Microfluidic assembly of red blood cell (RBC) lipids and components for engineering extracellular vesicles and nanoparticles

Abstract

Described herein are microfluidic approaches for the assembly of nanoparticles manufactured from isolated red blood cell (RBC) lipids and components. The engineered nanoparticles (e.g., vesicles) can be used to encapsulate cargo, including active agents such as proteins, nucleic acids (e.g., RNA), and other classes of therapeutic agents. Optionally, the vesicles can be further functionalized with targeting agents.

Description

[0001] MICROFLUIDIC ASSEMBLY OF RED BLOOD CELL (RBC) LIPIDS AND COMPONENTS FOR ENGINEERING EXTRACELLULAR VESICLES AND NANOPARTICLES

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims benefit of priority of U.S. Provisional Application No. 63 / 603,947, filed November 29, 2023. which is hereby incorporated herein by reference in its entiret .

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under U18 TR003807 and UG3 TR002884 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] REFERENCE TO SEQUENCE LISTING

[0007] The Sequence Listing submitted November 29, 2024, as a text filed named “103361- 625W01_ST26.xml” created November 29, 2024, and having a file size of 1,806 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).

[0008] BACKGROUND

[0009] Extracellular vesicles are secreted by a wide variety of cell types. In general, extracellular vesicles such as exosomes, microvesicles, and apoptotic bodies are membranebound and can be loaded with a therapeutic cargo. Exosomes are a ty pe of extracellular vesicle that are secreted by most eukaryotic cells. Exosomes are natural membranous vesicles with a diameter of 30-300 nm. They are generated through inward invagination of endosomal membranes which form multivesicular bodies (MVBs), followed by release into the extracellular milieu upon fusion of the MVBs with the plasma membrane.

[0010] As endogenous nanocarriers secreted by various ty pes of cells, exosomes play important roles in cell-cell communication through transfer of mRNA, miRNA. receptors, enzymes, cytokines, etc. Importantly, the membrane of exosome is characterized by a phospholipid bilayer and abundant tetraspanin CD9 on its surface, facilitating direct membrane fusion with target cells. This fusion mode circumvents the endosomal-lysosomal pathway required for the synthetic vehicles and promotes cellular delivery of therapeutic agents. Thus, exosomes and other extracellular vesicles are under investigation as therapeutic treatments.

[0011] Extracellular vesicles have been examined as carriers for therapeutic agents including nucleic acids. However, most current methods of producing extracellular vesicles and encapsulation of active agents within the extracellular vesicles have several drawbacks. First, the yield of producing extracellular vesicles incorporating active agents is generally low. Further, directing the extracellular vesicles to an in vivo target remains a challenge, as the majority of the extracellular vesicles in circulation accumulate and are metabolized in the liver, spleen, and kidney.

[0012] Therefore, there is a need for improved extracellular vesicles and methods for efficiently producing extracellular vesicles. There is also a need for systems and methods of incorporating active agents in such extracellular vesicles for use in biomedical applications.

[0013] SUMMARY

[0014] The present disclosure relates to materials and methods for the preparation of lipid based nanoparticles, including extracellular vesicles, for the delivery of cargo (e.g., active agents) to mammalian (e.g., human) cells. In particular, described herein are microfluidic approaches for the assembly of nanoparticles manufactured from isolated RBC lipids and components. The engineered nanoparticles can be used to encapsulate proteins, RNAs, and other therapeutic cargo.

[0015] For example, described herein are methods for generating red blood cell lipid nanoparticles. These methods can comprise mixing a first solution comprising a mixture of lipids isolated from red blood cells and a second solution comprising an aqueous buffer within a microfluidic device under conditions effective to form a crude mixture comprising the population of red blood cell lipid nanoparticles dispersed within an aqueous fluid; and filtering the crude mixture comprising the population of red blood cell lipid nanoparticles by microfiltration against a filtration membrane, thereby forming a retentate fraction comprising the population of red blood cell lipid nanoparticles and a permeate fraction comprising low molecular weight contaminants.

[0016] The red blood cell lipids can comprise a mixture of lipids isolated from the membranes red blood cells. In some embodiments, the mixture of lipids isolated from red blood cells comprises a mixture of lipids isolated from membranes of human red blood cells. In some embodiments, the mixture of lipids isolated from red blood cells comprises a mixture of lipids isolated from membranes of non-human red blood cells. In some embodiments, the mixture of lipids isolated from red blood cells comprises a mixture of lipids isolated from membranes of red blood cells derived from stem cells, ’‘recombinant red cells” cultured in bioreactors, or engineered red cells obtained from genetic engineering of stem cells.

[0017] In some embodiments, the mixture of lipids isolated from red blood cells can be substantially free of proteins. For example, in certain embodiments, the mixture of lipids isolated from red blood cells can comprise less than 1% by weight, less than 0.5% by weight, less than 0.1% by weight, less than 0.05% by weight, or less than 0.01% by weight of proteins.

[0018] The mixture of lipids isolated from red blood cells can comprise cholesterol, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), lysophosphatidylcholine (LysoPC), sphingomyelin-DSM (SM-DSM), ethylphosphatidylcholine (ePC), lysophosphatidylethanolamine (LysoPE), ethylphosphatidylethanolamine (ePE), ethylphosphatidylserine (ePS), phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylethanolamine-ceramide (PE-Cer), or any combination thereof.

[0019] In some embodiments, the mixture of lipids isolated from red blood cells comprises a mixture of phospholipids, and the mixture of phospholipids comprises from 40% by weight to 50% by weight phosphatidylcholine (PC), from 15% by weight to 25% by weight phosphatidylethanolamine (PE), from greater than 0% by weight to 0.5% by weight phosphatidylinositol (PI), from 1% by weight to 5% by weight phosphatidylserine (PS), from 0.2% by weight to 1% by weight lysophosphatidylcholine (LysoPC), from 20% by weight to 25% by weight sphingomyelin-DSM (SM-DSM), from 3% by weight to 6% by weight ethylphosphatidylcholine (ePC), from greater than 0% by weight to 0.5% by weight lysophosphatidylethanolamine (LysoPE), from 0.5% by weight to 3% by weight ethylphosphatidylethanolamine (ePE), from greater than 0% by weight to 0.2% by weight ethylphosphatidylserine (ePS), from greater than 0% by weight to 0.5% by weight phosphatidic acid (PA), from greater than 0% by weight to 0.5% by weight phosphatidylglycerol (PG), and from greater than 0% by weight to 0.2% by weight phosphatidylethanolamine-ceramide (PE- Cer). In certain embodiments, the mixture of lipids isolated from red blood cells comprises a mixture of phospholipids, and the mixture of phospholipids comprises -46.6% by weight phosphatidylcholine (PC), -20.4% phosphatidylethanolamine (PE), -0.2% phosphatidylinositol (PI), -2.5% phosphatidylserine (PS), -0.6 % lysophosphatidylcholine (LysoPC), 22.89% sphingomyelin-DSM (SM-DSM), 4.5% ethylphosphatidylcholine (ePC), 0.25% lysophosphatidylethanolamine (LysoPE), 1.7% ethylphosphatidylethanolamine (ePE), 0.04% ethylphosphatidylserine (ePS). 0.2% phosphatidic acid (PA), 0.1% phosphatidylglycerol (PG), and -0.01% phosphatidylethanolamine-ceramide (PE-Cer).

[0020] In some embodiments, the mixture of lipids isolated from red blood cells is obtained by a process that comprises extraction of lipids from a population of lysed red blood cells using a chloroform-methanol phase separation technique.

[0021] In certain embodiments, the method further comprises; lysing a population of red blood cells; extracting the lysed population of red blood cells with chloroform and methanol to obtain a biphasic mixture comprising an organic phase and an aqueous phase; separating the organic phase from the aqueous phase; filtering the organic phase to remove any suspended proteins; and isolating mixture of lipids isolated from red blood cells from the organic phase.

[0022] In some embodiments, the first solution comprises the mixture of lipids isolated from red blood cells dissolved or dispersed in water, a water-miscible solvent, or a combination thereof. In some embodiments, the first solution comprises the mixture of lipids isolated from red blood cells dissolved or dispersed in a water-miscible solvent. In certain embodiments, the water- miscible solvent comprises an alcohol, such as ethanol.

[0023] In some embodiments, the aqueous buffer comprises a citrate buffer.

[0024] In some embodiments, the aqueous buffer exhibits a pH of from 5.5 to 8.5, such as a pH of from 5.5 to 7.5, a pH of from 5.5 to 7.0, a pH of from 5.5 to 6.5, or a pH of about 6.

[0025] In some embodiments, the microfluidic device comprises a central fluid inlet channel and two outer fluid inlet channels, wherein the central fluid inlet channel and the outer fluid inlet channels converge in a mixing channel upstream of an outlet. In these embodiments, mixing the first solution comprising the mixture of lipids isolated from red blood cells and the second solution comprising the aqueous buffer can comprise flowing the first solution through the central fluid inlet and flowing the second solution through the two outer fluid inlet channels.

[0026] In some embodiments, the microfluidic comprises a first fluid inlet channel and a second fluid inlet channel that converge in a mixing channel upstream of an outlet, wherein the mixing channel further comprises one or more mixing elements, such as pillars or ridges, that increase chaotic mixing of fluids within the mixing channel. In these embodiments, mixing the first solution comprising the mixture of lipids isolated from red blood cells and the second solution comprising the aqueous buffer can comprise flowing the first solution through the first fluid inlet channel and flowing the second solution through the second fluid inlet channel.

