mapped total reads (Figure 33A). Furthermore, of the 260 total microRNAs detected, the top 11 microRNAs were identified as highly abundant in all samples, occupying a significant fraction of the total reads, with miR-451 from erythrocyte-enriched microRNA being the most abundant (Figure 33B). Pathway analysis of known targets of the top 11 microRNAs was associated with processes including regulation, transcription, biosynthesis, and senescence (Figure 33C). Venn diagrams showed no unique proteins between groups, suggesting that supplementation did not induce any changes in the RBC-EV microRNA signature (Figure 33D).

[0309] Overall, these results support the distinct protein signatures in the RBC and RBC-EV groups, with N1 / SOD combination supplementation yielding the most proteomic changes in the resulting EV population compared to other supplementation groups or controls. Furthermore, RBCs incubated in N1 / SOD supplementation were found to have an anti-inflammatory profile, which was reflected in RBC-EV(N1 / SOD).

[0310] Example 26: RBC-EV has anti-inflammatory properties and can reduce the release of inflammatory cytokines from peripheral bone mononuclear cells (PBMCs). To validate the immunomodulatory properties of RBC-EV identified from multi-omics analysis, RBC-EV was sent for independent validation of viability, safety, and anti-inflammatory properties, with and without supplementation. RBC-EV was incubated on peripheral bone mononuclear cells (PBMCs) with and without inflammatory stimulation (LPS). Dose-response was also evaluated. Cytokine output and viability measurements were compared to control groups (culture medium, PBS, PBS(N1 / SOD), and dexamethasone 10 nm). Results showed a dose-dependent increase in PBMC viability in response to increased RBC-EV concentrations in both control (Figure 34A) and LPS-stimulated (Figure 34B) cells, regardless of supplementation group. While no significant differences were observed between groups under control conditions, RBC-EV(N1 / SOD) generally promoted higher PBMC viability at each dose compared to RBC-EV(PBS). Under all conditions, no change in survival rate was observed in the control group, supporting the role of RBC-EV in promoting cell proliferation / survival.

[0311] We evaluated the safety profiles of RBC-EVs against PBMCs and measured cytokine output (MIP-1α(Ccl3), IL-1β, IL-6, IL-8, IL-10, TnFα, MCP-1(Ccl2), and IL-1α) after 48 hours of incubation. These cytokines play known pathogenic roles in both retinal and neurodegenerative diseases (Kauppinen et al., 2016; Wooff et al., 2019; Nagatsu et al., 2005). The overall trend showed a dose-dependent increase in cytokine production for RBC-EV(PBS), RBC-EV(N1), and RBC-EV(SOD), although mainly at low levels (Figure 35). Conversely, PBMCs treated with RBC-EV(N1 / SOD) showed a dose-dependent decrease in cytokine production. RBC-EV(N1 / SOD) did not induce the release of any robust cytokines above baseline levels (compared to the medium control and above LLOQ), with the exception of IL-8 and IL-1α. Overall, these results support the safety of RBC-EV(N1 / SOD) and suggest a strong dose-dependent response to cytokine reduction.

[0312] Finally, RBC-EVs were evaluated for their anti-inflammatory properties using the same cytokine output and after 48 hours of LPS stimulation and EV incubation. The results showed a clear anti-inflammatory response in all RBC-EV groups, with dose-dependent reductions in all cytokine production (Figure 36). Importantly, RBC-EV(N1 / SOD) was able to reduce cytokine output for MIP-1α(Ccl3), IL-6, IL-8, and MCP-1(Ccl2) to levels below that of the anti-inflammatory steroid dexamethasone (10 nM) (Figure 36A, C, D, and G). In summary, these results support a potent anti-inflammatory mechanism for RBC-EVs and their ability to reduce key cytokines known to be involved in both retinal and neurodegenerative diseases.