[0027] In some embodiments, the second solution comprising the aqueous buffer and the first solution comprising the mixture of lipids isolated from red blood cells are introduced at a flow rate ratio (FRR) of from 1:1 to 1:60, such as from 1:2 to 1 :20 or from 1:4 to 1 :20. In some embodiments, the first solution comprising the mixture of lipids isolated from red blood cells is introduced into the microfluidic device at a flow rate of from 100 pL / min to 1000 pL / min. In some embodiments, the second solution comprising the aqueous buffer is introduced into the microfluidic device at a flow rate of from 10 pL / min to 200 pL / min.

[0028] In some embodiments, the mixture of lipids isolated from red blood cells are present in the first solution at a concentration of from 0.5 rnM to 10 mM, such as from 1 mM to 4 mM.

[0029] In some embodiments, the filtration membrane is rated for a 500 kDa molecular weight cut-off. In some embodiments, filtering the crude mixture comprising the population of red blood cell lipid nanoparticles by microfiltration comprises filtering for at least 3 diafiltrations cycles, such as at least 4 diafiltration cycles, at least 5 diafiltration cycles, at least 6 diafiltration cycles, at least 7 diafiltrations cycle, at least 8 diafiltration cycles, at least 9 diafiltrations cycle, or at least 10 diafiltration cycles. In some embodiments, the microfiltration comprises tangential flow filtration.

[0030] In some embodiments, the first solution, the second solution, or a combination thereof further comprises an active agent, and the population of red blood cell lipid nanoparticles formed by the method comprise the active agent encapsulated therein. In certain embodiments, the second solution further comprises an active agent, and the population of red blood cell lipid nanoparticles formed by the method comprise the active agent encapsulated therein. In these embodiments, the active agent can comprise any suitable active agent. By way of example, in some embodiments, the active agent can comprise a small molecule, an organometallic compound, a nucleic acid (e.g., DNA, RNA), a protein (including multimeric proteins, protein complexes, etc ), a peptide, a lipid, a carbohydrate, a hormone, a metal, a radioactive element or compound, a drug, a vaccine, an immunological agent, a nanoparticle, or a combination thereof.

[0031] In some embodiments, the method further comprises covalently functionalizing the red blood cell lipid nanoparticles with a targeting moiety. In certain embodiments, the red blood cell lipid nanoparticles are covalently functionalized with the targeting moiety using a click chemistry reaction (e.g., an azide-alkyne Huisgen cycloaddition). In certain embodiments, the targeting moiety comprises a nucleic acid, a small molecule, or an antibody.

[0032] In some embodiments, the population of red blood cell lipid nanoparticles is monodisperse. In certain embodiments, the population of red blood cell lipid nanoparticles exhibits a PDI of 0. 1 or less.

[0033] In some embodiments, the population of red blood cell lipid nanoparticles exhibits an average particle size of from 50 nm to 500 nm, such as from 75 nm to 300 nm, as measured by dynamic light scattering (DLS). In certain embodiments, the population of red blood cell lipid nanoparticles comprise engineered red blood cell extracellular vesicles (eRBCEVs).

[0034] In some embodiments, the mixture of lipids isolated from red blood cells further comprises one or more additional lipids added to the mixture of lipids isolated from red blood cells. In certain embodiments, the one or more additional lipids can comprise a synthetic PEGylated lipid, such as DSPE-PEG-DBCO.

[0035] BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figures 1A-1G. Synthesis technique and computational analysis. Figure 1A is a schematic representation of (i) red blood cell (ii) extracted RBC lipids (iii) hollow bilayer EV structure, eRBCEVs encapsulated (iv) human hemoglobin (hHb) (v) Lumbricus terrestris erythrocruorin (LtEc) (vi) gold nanoparticles (Au-NPs) (vii) FAM oligonucleotides (FAM-O) and (iv) functional EVs with biotin on the surface of eRBCEVs. Figure IB is a plot shoring the RBC lipid profile from mass spectroscopy analysis, indicating the composition includes 22.89% sphingomyelin & detergent-soluble membranes (SM-DSM), 4.5% ethyl phosphatidylcholine (ePC), 46.59% phosphatidylcholine (PC), 20.4% Phosphatidylethanolamine (PE), 2.5% Phosphatidylserine (PS) and traces of LysoPE, LysoPC. PI, PA, and PG. Figure 1C is a schematic representation of automated pressure-driven microfluidic lipid nanoparticle synthesis system showing (1) air-filtered pressurized N2 inlet (2) digital pressure controller (3) digital flow sensor connected to pressure controller (4) narrow flow resistor (5) USB live camera focused on microfluidic chip (6) collection of eRBCEVs in low binding vial / tube with magnified schematic, (7) pressurized reservoir containing aqueous phase connected to controller and flow sensor (8) pressurized reservoir containing lipid phase connected to controller and flow sensor and (9) data acquisition and input USB connection to computer. Figure ID shows a tangential flow filtration (TFF) setup with 500 kDa membrane, schematic representing R - retentate flow containing eRBCEVs, P - permeate flow containing free lipids and proteins, and IN - inlet flow with 35 mL / min flow rate. Figure IE shows a surface gradient plot of flow focusing design showing lipid flow and aqueous phase interface at (i) ImM and (ii) 4mM lipid concentration at flow rate ratio (FRR) 1:20. Figure IF shows a surface gradient plot of flow focusing design showing lipid concentration diffusion with interface diffusion detection limit (i) 50 pM at 0 s and (ii) 500 pM at 10 s at 4mM initial concentration and FRR 1:20. Figure 1G shows comparisons of volume of flow with actual fluid flow in flow focusing (FF) and herringbone device (HB), (i) FF: FRR- 100: 400 pL / min (1:4) (ii) FF: FRR- 20:400 pL / min (1 :20) (iii) HB: FRR- 100: 400 pL / mm (1 :4) (iv) HB: FRR- 20:400 pL / min (1:20). Figures 2A-2I. Characterization and optimization of eRBCEVs. Figure 2A shows NTA size histograms from FF and HB designs at FRR 100:400 pL / min, 40:200 pL / min, 50:300 pL / min, 40:320 pL / min, and 40:400 pL / min. The concentration of eRBCEVs was found to be in the range of elO particles / mL. Illustrates the mean size from each FRR and design. Figure 2B shows the variation of mean eRBCEV size at FRR 1 :20, depicted as the violin graph with the statistical comparison of eRBCEV size produced on FF and HB designs (N = 2, n = 3, ** p<0.001). Figure 2C shows the variation of mean eRBCEV size at FRR 1:20, depicted by the size vs concentration histogram comparing empty eRBCEVs against dye-encapsulated eRBCEVs. Figure 2D shows the variation of mean eRBCEV size at FRR 1:20 for different lipid concentrations including 1 mM, 2 mM, and 4 mM is depicted by the histogram. Figure 2E is a bar graph representing statistically significant differences by comparison of the poly dispersity index (PDI) of hollow eRBCEVs against encapsulated eRBCEVs produced on HB at FRR 1 :20 (N = 2, n = 3, ****p<0.0001). Figure 2F is a stacked line marker graph depicting a statistical comparison of eRBCEVs PDI assessments produced on HB at FRR 4, 5,6, 8. 10. 20. and 40 at 1 mM, 2 mM, and 4 mM RBC lipid concentration (N =3, N = 7, n = 3). Figure 2G is a radar graph representing the impact of different loading cargo on PDI, including hHb -5 nm, spherical AuNPs ~30 nm, & -10 nm, LtEc -28 nm, and Oligos ~20 bp, (n = 3). Figure 2H shows a transmission electron microscopy (TEM) image of hollow eRBCEVs produced at FRR 1:20 using FF design. The zoomed images represent RBC lipid bilayer at 50 nm scale. Figure 21 us a bar histogram with continuous fit representing the quantification of hollow eRBCEV’s size determined from TEM image counts (n = 3).

[0037] Figures 3A-3D. Optical and qualitative characterization of eRBCEVs. Figure 3 A includes (i) a TEM image of eRBCEVs encapsulating hHb. the zoomed area representing the presence of hHb at electron dense core; and (ii) a visual quantification of hHb encapsulation efficiency of eRBCEVs at FRR 1:20 on HB design (N = n = 3). Figure 3B includes (i) a TEM image of eRBCEVs encapsulating AuNP, the zoomed area representing the presence of AuNPs as the dark core; and (ii) a visual quantification of AuNP encapsulation efficiency of eRBCEVs at FRR 1 :5 on HB design (N = n = 3). Figure 3C shows TEM-EDAX energy -intensity elemental mapping of Au, C, P, N, O in eRBCEV encapsulating -10 nm AuNP at FRR 1 :20 on HB design. Figure 3D includes (i) a TEM image of LtEc protein structure, the zoomed area representing the presence of hexagonal structure of LtEc at 10 nm scale; (ii) TEM image of eRBCEVs encapsulating LtEc, the zoomed area represents the presence of LtEc at the core at 100 nm scale; and (iii) a visual quantification of LtEc encapsulation efficiency of eRBCEVs at FRR 1 :5 on HB design (N = n = 3). Figures 4A-4E. Topology, co-localization. and purification of eRBCEVs. Figure 4A includes (i) Cryo-EM image representation of eRBCEVs depicting bilayer and ‘onion-like rings’; and (ii) continuous fit histogram representing the quantification of the size of eRBCEVs based on cryo-EM results (n = 3). Figure 4B shows the co-localization of FAM-0 within encapsulated eRBCEVs. (i) 488 nm channel FAM-0 signals (ii) 647 nm channel DiR-labelled eRBCEVs signals (iii) Co-localized signals with zoomed signal overlap. Figure 4C shows the co-localization of FITC-labelled LtEc within encapsulated eRBCEVs. (i) 488 nm channel FITC- labelled LtEc signals (ii) 647 nm channel DiR-labelled eRBCEVs signals (iii) Co-localized signals with zoomed signal overlap. Figure 4D shows a bar graph representing statistical comparisons of TIRFM signals from FAM-0, eRBCEVs, PBS, encapsulated particle, DiR, and FITC (n =100, N.S - not significant, ****p<0.0001, ***p<0.001). Figure 4E shows the optimization of TFF purification for eRBCEVs by considering permeate and retentate hHb concentration with BCA quantification after each Dia-cycle.

[0038] Figures 5A-5C. Immunogenicity studies using human neutrophils. Figure 5A includes a bar graph depicting quantification and analysis of percentage activation of neutrophils after 90 minutes incubation upon spiking PBS, BioParticle™, cationic lipid nanoparticle (cLNP), and empty eRBCEVs. (i) Neutrophil under bright field microscopy (BFM) with PBS control, (ii) Neutrophil under BFM with BioParticle™ positive control, (iii) Neutrophil under BFM with cLNP (iv) Neutrophil under BFM with eRBCEVs (N = 3, ****p<0.0001). Figure 5B includes a bar graph showing the statistical specific quantification and visual analysis of percentage activation of neutrophils after 90 minutes incubation upon spiking free hHb and encapsulated hHb within eRBCEVs at 0.5 mg / mL concentration, (v) Neutrophil under BFM with free hHb, (vi) Neutrophil under BFM with eRBCEVs encapsulating hHb (N = 3, ****p<0.0001). Figure 5C is a stacked line graph representing the percentage activation of neutrophil against time points considering various concentrations of free hHb including 225, 22.5, 2.25 mg / mL and eRBCEVs 10-fold dilutions including elO, e09, and e08 particles / mL (N = 6, n = 3).

[0039] Figures 6A-6C. Nanobody conjugation and dynamics on the surface of eRBCEVs. Figure 6A is a bar graph plotting the concentration and size distribution of eRBCEVs-DBCO conjugated particles. The population exhibited a mean size of 125 nm (SD + / - 23.5 nm). The inset includes a TEM micrograph showing the structure of the eRBCEVs after the modification with DSPE-PEG2000-DBCO. Figure 6B is a TIRFM image showing the colocalization of PDL1 nanobodies on the surface of modified eRBCEVs. The red dye represents the eRBCEVs labelled with DiR; the green dots represent the PDL1 nanobodies labelled with NHSF (degree of labelling = 1.2, >1). Figure 6C is a bar graph showing the quantification of colocalization of PDL1 nanobodies on the eRBCEV surface (n =100. N.S - not significant, ****p<0.0001, ***p<0.001).

[0040] Figures 7A-7D. Internalization of eRBCEVs with PDL1 in EMT6 cells. Figure 7A shows confocal microscopy images representing (i) DAPI stained EMT6 cell nucleus, (ii) DiR stained eRBCEVs modified with DBCO (hi) NHSF stained PDL1 nanobodies on the surface of eRBCEVs, (iv) RFP-stained lysosome inside the EMT6 cells. Figure 7B is a plot showing a colocalization merged confocal image representing the eRBCEVs-DBCO-PDLl internalization in the EMT6 cells (n = 3, N = 100) with orthogonal projections representing side and front views of cell uptake at 37 °C. The highlighted cell is zoomed with nucleus boundary and visualized using z-stacking in three dimensions. Figure 7C is a plot showing a colocalization merged confocal image representing the eRBCEVs-DBCO-PDLl internalization in the EMT6 cells (n = 3, N = 100) with orthogonal projections representing side and front views of cell uptake at 4 °C. The highlighted cell is zoomed with nucleus boundary and visualized using z-stacking in three dimensions. Figure 7D is a plot showing a quantification of the florescence intensity / area as a function of temperature 37°C (averaged median intensity / area - 68) and 4°C (averaged median intensity / area - 48) with DiR, NHSF - GFP, and RFP labels.

[0041] Figures 8A-8E. Evaluation of the acute toxicity of eRBCEVs in vivo. Figure 8 A includes photographs of mice used to assess acute toxicity and the physiological responses to the administered substances, C57BL6 / J mice (n = 6) were retro-orbitally injected with eRBCEVs at a concentration equivalent to E10 particles (n = 3) or with PBS as a control (n = 3). Figure 8B is a line graph illustrates the percentage survival of C57BL6 / J mice following retro-orbital injections of eRBCEVs (purple line, n = 3) and PBS (black line, n = 3). Mice were monitored for survival over the course of the study to evaluate the potential toxicity of eRBCEVs compared to the control. Figure 8C is a graph showing the weight loss trends in mice retro-orbitally injected with eRBCEVs (purple solid line, n = 3) and PBS (black solid line, n = 3) after the first and second doses. Figure 8D includes photographs showing the spleens of C57BL6 / J mice 48 hours post-injection with eRBCEVs and PBS. A measuring scale is included for size reference, highlighting potential morphological changes in response to the treatments. Figure 8E is a bar graph presenting a statistical comparison of spleen surface area measurements between eRBCEV -treated mice and PBS-treated controls 48 hours post-injection. Data indicate no significant difference in spleen size (n = 3 per group, p > 0.05), suggesting a physiological response to eRBCEV administration.

[0042] Figures 9A-9D. Human RBC lipid isolation and multi-physics simulation. Figure 9A is a schematic representation of the method used for the extraction of membrane lipids from 0.8 mL red blood cells: (1) RBC is lysed with 0.8 mL DI water. (2) Chloroform: methanol (2: 1) 6mL solution is added and mixed (3) Additional 2 mL chloroform is added (4) 2 mL DI water is added, Vortex mixed for 2 mins, furthermore the solution is centrifuged for phase separation and purified, lastly chloroform is aspirated by N2 gas under sterile conditions. Dried lipid pellet is packed into the vial and weighed. Figure 9B shows 2D microfluidic chip designs on COMSOL Multiphysics with defined meshing configurations, (i) Flow focusing design representation (ii) tetrahedral finer mesh configuration for FF design (iii) Staggered herringbone design representation (iv) adaptive triangular fine curvature mesh configuration for HB design. Figure 9C includes a visualization of phase mixing with simulation of volume of fluid (VoF) in FF chip at 4 mM lipid concentration and FRR (i) 1: 2 - 100:200 pL / min (ii) 1 :6 - 50:300 pL / min (iii) 1:8 - 40:320 pL / min (iv) 1: 16 - 20:320 pL / min. Figure 9D is a comparison of simulated interface visuals against real-time capture on FF chip at (i) FRR 1:5 - 50:250 pL / min and (ii) 1 : 10 - 50:500 pL / min respectively.

[0043] Figures 10A-10C. Quantification of diffusion of lipid phase across the microfluidic channel length and height. Figure 10A includes (i) a COMSOL line graph execution using 2D cut X function segmented every 50 pm; and (ii) quantification of normalized concentration of lipid phase across each line cut throughout the channel. Figure 10B shows a quantification of diffusion overlapping of lipid phase, aqueous phase, and interface phase across the channel length and height, including (i) COMSOL line graph execution using 2D cut Y function segmented every 10 pm, (ii) Quantification of diffusion of LG1 lipid phase, LG2 aqueous phase and LG3 interphase across the channel length. Figure 10C shows a visualization of phase mixing with simulation of volume of fluid (VoF) in HB chip at 4 mM lipid concentration and FRR (i) 1 : 1 - 100: 100 pL / min (11) 1:6 - 50:300 pL / min (111) 1 : 10- 40:400 pL / min (iv) 1:40 - 15:600 pL / min.

[0044] Figure 11A-11D. Statistical analysis for analytically characterized eRBCEVs. Figure 11 A is a bar graph revealing the turkey statistical differences among FRR 4, 5, 6, 8, 10, 20, and 40 against the mean average size of eRBCEVs produced at 4mM lipid concentration on HB chip. Figure 1 IB is a bar graph representing the statistical comparisons on the effect of encapsulation of cargo into eRBCEVs against mean size (N.S. - not significant). Figure 11C is a bar graph showing the statistical comparisons of dependencies of eRBCEV size on various RBC lipid compositions including 1 mM, 2 mM, and 4 mM at FRR 20 (n=3, **p<0.001, *p<0.01). Figure 1 ID is a bar graph depicting statistical comparisons of surface charge as zeta potential of eRBCEVs against negative standard (N=2, n = 3, ****p<0.0001). Figure 12A-12B. Structure comparison of eRBCEVs and lipid aggregation on fluidic devices. Figure 12A shows qualitative structural comparisons of (i) natural RBCEVs on TEM, (ii) the concentration and size distribution (mean - 121.4 nm, SD + / - 68.7 nm) (iii) engineered RBCEVs on TEM, and (iv) concentration and size distribution (mean - 132 nm, SD + / - 9.2 nm) measured on NTA. Figure 12B includes a visualization of fluid-particle aggregation near the interface on FF and HB devices at FRR 1 :20, and lipid concentration 4 mM recorded at (i) 0 min, and (ii) 15 min.