[0313] Example 27: Measurement of RBCs, platelets, leukocytes, and reticulocytes in the RBC-enriched fraction of whole mouse blood. Flow cytometry was used to determine the proportion of red blood cells (RBCs) after plasma removal and leukocyte depletion. Cells were labeled using fluorescent conjugate antibodies specific to various cell lineages: TER119 for erythroid cells, CD41 for megakaryocytes, CD71 for reticulocytes, and CD45 for lymphoid cells. A gating strategy was applied to isolate populations of TER119+, CD41+, CD71+, and CD45+ cells, as shown in Figure 37A. This analysis showed that mature RBCs and reticulocytes together constituted approximately 83% of the total cell population. Additionally, as illustrated in Figure 37B, CD41+ platelets represented 15–19%, and leukocytes accounted for less than 0.04% of the cells. This suggests that the EV population induced after incubation consisted of both RBC and platelet-derived vesicles.

[0314] Those skilled in the art will understand that numerous variations and / or modifications may be made to the present invention without departing from the spirit or scope of the invention as broadly described, as shown in the specific embodiments. Accordingly, these embodiments should be considered illustrative and not restrictive in all respects.

[0315] This application claims priority to Australian Provisional Application No. 2023901479, entitled “Methods of producing extracellular vesicles and uses thereof,” filed on 15 May 2023, and Australian Provisional Application No. 2023901480, entitled “Extracellular vesicles and uses thereof,” filed on 15 May 2023, the entire contents of which are incorporated herein by reference.

[0316] All publications discussed and / or referenced herein are incorporated in their entirety herein.

[0317] Any discussion of documents, actions, materials, devices, articles, etc., contained herein is for the sole purpose of providing context to the invention. It should not be construed as an acknowledgment that any or all of these matters constitute part of the foundation of the prior art or were common general knowledge in the art related to the invention that existed prior to the priority date of each claim of this application.

[0318] References Al-Ubaidi et al (1992) Proceedings of the National Academy of Sciences of the United States of America 89(4):1194-1198 Al-Ubaidi et al(2013)Investigative Ophthalmology & Visual Science 54(13):8119-24 Amin et al (2020) Translation Psychiatry 10:267. Araujo et al (2022) Cells 11(18):2908. Averill-Bates (2023) Vitamin Horm 121:109-41. Benatar et al (2022) Brain 145(1):27-44. Bjelobaba et al(2017)Current Pharmaceutical Design 23(5):693-730. Blasi et al (1990) Journal of Neuroimmunology 27(2-3):229-37. Cherry et al (2022) Journal of Neuroinflammation 19:278. Chiangjong et al (2021) Frontiers in Medicine doi:10.3389 / fmed.2021.761362 Deacon(2013)J Vis Exp(75):e2609. Dellar et al(2022)Journal of Extracellular Biology 1:e40. Dunn et al(1996)Experimental Eye Rsearch 62(2):155-169. Fernando et al(2018)Investigative Ophthalmology & Visual Science 59(11):4362-74. Gangadaran et al(2018)Frontiers in Pharmacology doi:10.3389 / fphar.2018.00817 Jacob et al(1991). Cytometry,12(6),550-558. doi:10.1002 / cyto.990120612 Jayaraj et al(2019)Journal of Neuroinflammation 16:142. Josefsberg et al.(2012)Biotech and Bioengineering.97(1):73-85. Kauppinen et al(2016)Cellular and molecular life sciences 73(9):1765-86. Koster et al(2022)International Journal of Molecular Sciences 23(6). Lamichhane and Jay(2018)Methods Molecular Biology 1831:37-47. Laurent et al(2018)Biomedical Journal 41(1):21-33. Lee et al(2021)doi:10.1007 / 978-1-0716-1712-0_9 Leng et al(2021)Nature Reviews Neurology 17:157-172. Lieto et al(2022)International Journal of Molecular Science 23(16):9158. Limb et al(2002)Investigative Ophthalmology & Visual Science 43(3):864-869. Lu et al(2017)Experimental Eye Research 165:78-89. Masini et al(2021)Biomedicines 9(6):598. Mead et al(2023)Nature Reviews Drug Discovery 22:185-212. Naegele et al(2014)Handbook Clinical Neurology 122:59-87 Nagatsu and Sawada(2005)Curr Pharm Des 11(8):999-1016. Natoli et al(2010)Molecular Vision 16:1801-22. Natoli et al(2016)Experimental Eye Research 147:114-127. Nederveen et al(2021)Frontiers in Physiology 11:2020. Palpagama et al(2019)Frontiers in Molecular Neuroscience 12:258. Rojas et al(2020)doi:10.3389 / fnins.2020.566858 Rutar et al(2015)Journal of Neuroinflammation 12(1):8. Salehi et al(2018)Biomedicine 6(3). Sanchez-Sarasua et al(2020)International Journal of Molecular Science 21(22):8751. Silva(2012)Genes Chromosom Cancer 51. Sreekumar et al(2021)Antioxidants 10(5). Jacob et al(1991)Cytometry,12(6),550-558. Usman et al(2018)Nature Communications 9(1):2359. Wolf et al(2011)Expert Rev Vaccine.10(10):1451-1475. Wooff et al(2019)Frontiers in Immunology 10:1618. Wooff et al(2020)Frontiers in Neuroscience 14.160.