[0045] Figure 13A-13C. High-resolution optical characterization and quantification of eRBCEVs. Figure 13A (i - iii) TEM images of eRBCEVs encapsulated hHb at FRR 20 lipid concentration 4 mM, (iv) Histogram depicting quantification of eRBCEVs size from TEM images. Figure 13B (i - iii) TEM images of eRBCEVs encapsulated AuNPs at FRR 20 lipid concentration 4 mM, (iv) Histogram depicting quantification of eRBCEVs size from TEM images. Figure 13C shows elemental mapping on TEM ED AX: (i) Negative stain of eRBCEVs encapsulating AuNPs, (ii) Oxygen in K shell energy band, (iii) Phosphorous in K shell energy band, (iv) CPS mapping, (v) Silica in K shell energy band, (vi) Nikel in K shell energy band, (vii) Density mapping, (viii) Carbon in K shell energy' band and (ix) elemental overlay.

[0046] Figure 14A-14B. High-resolution characterization of eRBCEVs. Figure 14A shows a cryo-EM border view for quantification of size and structure of eRBCEVs. Figure 14B shows a visualization of TIRFM signals for surface modification, (i) PBS (ii) Channel 561, and (iii) eRBCEVs - Neu488 captured Biotin signals on channel 488.

[0047] Figure 15A-15G. Single-EV resolution characterization and quantification of eRBCEVs. Figure 15A includes a visualization of TIRFM signals from (i) free FAM-O, (ii) PBS, (iii) empty eRBCEVs labelled with DiR in 657 nm channel, and empty eRBCEVs labelled with DiR in 488 nm channel. Figure E5B includes a quantification of TIRFM signals from (i) Mean intensities against counts of FAM-0 signals, and (ii) Mean intensities against counts of DiR labelled eRBCEVs. Figure 15C is a histogram representing mean size distribution of eRBCEVs with continuous fit at FRR 20, lipid concentration 2 mM produced on HB chip. Figure 15D is a visual representation of TIRFM signals from (i) free LtEc protein labelled with FITC, (ii) PBS, (iii) empty eRBCEVs labelled with DiR in 657 nm channel, and empty eRBCEVs labelled with DiR in 488 nm channel. Figure 15E includes a quantification of TIRFM signals from (i) Mean intensities against the area of FITC-LtEc signals, and (ii) Mean intensities against the area of DiR labelled eRBCEVs. Figure 15F is a histogram depicting mean size distribution of eRBCEVs with continuous fit at FRR 5, lipid concentration 4 mM produced on HB chip. Figure 15G is a qualitative estimation of free FITC leak and unlabelled LtEc presence using Nano- Nano- Spectrophotometer after purification, (i) absorbance peaks comparing free F1TC. free LtEc, and PBS, (ii) Absorbance peaks comparing eRBCEVs encapsulated with LtEc, purified sample, WFI and PBS, (iii) Absorbance peaks comparing free FITC, retentate containing eRBCEVs (LtEc), and permeate containing any leaked FITC or free LtEc.

[0048] Figure 16A-16B. Quantification of immunogenic response on brightfield microscopy. Figure 16A is a spline fit line graph representing neutrophil activation percentage to estimate the rate of activation at various time points from 5 mins to 360 minutes with samples including BioParticles™. hHb at various dilutions including 0.5, 1, 2, 20 and 225 mg / mL and PBS controls. Figure 16B includes brightfield images showing the neutrophil structure after 360 minutes with induced (i) PBS, (ii) BioParticles™, (iii) cLNP, and (iv) eRBCEVs encapsulating hHb (0.5 mg / mL).

[0049] Figure 17. Qualitative assessment of eRBCEVs surface functionalization by PDL1 nanobodies. TIRFM images for the channels 488 and 647 nm represents (i) PBS - no evidence of significant fluorescence signal on either of the channels (ii) DiR labelled eRBCEVs was evident on the 647 nm channel only (iii) The fluorescent signals from NHSF labelled PDL1 nanobodies with DiR was observed only on the 488 nm channel, confirming no significant evidence of cross channel signals and presence of eRBCEVs and (iv) The surface modified eRBCEVs with nanobodies show colocalized signals forming yellow spots on the merged image.

[0050] Figures 18A-18B. Qualitative assessment of eRBCEVs uptake by EMT6 cells on confocal microscopy. Figure 18A shows confocal imaging EMT6 cells represents (i) DAPI stained nucleus incubated at 4 °C, (ii) RFP stained lysosome at 4 °C, (iii) NHSF-GFP stained PDL1 nanobodies at 4 °C, (iv) DiR stained eRBCEVs at 4 °C, assigned blue color to differentiate with RFP. (v) Merged image of nucleus, lysosome and PDL1 showing localized signals around the cells at 4 °C with orthogonal projections (vi) Merged image of nucleus, lysosome and PDL1 showing localized signals around the cells at 37 °C with orthogonal projections. Figure 18B shows a large ROI view of control images representing (i) Merged signals from PBS at 37 °C, (ii) Merged signals of PDL1, DAPI and lysosome at 37 °C. (iii) Merged signals from eRBCEVs, DAPI and lysosome at 37 °C, and colocalized signals from natural RBCEVs, DAPI and lysosome at (v) 4 °C, and (vi) 37 °C.

[0051] DETAILED DESCRIPTION

[0052] The present invention can be understood more readily by reference to the following detailed description, examples, drawings, and claims, and their previous and following description. However, before the present articles, systems, and / or methods are disclosed and described, it is to be understood that this invention is not limited to the specific or exemplary- aspects of articles, systems, and / or methods disclosed unless otherwise specified, as such can, of course, vary. It is also to be understood that the terminology' used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

[0053] The following description of the invention is provided as an enabling teaching of the invention in its best, currently known aspect. To this end, those skilled in the relevant art will recognize and appreciate that many changes can be made to the various aspects of the invention described herein, while still obtaining the beneficial results of the present invention. It will also be apparent that some of the desired benefits of the present invention can be obtained by selecting some of the features of the present invention without utilizing other features. Accordingly, those of ordinary- skill in the pertinent art will recognize that many modifications and adaptations to the present invention are possible and may even be desirable in certain circumstances and are a part of the present invention. Thus, the following description is again provided as illustrative of the principles of the present invention and not in limitation thereof.

[0054] Definitions

[0055] It is appreciated that certain features of the disclosure, which are, for clarity-, described in the context of separate aspects, can also be provided in combination in a single aspect. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single aspect, can also be provided separately or in any suitable subcombination.

[0056] As used in the description and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0057] As used herein, the terms “optional” or “optionally” mean that the subsequently- described event or circumstance may or may not occur and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0058] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. As used in the specification and in the claims, the term “comprising” can include the aspects “consisting of’ and “consisting essentially of.” Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly' understood by one of ordinary skill in the art to which this invention belongs. In this specification and in the claims which follow, reference will be made to a number of terms that shall be defined herein.

[0059] For the terms "for example" and "such as," and grammatical equivalences thereof, the phrase "and without limitation" is understood to follow unless explicitly stated otherwise. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Furthermore, when numerical ranges of varying scope are set forth herein, it is contemplated that any combination of these values inclusive of the recited values may be used. Further, ranges can be expressed herein as from “about” one particular value and / or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value.

[0060] Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint. Unless stated otherwise, the term “about” means within 5% (e.g., within 2% or 1%) of the particular value modified by the term “about.” Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5. 5.3, 6 and any whole and partial increments therebetween. This applies regardless of the breadth of the range.

[0061] It will be understood that, although the terms "first," "second," etc., may be used herein to describe various elements, components, regions, layers, and / or sections. These elements, components, regions, layers, and / or sections should not be limited by these terms. These terms are only used to distinguish one element, component, region, layer, or section from another element, component, region, layer, or a section. Thus, a first element, component, region, layer, or section discussed below could be termed a second element, component, region, layer, or section without departing from the teachings of example embodiments.

[0062] As used herein, the term "substantially" means that the subsequently described event or circumstance completely occurs or that the subsequently described event or circumstance generally, typically, or approximately occurs. As used herein, the term "substantially,” in, for example, the context “substantially identical” or “substantially similar” refers to a method or a system, or a component that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%. at least about 94%, at least about 95%, at least about 96%, at least about 97%. at least about 98%. at least about 99%, or about 100% by similar to the method, system, or the component it is compared to.

[0063] As used herein, the term “substantially,” in, for example, the context “substantially free from” refers to a composition that includes less than 5% by weight, less than 2.5% by weight, less than 2% by weight, less than 1% by weight, less than 0.5% by weight, less than 0.1% byweight, less than 0.05% by weight, or less than 0.01% by weight of the component described as substantially absent from the composition.

[0064] “Nanoparticle”, as used herein, refers to any entity having a diameter of less than 100 microns (pm). Typically, particles have a greatest dimension (e g., diameter) of 1000 nm or less. In some embodiments, particles have a diameter of 300 nm or less. In some embodiments, nanoparticles have a diameter of 200 nm or less. In some embodiments, nanoparticles have a diameter of 100 nm or less. In general, particles are greater in size than the renal excretion limit, but are small enough to avoid accumulation in the liver.

[0065] “Mean particle size,” as used herein, generally refers to the statistical mean particle size (diameter) of the particles in a population of particles. The diameter of an essentially spherical particle may be referred to as the physical or hydrodynamic diameter. The diameter of a non- spherical particle may refer preferentially to the hydrodynamic diameter. As used herein, the diameter of a non-spherical particle may refer to the largest linear distance between two points on the surface of the particle. Mean particle size can be measured using methods known in the art, such as dynamic light scattering.

[0066] “Monodisperse” and “homogeneous size distribution,” are used interchangeably herein and describe a plurality of liposomal nanoparticles or microparticles where the particles have the same or nearly the same diameter or aerodynamic diameter. As used herein, a monodisperse distribution refers to particle distributions in which 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 86, 88, 89, 90, 91, 92, 93, 94, 95% or greater of the distribution lies within 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, or 10% of the mass median diameter or aerodynamic diameter.

[0067] “Small molecule”, as used herein, generally refers to an organic molecule that is less than about 2000 g / mol in size. In some embodiments, the small molecule is less than about 1500 g / mol or less than about 1000 g / mol. In some embodiments, the small molecule is less than about 800 g / mol or less than about 500 g / mol. In some embodiments, small molecules are non- polymeric and / or non-oligomeric. In some embodiments, small molecules are not proteins, peptides, or amino acids. In some embodiments, small molecules are not nucleic acids or nucleotides. In some embodiments, small molecules are not saccharides or polysaccharides.

[0068] The term “active agent” refers to a therapeutic agent, a diagnostic agent, or a prophylactic agent that can be administered to a subject for to elicit a benefit or effect (e.g., to prevent or treat a disease or disorder). Examples of active agents can include a nucleic acid, a nucleic acid analog, a small molecule, a peptidomimetic, a protein, peptide, carbohydrate or sugar, lipid, or surfactant, or a combination thereof.

[0069] The terms “incorporated” and “encapsulated” refers to incorporating, formulating, or otherwise including an active agent into and / or onto a composition that allows for release, such as sustained release, of such agent in the desired application. The terms contemplate any manner by which an active agent or other material is incorporated into a lipid particle (e.g., a vesicle), including, for example: attached to a particle (by covalent, ionic, or other binding interaction), physical admixture, enveloping the agent in a coating layer of lipid, incorporated into the lipid, distributed throughout a lipid matrix, appended to the surface of the lipid matrix (by covalent or other binding interactions), encapsulated inside the lipid matrix or vesicle, etc. The term “coincorporation” or “co-encapsulation” refers to-the incorporation of an active agent or other material and at least one other active agent or other material in a subject composition.

[0070] The term “targeting moiety" as used herein refers to a moiety that localizes to or away from a specific locale. The moiety may be, for example, a protein, nucleic acid, nucleic acid analog, carbohydrate, or small molecule. Said entity may be, for example, a therapeutic compound such as a small molecule, or a diagnostic entity’ such as a detectable label. Said locale may be a tissue, a particular cell type, or a subcellular compartment. In one embodiment, the targeting moiety directs the localization of an active entity. The active entity may be a small molecule, protein, polymer, or metal. The active entity may be useful for therapeutic, prophylactic, or diagnostic purposes.

[0071] It is understood that in aspects disclosed herein, the terms “diacycle” and “diafiltration cycles” are used interchangeably.

[0072] As used herein, the term "tangential-flow filtration" refers to a process in which the fluid mixture containing the components to be separated by fdtration is recirculated at velocities tangential to the plane of the filtration membrane to reduce fouling of the filter. In such filtrations a pressure differential is applied along the length of the filtration membrane to cause the fluid and filterable solutes to flow through the membrane (i.e., filter). This filtration is suitably conducted as a batch process as well as a continuous-flow process. For example, the solution may be passed repeatedly over the membrane while that fluid which passes through the filter is continually rawn off into a separate unit or the solution is passed once over the membrane and the fluid passing through the filter is continually processed downstream.

[0073] While aspects of the present invention can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only, and one of ordinary skill in the art will understand that each aspect of the present invention can be described and claimed in any statutory class. Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that an order be inferred in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0074] Methods for Generating Red Blood Cell Lipid Nanoparticles

[0075] Red blood cells (RBCs) are a unique type of oxygen-carrying cell, with lipids — specifically phospholipids — on their cell membrane. These lipids form a semipermeable barrier that separates the internal cell contents from the external circulatory stream. Traditionally, RBC nanoparticles are synthesized using a top-down approach through hemolysis to create erythrocyte ghosts. These ghosts then undergo several physical processes to form nanoparticles. The most common techniques for synthesizing these RBC-LNPs include sonication, the ethanol- inj ection method, hand mixing, and extrusion methods. However, sonication carries the risk of heat generation and particle aggregation; the ethanol-injection method faces challenges in control and solvent impact; hand mixing lacks uniformity and consistency; and extrusion involves shear forces and limited size control.

[0076] Described herein are methods for generating red blood cell lipid nanoparticles that overcome the draw backs of these existing methods. These methods can comprise mixing a first solution comprising a mixture of lipids isolated from red blood cells and a second solution comprising an aqueous buffer within a microfluidic device under conditions effective to form a crude mixture comprising the population of red blood cell lipid nanoparticles dispersed within an aqueous fluid; and filtering the crude mixture comprising the population of red blood cell lipid nanoparticles by microfiltration against a filtration membrane, thereby forming a retentate fraction comprising the population of red blood cell lipid nanoparticles and a permeate fraction comprising low molecular weight contaminants.

[0077] The red blood cell lipids can comprise a mixture of lipids isolated from the membranes red blood cells. The red blood cells can comprise human red blood cells or non-human red blood cells. In some embodiments, the red blood cells can comprise human red blood cells isolated from whole blood. In other embodiments, the red blood cells comprise red blood cells derived from stem cells, “recombinant red cells” cultured in bioreactors, or engineered red cells obtained from genetic engineering of stem cells.

[0078] In some embodiments, the mixture of lipids isolated from red blood cells can be substantially free of proteins. For example, in certain embodiments, the mixture of lipids isolated from red blood cells can comprise less than 1% by weight, less than 0.5% by weight, less than 0.1% by weight, less than 0.05% by w eight, or less than 0.01% by w eight of proteins. In some embodiments, the mixture of lipids isolated from red blood cells can comprise less than 1% by weight (e.g., less than 0.75% by weight, less than 0.5% by weight, less than 0.4% by weight, less than 0.3% by weight, less than 0.25% by weight, less than 0.2% by weight, less than 0.1% by w eight, less than 0.05% by weight, or less than 0.01% by weight) free hemoglobin and free heme. In some embodiments, the mixture of lipids isolated from red blood cells can comprise no detectable free hemoglobin and free heme.

[0079] The mixture of lipids isolated from red blood cells can comprise cholesterol, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), lysophosphatidylcholine (LysoPC), sphingomyelin-DSM (SM-DSM), ethylphosphatidylcholine (ePC), lysophosphatidylethanolamine (LysoPE), ethylphosphatidylethanolamine (ePE). ethylphosphatidylserine (ePS), phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylethanolamine-ceramide (PE-Cer), or any combination thereof.

[0080] In some embodiments, the mixture of lipids isolated from red blood cells comprises a mixture of phospholipids, and the mixture of phospholipids comprises from 40% by weight to 50% by weight phosphatidylcholine (PC), from 15% by weight to 25% by weight phosphatidylethanolamine (PE), from greater than 0% by weight to 0.5% by weight phosphatidylinositol (PI), from 1% by weight to 5% by weight phosphatidylserine (PS), from 0.2% by weight to 1% by weight lysophosphatidylcholine (LysoPC), from 20% by weight to 25% by weight sphingomyelin-DSM (SM-DSM), from 3% by weight to 6% by weight ethylphosphatidylcholine (ePC), from greater than 0% by weight to 0.5% by w eight lysophosphatidylethanolamine (LysoPE), from 0.5% by w eight to 3% by weight ethylphosphatidylethanolamine (ePE). from greater than 0% by weight to 0.2% by weight ethylphosphatidylserine (ePS), from greater than 0% by weight to 0.5% by weight phosphatidic acid (PA), from greater than 0% by weight to 0.5% by w eight phosphatidylglycerol (PG), and from greater than 0% by weight to 0.2% by weight phosphatidylethanolamine-ceramide (PE- Cer). In certain embodiments, the mixture of lipids isolated from red blood cells comprises a mixture of phospholipids, and the mixture of phospholipids comprises -46.6% by weight phosphatidylcholine (PC), -20.4% phosphatidylethanolamine (PE), -0.2% phosphatidylinositol (PI), -2.5% phosphatidylserine (PS), -0.6 % lysophosphatidylcholine (LysoPC), 22.89% sphingomyelin-DSM (SM-DSM), 4.5% ethylphosphatidylcholine (ePC), 0.25% lysophosphatidylethanolamine (LysoPE), 1.7% ethylphosphatidylethanolamine (ePE), 0.04% ethylphosphatidylserine (ePS), 0.2% phosphatidic acid (PA), 0.1% phosphatidylglycerol (PG), and -0.01% phosphatidylethanolamine-ceramide (PE-Cer).

[0081] In some embodiments, the mixture of lipids isolated from red blood cells is obtained by a process that comprises extraction of lipids from a population of lysed red blood cells using a chloroform-methanol phase separation technique. In certain embodiments, the method further comprises; lysing a population of red blood cells; extracting the lysed population of red blood cells with chloroform and methanol to obtain a biphasic mixture comprising an organic phase and an aqueous phase; separating the organic phase from the aqueous phase; filtering the organic phase to remove any suspended proteins; and isolating mixture of lipids isolated from red blood cells from the organic phase.