Claims

1. A method for producing extracellular vesicles (EVs), comprising incubating or culturing EV-producing cells in a medium containing an antioxidant and / or an N1 medium component or equivalent thereof.

2. The method according to claim 1, wherein the antioxidant comprises one or both of an enzymatic antioxidant and a non-enzymatic antioxidant.

3. The method according to claim 2, wherein the enzymatic antioxidant is selected from one or more of superoxide dismutase (SOD), manganese superoxide dismutase (SOD2), catalase, peroxiredoxin, glutathione peroxidase, and glutathione reductase.

4. The method according to any one of claims 1 to 3, wherein the enzymatic antioxidant is superoxide dismutase (SOD).

5. The method according to any one of claims 1 to 4, wherein the non-enzymatic antioxidant is selected from one or more of hemoglobulin, kaempferol, glutathione, vitamin E, vitamin A, vitamin C, tocopherol, carotenoids, glutathione, and curcumin.

6. The method according to any one of claims 1 to 5, wherein the N1 culture medium component is selected from one or more of transferrin, insulin, sodium selenite, putrescine, and progesterone, or equivalents thereof.

7. The method according to claim 6, wherein the N1 culture medium component is transferrin or its equivalent.

8. The method according to any one of claims 1 to 7, wherein the culture medium includes N1 medium.

9. The method according to any one of claims 1 to 8, wherein the culture medium comprises N1 medium and SOD.

10. The method according to any one of claims 1 to 9, wherein the EV-producing cells are human cells.

11. The method according to any one of claims 1 to 10, wherein the EV-producing cells are selected from erythrocytes, reticulocytes, mesenchymal stem cells, epithelial cells, endothelial progenitor cells, umbilical cord cells, ophthalmic cell lines, nerve cell lines, dental pulp cells, dendritic cells, leukocytes, cancer cells, microglia, glial cells, astrocytes, photoreceptor cells, and embryonic fibroblasts.

12. The method according to claim 11, wherein the cell is a red blood cell.

13. The method according to claim 11 or 12, wherein the cells are red blood cells and reticulocytes.

14. The method according to any one of claims 1 to 13, wherein the EV-producing cells are present together with EV-producing cell fragments.

15. The method according to claim 14, wherein the EV-producing cell fragment is a platelet.

16. The method according to any one of claims 12 to 15, wherein the red blood cells are diluted by about 1:10 before being added to the culture medium.

17. The method according to claim 11, wherein the ophthalmic cell line is a retinal cell line selected from aRPE19, BV2, MIO-M1, D407, and iMG.

18. The method according to any one of claims 1 to 17, wherein the EV is isolated by centrifugation after incubation / culturing.

19. The method according to any one of claims 1 to 18, wherein the culture medium comprises phosphate-buffered saline (PBS) when the method includes incubation.

20. The method according to any one of claims 1 to 19, wherein the method includes incubating or culturing for about 10 to about 24 hours, or about 12 to about 20 hours, or about 12 to about 18 hours, or about 18 hours.

21. The method according to claim 20, wherein the method comprises incubating for about 16 hours.

22. The method according to any one of claims 1 to 21, wherein the method increases the quality and / or quantity and / or effectiveness of the EV produced.

23. The method according to claim 22, wherein increasing quality includes increasing the uniformity of EV vesicle size.

24. The method according to claim 23, wherein increasing quality includes increasing the level of one or both of the endogenous antioxidants and exogenous antioxidants in the EV.