[0082] Lysing the population of RBCs can comprise any suitable method for inducing lysis of cells. In some embodiments, this can comprise contacting the population of RBCs with a lysing agent, such as water or a buffer solution having an appropriate ionic strength to induce lysis of the RBCs. Suitable lysing agents are known in the art, including commercially available RBC lysis buffers, which are ammonium chloride-based buffer systems designed for the preferential lysis of red blood cells from whole blood.

[0083] Filtering the organic phase to remove any suspended proteins can be performed using any suitable filter or membrane having an appropriate pore size to retain the proteins while allowing impurities to pass to the permeate size of the filter / membrane. In some examples, the filter or membrane can be rated as having a pore size of 0. 1 pm, 0.2 pm, 0.45 pm, 0.65 pm, 0.8 pm, 1 pm, or 1.2 pm. In certain examples, the filter or membrane can be rated as having a pore size of 0.45 pm.

[0084] If desired, one or more additional lipids can be added to the mixture of lipids isolated from red blood cells, including fatty7acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids. and polyketides (derived from condensation of ketoacyl subunits); and sterol lipids and prenol lipids (derived from condensation of isoprene subunits). Examples of lipids that can be added to the mixture of lipids isolated from red blood cells include those described in U.S. Patent No. 10,864,162, which is incorporated herein by reference in its entirety.

[0085] In some embodiments, the mixture of lipids isolated from red blood cells further comprises one or more additional lipids added to the mixture of lipids isolated from red blood cells. In certain embodiments, the one or more additional lipids can comprise a synthetic PEGylated lipid, such as DSPE-PEG-DBCO.

[0086] In some embodiments, the first solution comprises the mixture of lipids isolated from red blood cells dissolved or dispersed in water, a water-miscible solvent, or a combination thereof. In some embodiments, the first solution comprises the mixture of lipids isolated from red blood cells dissolved or dispersed in a water-miscible solvent. In certain embodiments, the water- miscible solvent comprises an alcohol, such as ethanol.

[0087] In some embodiments, the aqueous buffer comprises a citrate buffer.

[0088] In some embodiments, the aqueous buffer exhibits a pH of from 5.5 to 8.5, such as a pH of from 5.5 to 7.5, a pH of from 5.5 to 7.0, a pH of from 5.5 to 6.5, or a pH of about 6.

[0089] In some embodiments, the microfluidic device comprises a central fluid inlet channel and two outer fluid inlet channels, wherein the central fluid inlet channel and the outer fluid inlet channels converge in a mixing channel upstream of an outlet. In these embodiments, mixing the first solution comprising the mixture of lipids isolated from red blood cells and the second solution comprising the aqueous buffer can comprise Plowing the first solution through the central fluid inlet and flowing the second solution through the two outer fluid inlet channels. Examples of such devices and methods include those described in U.S. Patent No. 10,864.162. which is incorporated herein by reference in its entirety.

[0090] In some embodiments, the microfluidic comprises a first fluid inlet channel and a second fluid inlet channel that converge in a mixing channel upstream of an outlet, wherein the mixing channel further comprises one or more mixing elements, such as pillars or ridges, that increase chaotic mixing of fluids within the mixing channel. In these embodiments, mixing the first solution comprising the mixture of lipids isolated from red blood cells and the second solution comprising the aqueous buffer can comprise flowing the first solution through the first fluid inlet channel and flowing the second solution through the second fluid inlet channel.

[0091] In some embodiments, the second solution comprising the aqueous buffer and the first solution comprising the mixture of lipids isolated from red blood cells are introduced at a flow' rate ratio (FRR) of from 1:1 to 1:60, such as from 1:2 to 1 :20 or from 1:4 to 1 :20. In some embodiments, the first solution comprising the mixture of lipids isolated from red blood cells is introduced into the microfluidic device at a flow rate of from 100 pL / min to 1000 pL / min. In some embodiments, the second solution comprising the aqueous buffer is introduced into the microfluidic device at a flow rate of from 10 pL / min to 200 pL / min.

[0092] In some embodiments, the mixture of lipids isolated from red blood cells are present in the first solution at a concentration of from 0.5 rnM to 10 mM, such as from 1 mM to 4 mM.

[0093] In some embodiments, the filtration membrane is rated for a 500 kDa molecular weight cut-off. In some embodiments, filtering the crude mixture comprising the population of red blood cell lipid nanoparticles by microfiltration comprises filtering for at least 3 diafiltrations cycles, such as at least 4 diafiltration cycles, at least 5 diafiltration cycles, at least 6 diafiltration cycles, at least 7 diafiltrations cycle, at least 8 diafiltration cycles, at least 9 diafiltrations cycle, or at least 10 diafiltration cycles. In some embodiments, the microfiltration comprises tangential flow filtration.

[0094] In some embodiments, the population of red blood cell lipid nanoparticles is monodisperse. In certain embodiments, the population of red blood cell lipid nanoparticles exhibits a PDI of 0. 1 or less.

[0095] In some embodiments, the population of red blood cell lipid nanoparticles exhibit an average particle size of at least 10 nm (e.g., at least 20 nm, at least 25 nm, at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 75 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, at least 120 nm, at least 125 nm, at least 130 nm, at least 140 nm, at least 150 nm, at least 160 nm, at least 170 nm, at least 175 nm, at least 180 nm, at least 190 nm, at least 200 nm, at least 210 nm, at least 220 nm, at least 225 nm, at least 230 nm, at least 240 nm, at least 250 nm. at least 275 nm, at least 300 nm. at least 325 nm, at least 350 nm, at least 375 nm, at least 400 nm, at least 425 nm, at least 450 nm, or at least 475 nm), as measured by dynamic light scattering (DLS). In some embodiments, the population of red blood cell lipid nanoparticles exhibit an average particle size of 500 nm or less (e g., 475 nm or less, 450 nm or less. 425 nm or less, 400 nm or less, 375 nm or less, 350 nm or less. 325 nm or less,

[0096] 300 nm or less, 275 nm or less, 250 nm or less, 240 nm or less, 230 nm or less, 225 nm or less,

[0097] 220 nm or less, 210 nm or less, 200 nm or less, 190 nm or less, 180 nm or less, 175 nm or less,

[0098] 170 nm or less. 160 nm or less, 150 nm or less, 140 nm or less, 130 nm or less, 125 nm or less,

[0099] 120 nm or less. 110 nm or less, 100 nm or less, 90 nm or less. 80 nm or less, 75 nm or less, 70 nm or less, 60 nm or less, 50 nm or less, 40 nm or less, 30 nm or less, 25 nm or less, or 20 nm or less), as measured by dynamic light scattering (DLS). In some embodiments, the population of red blood cell lipid nanoparticles can exhibit a zeta potential close to 0 mV at low to zero ionic strengths (1 to 10 mM), with zeta potential values between +5 to -5 mV, and a zero / neutral or a small surface charge.

[0100] The population of red blood cell lipid nanoparticles can exhibit an average particle size ranging from any of the minimum values described above to any of the maximum values described above. For example, in some embodiments, the population of red blood cell lipid nanoparticles exhibit an average particle size of from 10 nm to 500 nm, such as from 75 nm to 300 nm, as measured by dynamic light scattering (DLS).

[0101] In certain embodiments, the population of red blood cell lipid nanoparticles comprise engineered red blood cell extracellular vesicles (eRBCEVs).

[0102] Active Agents

[0103] In some embodiments, the first solution, the second solution, or a combination thereof further comprises an active agent including a therapeutic agent / drug, a diagnostic agent (e.g. contrast agent; radionuclides and / or fluorescent, luminescent, or magnetic moiety), prophylactic agent (e.g. vaccines), and / or nutraceutical agent (e.g. vitamin, mineral, etc.) to be associated with the surface of, encapsulated within, surrounded by, and / or dispersed throughout the particles produced using the methods described herein. The particles may provide for delivery and / or release of the active agent.

[0104] In some embodiments, the first solution, the second solution, or a combination thereof further comprises an active agent, and the population of red blood cell lipid nanoparticles formed by the method comprise the active agent encapsulated therein. In certain embodiments, the second solution further comprises an active agent, and the population of red blood cell lipid nanoparticles formed by the method comprise the active agent encapsulated therein.

[0105] The active agent can comprise any suitable active agent. By way of example, in some embodiments, the active agent can comprise a small molecule (e.g. a cytotoxic agent), an organometallic compound, a nucleic acid (e.g., DNA or RNA, such as a siRNA, RNAi, or mircoRNA agent), a protein (including multimeric proteins, protein complexes, etc., such as an antibody), a peptide, a lipid, a carbohydrate, a hormone, a metal, a radioactive element or compound, a drug, a vaccine, an immunological agent, a nanoparticle, or a combination thereof. In some embodiments, the active agent comprises an active agent useful in the treatment of cancer. In some embodiments, the active agent can comprise an oxygen-binding protein, such as hemoglobin, myoglobin, erythrocruorin, chlorocruorin, or hemocyanin.

[0106] In some embodiments, the active agent is a small molecule and / or organic compound with pharmaceutical activity7. In some embodiments, the active agent is a clinically-used drug. In some embodiments, the drug is an anti-cancer agent, antibiotic, anti-viral agent, anti-HIV agent, anti-parasite agent, anti-protozoal agent, anesthetic, anticoagulant, inhibitor of an enzyme, steroidal agent, steroidal or non-steroidal anti-inflammatory agent, antihistamine, immunosuppressant agent, anti-neoplastic agent, antigen, vaccine, antibody, decongestant, sedative, opioid, analgesic, anti-pyretic, birth control agent, hormone, prostaglandin, progestational agent, anti-glaucoma agent, ophthalmic agent, anti-cholinergic, analgesic, antidepressant, anti-psychotic, neurotoxin, hypnotic, tranquilizer, anti-convulsant, muscle relaxant, anti-Parkinson agent, anti-spasmodic, muscle contractant, channel blocker, miotic agent, anti- secretory agent, anti -thrombotic agent, anticoagulant, anti-cholinergic, beta. -adrenergic blocking agent, diuretic, cardiovascular active agent, vasoactive agent, vasodilating agent, antihypertensive agent, angiogenic agent, modulators of cell-extracellular matrix interactions (e.g. cell growth inhibitors and anti-adhesion molecules), inhibitor of DNA, RNA, or protein synthesis, etc.

[0107] Exemplary active agents that can be incorporated into the particles include, but are not limited to. tumor antigens, CD4+ T-cell epitopes, cytokines, chemotherapeutic agents, radionuclides, small molecule signal transduction inhibitors, photothermal antennas, monoclonal antibodies, immunologic danger signaling molecules, other immunotherapeutics, enzymes, antibiotics, antivirals (especially protease inhibitors alone or in combination with nucleosides for treatment of HIV or Hepatitis B or C), anti-parasitics (helminths, protozoans), growth factors, growth inhibitors, hormones, hormone antagonists, antibodies and bioactive fragments thereof (including humanized, single chain, and chimeric antibodies), antigen and vaccine formulations (including adjuvants), peptide drugs, anti-inflammatories, immunomodulators (including ligands that bind to Toll-Like Receptors to activate the innate immune system, molecules that mobilize and optimize the adaptive immune system, molecules that activate or up-regulate the action of cytotoxic T lymphocytes, natural killer cells and helper T-cells, and molecules that deactivate or down-regulate suppressor or regulatory T-cells), agents that promote uptake of the particles into cells (including dendritic cells and other antigen-presenting cells), nutraceuticals such as vitamins, and oligonucleotide drugs (including DNA, RNAs, antisense, aptamers, small interfering RNAs, ribozymes, external guide sequences for ribonuclease P, and triplex forming agents).

[0108] In some embodiments, the active agent or drug may be a therapeutic agent such as mTor inhibitors (e.g., sirolimus, temsirohmus, or everolimus), vinca alkaloids (e.g. vinorelbine or vincristine), a diterpene derivative, a taxane (e.g. paclitaxel or its derivatives such as DHA- paclitaxel or PG-paxlitaxelor, or docetaxel), a boronate ester or peptide boronic acid compound (e.g. bortezomib), a cardiovascular agent (e.g. a diuretic, a vasodilator, angiotensin converting enzyme, a beta blocker, an aldosterone antagonist, or a blood thinner), a corticosteroid (e.g. budensonide, fluocinonide, triamcinolone, mometasone, amcinonide, halcinonide, ciclesonide, beclomethansone), an antimetabolite or antifolate agent (e.g. methotrexate), a chemotherapeutic agent (e.g. epothilone B), a nitrogen mustard agent (e.g. bendamustine), or the active agent or drug may be an siRNA.

[0109] Example active agents include chemotherapeutic agents such as doxorubicin (adriamycin), gemcitabine (gemzar), daunorubicin, procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5 -fluorouracil (5-FU), vinca alkaloids such as vinblastine, vinoelbine, vindesine, or vincristine; bleomycin, taxanes such as paclitaxel (taxol) or docetaxel (taxotere), mTOR inhibitors such as sirolimus, temsirolimus, or everolimus, aldesleukin, asparaginase, boronate esters or peptide boronic acid compounds such as bortezomib, busulfan, carboplatin, cladribine, camptothecin, CPT-11. 10-hydroxy-7-ethylcamptothecin (SN38), dacarbazine, S-I capecitabine. ftorafur, 5 'deoxy flurouri dine, UFT, eniluracil. deoxy cytidine. 5-azacytosine, 5- azadeoxycytosine, allopurinol, 2-chloroadenosine, trimetrexate, aminopterin, methylene-10- deazaaminopterin (MDAM), oxaplatin, picoplatin, tetraplatin, satraplatin, platinum-DACH, ormaplatin, CI-973, JM-216. epirubicin, etoposide phosphate, 9-aminocamptothecin, 10,11- methylenedioxy camptothecin, karenitecin, 9-nitrocamptothecin, TAS 103, L-phenylalanine mustard, ifosphamidemefosphamide, perfosfamide, trophosphamide carmustine, semustine, bendamustine, epothilones A-E, tomudex, 6-mercaptopurine, 6-thioguanine, amsacrine, karenitecin, acyclovir, valacyclovir, ganciclovir, amantadine, rimantadine, lamivudine, zidovudine, bevacizumab, trastuzumab, rituximab, budesonide, and combinations thereof, or the therapeutic agent may be an siRNA.