25. The method according to any one of claims 1 to 24, wherein the method increases the level of one or both of the endogenous antioxidant and the exogenous antioxidant in the EV-producing cells.

26. The method according to any one of claims 1 to 25, wherein the method reduces the percentage of EV-producing cells having abnormal morphology compared to EV-producing cells incubated or cultured in a medium that does not contain the antioxidant and / or N1 medium component or equivalent thereof.

27. The method according to claim 26, wherein less than 10% of the EV-producing cells have an abnormal morphology.

28. The method according to claim 24 or 25, wherein the endogenous antioxidant is selected from one or more of hemoglobin, SOD, glutathione, vitamin C, vitamin E, catalase, and glutathione peroxidase.

29. The method according to claim 24 or 25, wherein the exogenous antioxidant is selected from one or more of SOD and kaempferol.

30. A population of extracellular vesicles (EVs) derived from cells containing increased levels of one or both endogenous and exogenous antioxidants.

31. A group of extracellular vesicles (EVs) produced by the method described in any one of claims 1 to 29.

32. The EV includes the increased expression of one or more proteins compared to the control, and the one or more proteins are ARIH1, RPL22, CFI, LNPK, RPS17, RPL23A, RPS21, CPOX, METAP2, FARSB, RPL18, MYG1, RPS3, RPS15A, HSP90B1, PHB1, EEF1D, DDX1, RPS4X, EIF2S3Y, AHSG, RPL12, NDUFA4, SACM1L, RPS5, RPL18A, RPL14, CALR, RPL26, RPLP0 A group of EVs according to claim 30 or 31, selected from HYOU1, ENO3, CTSE, ALDH1A7, PDIA6, PRKCSH, CLNS1A, RPS6, HACE1, RPL17, RPL27A, EPRS1, PRXL2A, TRIM56, CANX, NPEPL1, PDIA3, PPIB, EEF2, SPR, NGP, HSPA5, BAG2, SND1, RANGAP1, ENO1, RPS14, TFRC, THG1L, PFAS, PPP2R1A, GLO1, and SCAMP3.

33. The EV includes the expression of one or more proteins that are reduced compared to the control, and the one or more proteins are Fcho2, Crlf3, Psma5, Gspt1, Psmb4, Acp1, Ccdc6, Uros, Psmd3, Tgm2, Psmc2, Psma3, Glurx3, Rnh1, Ppid, Usp25, Gmpr, Usp5, Pgls, Ostf1, Psmb6, Eif5, Tbcb, Oxsr1, Stip1, Usp14, Psmd5, Tollip, Psmd8, Gpi, Otub1, Sri, A A group of EVs according to any one of claims 30 to 32, selected from gfg1, Cfap157, Psmd6, Chordc1, Phpt1, Psmd7, Psmd11, Psmd12, Psmd13, Gstm5, Synj1, Sh3glb1, Swap70, H4c1, Pzp, Psmd14, Wnk1, Gdi1, Pf4, Snx15, Coro1c, Ptgr2, Aldoart2, Ighg3, Lzic, Epn1, Pacs1, Skic2, Kyat3, Rnf213A, and Anxa6.

34. A population of EVs according to any one of claims 30 to 33, wherein the EV comprises the expression of one or more proteins increased compared to a control, and the one or more proteins mediate one or more pathways selected from Rab regulation of transport, membrane-targeting Srp-dependent cotranslating proteins, major pathways of rRNA processing in the nucleolus and cytoplasm, exon junction-independent nonsense mutation-dependent degradation mechanisms, nonsense mutation-dependent degradation mechanisms nmd, raf gefs exchange of gtp to gdp on rab, cytoplasmic ribosomal proteins, trans-Golgi network vesicle budding, formation of a pool of free 40s subunits, Golgi-associated vesicle biosynthesis, MHC class II antigen presentation, Rab geranylgeranylation, signaling by ntrk1 trka, signaling by ntrk, vesicle-mediated transport, eukaryotic translation initiation, signaling by receptor tyrosine kinase, neurotransmitter receptor and postsynaptic signaling, membrane transport, and cratrin-mediated endocytosis.

35. The EV includes the expression of one or more proteins that are reduced compared to the control, and the one or more proteins are involved in interleukin-1 family signaling, runx1-mediated transcriptional regulation, hedgehog ligand biosynthesis, hedgehog-on state, ubiquitin-mediated degradation of cdc25a, runx3-mediated transcriptional regulation, p53 stabilization, runx1-mediated regulation of gene transcription in HSCS differentiation, regulation of runx3 expression and activity, degradation of axin, cross-presentation of soluble exogenous antigen endosomes, asymmetric localization of pcp proteins, Apc c c cdh1-mediated degradation of cdc20 target proteins in late mitosis G1, GAPS-mediated regulation of ras, degradation of dvl, keap1 nef212 pathway, activation of Apc c and Apc c c cdc20-mediated degradation of mitotic proteins, pcp A population of EVs according to any one of claims 30 to 34, mediating one or more pathways selected from the ce pathway, transcriptional regulation by runx2, regulation of runx2 expression and activity, regulation of pten stability and activity, processing of gli3 to gli3r by the proteasome, G2 m checkpoint, auf1 hnrnp d0 binding to mRNA and destabilizing it, switching of origin to post-replication state, removal of Orc1 from chromatin, apc c-mediated degradation of cell cycle proteins, Tnfr2 non-canonical NF KB pathway, Ub-specific processing proteases, and beta-catenin-independent WNT signaling.

36. The group of vitamin E according to any one of claims 30 to 35, wherein the endogenous antioxidant is selected from one or more of hemoglobin, SOD, glutathione, vitamin C, vitamin E, catalase, and glutathione peroxidase.

37. The group of EVs according to any one of claims 30 to 36, wherein the exogenous antioxidant is selected from one or more of SOD and kaempferol.

38. The group of EVs according to any one of claims 30 to 37, wherein the EVs have an average diameter of less than 200 nm.

39. A population of EVs according to any one of claims 30 to 38, wherein a portion of the EVs localizes to the eye when administered systemically or locally.

40. A population of EVs according to any one of claims 30 to 39, wherein a portion of the EVs localizes to the retina when administered systemically or locally.

41. A population of EVs according to any one of claims 30 to 40, wherein a portion of the EVs localizes to the brain when administered systemically or locally.

42. A population of EVs according to any one of claims 30 to 41, wherein, when administered systemically or locally, a portion of the EVs localizes to one or more of microglia, glial cells, and neurons.

43. A group of EVs according to any one of claims 30 to 42, wherein the EV further comprises an exogenous cargo.

44. The population of EVs according to claim 43, wherein the exogenous cargo is selected from one or more of the following: drugs, antioxidants, chemotherapy, proteins, lipids, nucleic acids (such as DNA, mRNA, miRNA, siRNA, circular RNA, long non-coding RNA, and snoRNA), CRISPR / Cas9, nanoparticles, and exogenous targeting molecules.

45. A group of EVs according to any one of claims 30 to 44, wherein the EV is privately owned.

46. The group of EVs according to any one of claims 30 to 45, wherein the EV comprises an endogenous cargo, and the endogenous cargo is a miRNA selected from one or more of the following: mmu-miR-142a-3p, mmu-miR-486b-3p, mmu-let-7c-5p, mmu-miR-16-5p, mmu-miR-25-3p, mmu-miR-486a-3p, mmu-miR-486b-5p, mmu-miR-486a-5p, mmu-let-7f-5p, mmu-let-7a-5p, and mmu-miR-451a.

47. A composition comprising EVs produced by the method of any one of claims 1 to 29, or a population of EVs according to any one of claims 30 to 46, for use in treating and / or preventing a disease or condition in a subject.

48. A composition comprising EVs produced by the method of any one of claims 1 to 29, or a population of EVs according to any one of claims 30 to 46, for use in treating and / or preventing neurodegeneration in a subject.

49. A method for treating and / or preventing neurodegeneration in a subject, comprising administering to a population of EVs produced by the method of any one of claims 1 to 29, or of EVs as described in any one of claims 30 to 46.

50. Use of EVs produced by the method of any one of claims 1 to 29, or a population of EVs according to any one of claims 30 to 46, for the treatment and / or prevention of neurodegeneration in a subject.

51. Use of EVs produced by the method of any one of claims 1 to 29, or a population of EVs according to any one of claims 30 to 46, in the manufacture of a pharmaceutical product for treating and / or preventing neurodegeneration in a subject.

52. A composition comprising EVs produced by the method of any one of claims 1 to 29, or a population of EVs according to any one of claims 30 to 46, for use in reducing oxidative stress and / or inflammation in the eye of a subject.

53. A method for reducing oxidative stress and / or inflammation in a target eye, comprising administering to a population of EVs produced by the method of any one of claims 1 to 29, or EVs as described in any one of claims 30 to 46.

54. Use of EVs produced by the method of any one of claims 1 to 29, or a population of EVs according to any one of claims 30 to 46, to reduce oxidative stress and / or inflammation in the eye of a subject.

55. Use of EVs produced by the method of any one of claims 1 to 29, or a population of EVs according to any one of claims 30 to 46, in the manufacture of a pharmaceutical product for reducing oxidative stress and / or inflammation in a target eye.

56. A composition comprising EVs produced by the method of any one of claims 1 to 29, or a population of EVs according to any one of claims 30 to 46, for use in treating and / or preventing retinal degeneration in a subject.

57. A method for treating and / or preventing retinal degeneration in a subject, comprising administering to a population of EVs produced by the method of any one of claims 1 to 29, or EVs as described in any one of claims 30 to 46.

58. Use of EVs produced by the method of any one of claims 1 to 29, or a population of EVs according to any one of claims 30 to 46, for the treatment and / or prevention of retinal degeneration in a subject.

59. Use of EVs produced by the method of any one of claims 1 to 29, or a population of EVs according to any one of claims 30 to 46, in the manufacture of a pharmaceutical product for treating and / or preventing retinal degeneration in a subject.

60. The composition, use, or method according to any one of claims 47 to 59, wherein the EV is administered systemically or will be administered systemically.

61. The composition, use, or method according to claim 60, wherein systemic administration includes intravenous administration or infusion.

62. The composition, use, or method according to any one of claims 47 to 59, wherein the EV is administered or will be administered locally.

63. The composition, use, or method according to claim 62, wherein the local administration is selected from local administration to the eye, intraocular administration, subretinal administration, intravitreous administration, intracranial injection, subarachnoid injection, intracerebral injection, and intracerebral implantation.

64. The composition, use, or method according to any one of claims 47 to 63, wherein the neurodegeneration includes neuroinflammation.

65. The composition, use, or method according to any one of claims 47 to 64, wherein the neurodegeneration is selected from Parkinson's disease, amyotrophic lateral sclerosis, Alzheimer's disease, tauopathy, multiple sclerosis, Lewy body dementia, stroke, transient ischemic attack, and Huntington's disease.

66. The composition, use, or method according to any one of claims 47 to 65, wherein the neurodegeneration is Parkinson's disease.

67. The composition, use, or method according to any one of claims 47 to 66, wherein the neurodegeneration is Alzheimer's disease.

68. The composition, use, or method according to any one of claims 48 to 51, wherein the retinal degeneration is selected from macular degeneration, retinitis pigmentosa, and diabetic retinopathy.

69. The composition, use, or method according to claim 68, wherein the macular degeneration is selected from exudative macular degeneration and dry macular degeneration.

70. The composition, use, or method according to any one of claims 52 to 55, wherein the oxidative stress and / or inflammation is caused and / or a result of a disease or condition selected from macular degeneration, retinitis pigmentosa, diabetic retinopathy, Stargardt disease, Leber congenital amaurosis, cone-rod dystrophy, Usher syndrome, choroideremia, Valde-Vidl syndrome, macular telangiectasia, macular edema, retinal detachment, retinal ischemia, uveitis, scleritis, conjunctivitis, keratitis, corneal ulcer, glaucoma trachoma, choroidal melanoma, ocular melanoma, glaucoma retinal dystrophy, strabismus, and cataract.

71. A method for producing extracellular vesicles (EVs), comprising incubating or culturing EV-producing cells and fragments of EV-producing cells in a medium containing an antioxidant and / or N1 medium component or equivalent thereof.

72. The method according to claim 71, wherein the EV-producing cell fragment is a platelet.

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