[0110] Non-limiting examples of potentially suitable active agents include anti-cancer agents, including, for example, docetaxel, mitoxantrone, and mitoxantrone hydrochloride. In another embodiment, the payload may be an anti-cancer drug such as 20-epi-l, 25 dihydroxyvitamin D3, 4-ipomeanol. 5-ethynyluracil, 9-dihydrotaxol. abiraterone, acivicin. aclarubicin, acodazole hydrochloride, acronine, acylfiilvene, adecypenoL adozelesin, aldesleukin, all-tk antagonists, altretamine, ambamustine, ambomycin, ametantrone acetate, amidox, amifostine, aminoglutethimide, aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole, andrographolide. angiogenesis inhibitors, antagonist D, antagonist G. antarelix, anthramycin, anti-dorsalizdng morphogenetic protein- 1, antiestrogen, antineoplaston, antisense oligonucleotides, aphidicolin glycinate, apoptosis gene modulators, apoptosis regulators, apurinic acid, ARA-CDP-DL-PTBA, arginine deaminase, asparaginase, asperlin, asulacrine, atamestane, atnmustine. axinastatin 1. axinastatin 2, axinastatin 3, azacitidine, azasetron. azatoxin, azatyrosine, azetepa, azotomycin, baccatin III derivatives, balanol, batimastat, benzochlorins, benzodepa, benzoylstaurosporine, beta lactam derivatives, beta-alethine, betaclamycin B, betulinic acid, BFGF inhibitor, bicalutamide, bisantrene, bisantrene hydrochloride, bisazuidinylspermine, bisnafide. bisnafide dimesylate, bistratene A. bizelesin, bleomycin, bleomycin sulfate, BRC / ABL antagonists, breflate, brequinar sodium, bropirimine, budotitane, busulfan, buthionine sulfoximine, cactinomycin, calcipotriol, calphostin C, calusterone, camptothecin derivatives, canarypox IL-2, capecitabine, caraceraide, carbetimer, carboplatin, carboxamide-amino-triazole, carboxyamidotriazole, carest M3, carmustine, earn 700, cartilage derived inhibitor, carubicin hydrochloride, carzelesin, casein kinase inhibitors, castanospermine, cecropin B, cedefingol, cetrorelix, chlorambucil, chlorins, chloroquinoxaline sulfonamide, cicaprost, cirolemycin, cisplatin, cis-porphyrin, cladribine, clomifene analogs, clotrimazole, collismycin A, collismycin B, combretastatin A4, combretastatin analog, conagenin. crambescidin 816, crisnatol, crisnatol mesylate, cr ptophycin 8, cryptophycin A derivatives, curacin A, cyclopentanthraquinones, cyclophosphamide, cyclosporine, cycloplatam, cypemycin, cytarabine, cytarabine ocfosfate, cytolytic factor, cytostatin, dacarbazine, dacliximab, dactinomycin, daunorubicin hydrochloride, decitabine, dehydrodidemnin B, deslorelin, dexifosfamide, dexormaplatin, dexrazoxane. dexverapamil, dezaguanine, dezaguanine mesylate, diaziquone, didemnin B, didox, diethyhiorspermine, dihydro-5- azacytidine, dioxamycin, diphenyl spiromustine, docetaxel, docosanol, dolasetron, doxifluridine, doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifene citrate, dromostanolone propionate, dronabinol, duazomycin, duocannycin SA, ebselen. ecomustine, edatrexate, edelfosine. edrecolomab, eflomithine. eflomithine hydrochloride, elemene. elsamitrucin. emitefur, enloplatin, enpromate, epipropidine, epirubicin, epirubicin hydrochloride, epristeride, erbulozole, ery throcyte gene therapy vector system, esorubicin hydrochloride, estramustine, estramustine analog, estramustine phosphate sodium, estrogen agonists, estrogen antagonists, etanidazole, etoposide, etoposide phosphate, etoprine. exemestane, fadrozole, fadrozole hydrochloride, fazarabine, fenretinide, filgrastim, finasteride, flavopiridol, flezelastine, floxuridine, fluasterone, fludarabine, fludarabine phosphate, fluorodaunorunicin hydrochloride, fluorouracil, flurocitabine, forfenimex, formestane, fosquidone, fostriecin, fostriecin sodium, fotemustine, gadolinium texaphyrin. gallium nitrate, galocitabine, ganirelix, gelatinase inhibitors, gemcitabine, gemcitabine hydrochloride, glutathione inhibitors, hepsulfam, heregulin, hexamethylene bisacetamide, hydroxyurea, hypericin, ibandronic acid, idarubicin, idarubicin hydrochloride, idoxifene, idramantone, ifosfamide, ihnofosine, ilomastat, imidazoacridones, imiquimod, immunostimulant peptides, insulin-like growth factor- 1 receptor inhibitor, interferon agonists, interferon alpha-2A, interferon alpha-2B, interferon alpha-Nl, interferon alpha-N3, interferon beta-IA, interferon gamma-IB, interferons, interleukins, iobenguane, iododoxorubicin, iproplatm, irinotecan, irinotecan hydrochloride, iroplact, irsogladine, isobengazole. isohomohalicondrin B. itasetron, jasplakinolide. kahalalide F. lamellarin-N triacetate, lanreotide, lanreotide acetate, leinamycin, lenograstim, lentinan sulfate, leptolstatin, letrozole, leukemia inhibiting factor, leukocyte alpha interferon, leuprolide acetate, leuprolide / estrogen / progesterone, leuprorelin, levamisole, liarozole, liarozole hydrochloride, linear polyamine analog, lipophilic disaccharide peptide, lipophilic platinum compounds, lissoclinamide, lobaplatin, lombricine, lometrexol, lometrexol sodium, lomustine, lonidamine, losoxantrone, losoxantrone hydrochloride, lovastatin, loxoribine, lurtotecan, lutetium texaphyrin lysofylline, lytic peptides, maitansine, mannostatin A, marimastat, masoprocol, maspin, matrilysin inhibitors, matrix metalloproteinase inhibitors, maytansine, mechlorethamine hydrochloride, megestrol acetate, melengestrol acetate, melphalan, menogaril, merbarone, mercaptopurine, meterelin, methioninase, methotrexate, methotrexate sodium, metoclopramide, metoprine, meturedepa, microalgal protein kinase C inhibitors, MIF inhibitor, mifepristone, miltefosine, mirimostim, mismatched double stranded RNA, mitindomide, mitocarcin, mitocromin, mitogillin, mitoguazone, mitolactol, mitomalcin. mitomycin, mitomycin analogs, mitonafide, mitosper, mitotane, mitotoxin fibroblast grow th factor-saporin, mitoxantrone, mitoxantrone hydrochloride, mofarotene, molgramostim, monoclonal antibody, human chorionic gonadotrophin, monophosphoryl lipid a / myobacterium cell wall SK, mopidamol, multiple drug resistance gene inhibitor, multiple tumor suppressor 1-based therapy, mustard anticancer agent, mycaperoxide B. mycobactenal cell wall extract, mycophenolic acid, myriaporone, n- acetyldinaline, nafarelin, nagrestip, naloxone / pentazocine, napavin, naphterpin, nartograstim, nedaplatin, nemorubicin, neridronic acid, neutral endopeptidase, nilutamide, nisamycin, nitric oxide modulators, nitroxide antioxidant, nitrullyn. nocodazole, nogalamycin, n-substituted benzamides, O6-benzylguanine, octreotide, okicenone, oligonucleotides, onapristone, ondansetron, oracin, oral cytokine inducer, ormaplatin, osaterone, oxaliplatin, oxaunomycin, oxisuran, paclitaxel, paclitaxel analogs, paclitaxel derivatives, palauamine, palmitoylrhizoxin, pamidronic acid, panaxytriol, panomifene, parabactin, pazelliptine, pegaspargase, peldesine, peliomycin, pentamustine, pentosan polysulfate sodium, pentostatin, pentrozole, peplomycin sulfate, perflubron, perfosfamide, perillyl alcohol, phenazinomycin, phenylacetate, phosphatase inhibitors, picibanil, pilocarpine hydrochloride, pipobroman, piposulfan, pirarubicin, piritrexim, piroxantrone hydrochloride, placetin A, placetin B, plasminogen activator inhibitor, platinum complex, platinum compounds, platinum-triamine complex, plicamycin, plomestane. porfimer sodium, porfiromycin, prednimustine, procarbazine hydrochloride, propyl bis-acridone, prostaglandin J2, prostatic carcinoma antiandrogen, proteasome inhibitors, protein A-based immune modulator, protein kinase C inhibitor, protein tyrosine phosphatase inhibitors, purine nucleoside phosphorylase inhibitors, puromycin, puromycin hydrochloride, purpurins, pyrazorurin, pyrazoloacridine, pyridoxylated hemoglobin polyoxyethylene conjugate, RAF antagonists, raltitrexed, ramosetron, RAS famesyl protein transferase inhibitors, RAS inhibitors, RAS-GAP inhibitor, retelliptine demethylated, rhenium RE 186 etidronate, rhizoxin, riboprine, ribozymes, RH retinamide, RNAi. rogletimide, rohitukine, romurtide, roquinimex, rubiginone Bl, ruboxyl, safingol, safingol hydrochloride, saintopin, sarcnu, sarcophytol A, sargramostim, SDI1 mimetics, semustine, senescence derived inhibitor 1, sense oligonucleotides, signal transduction inhibitors, signal transduction modulators, simtrazene, single chain antigen binding protein, sizofiran, sobuzoxane, sodium borocaptate, sodium phenyl acetate, solverol, somatomedin binding protein, sonermin, sparfosafe sodium, sparfosic acid, sparsomycin, spicamycin D, spirogermanium hydrochloride, spiromustine, spiroplatin, splenopentin, spongistatin 1, squalamine, stem cell inhibitor, stem-cell division inhibitors, stipiamide, streptonigrin, streptozocin. stromelysin inhibitors, sulfinosine, sulofenur, superactive vasoactive intestinal peptide antagonist, suradista, suramin, swainsonine. synthetic glycosaminoglycans, talisomycin, tallimustine, tamoxifen methiodide, tauromustine, tazarotene, tecogalan sodium, tegafur, tellurapyrylium, telomerase inhibitors, teloxantrone hydrochloride, temoporfin, temozolomide, teniposide, teroxirone, testolactone, tetrachlorodecaoxide, tetrazomine, thaliblastine, thalidomide, thiamiprine, thiocoraline. thioguanine, thiotepa, thrombopoietin, thrombopoietin mimetic, thymalfasin. thymopoietin receptor agonist, thymotrinan. thyroid stimulating hormone, tiazofurin, tin ethyl etiopurpurin, tirapazamine, titanocene dichloride, topotecan hydrochloride, topsentin, toremifene, toremifene citrate, totipotent stem cell factor, translation inhibitors, trestolone acetate, tretinoin, triacetyluridine, triciribine, triciribine phosphate, trimetrexate, trimetrexate glucuronate. triptorelin, tropisetron. tubulozole hydrochloride, turosteride, tyrosine kinase inhibitors, tyrphostins, UBC inhibitors, ubenimex, uracil mustard, uredepa, urogenital sinus-derived grow th inhibitory factor, urokinase receptor antagonists, vapreotide, variolin B, velaresol, veramine, verdins, verteporfin, vinblastine sulfate, vincristine sulfate, vindesine, vindesine sulfate, vinepidine sulfate, vinglycinate sulfate, vinleurosine sulfate, vinorelbine or vinorelbine tartrate, vinrosidine sulfate, vinxaltine, vinzolidine sulfate, vitaxin, vorozole, zanoterone, zeniplatin, zilascorb, zinostatin, zinostatin stimalamer, and zorubicin hydrochloride. Exemplary immunomodulatory agents include cytokines, xanthines, interleukins, interferons, oligodeoxynucleotides, glucans, growth factors (e.g., TNF, CSF, GM-CSF and G- CSF), hormones such as estrogens (diethylstilbestrol, estradiol), androgens (testosterone, HALOTESTIN® (fluoxymesterone)), progestins (MEGACE® (megestrol acetate), PROVERA® (medroxyprogesterone acetate)), and corticosteroids (prednisone, dexamethasone, hydrocortisone).

[0111] Examples of immunological adjuvants that can be associated with the particles include, but are not limited to, TLR ligands, C-Type Lectin Receptor ligands, NOD-Like Receptor ligands, RLR ligands, and RAGE ligands. TLR ligands can include lipopolysaccharide (LPS) and derivatives thereof, as well as hpid A and derivatives there of including, but not limited to, monophosphoryl hpid A (MPL), glycopyranosyl lipid A, PET-lipid A, and 3-O-desacyl-4'- monophosphoryl lipid A.

[0112] The active agent may also include antigens and / or adjuvants (i.e., molecules enhancing an immune response). Peptide, protein, and DNA based vaccines may be used to induce immunity to various diseases or conditions. Cell-mediated immunity is needed to detect and destroy virus -infected cells. Most traditional vaccines (e.g. protein-based vaccines) can only induce humoral immunity. DNA-based vaccine represents a unique means to vaccinate against a virus or parasite because a DNA based vaccine can induce both humoral and cell-mediated immunity. In addition, DNA based vaccines are potentially safer than traditional vaccines. DNA vaccines are relatively more stable and more cost-effective for manufacturing and storage. DNA vaccines consist of two major components — DNA carriers (or delivery vehicles) and DNAs encoding antigens. DNA carriers protect DNA from degradation, and can facilitate DNA entry to specific tissues or cells and expression at an efficient level.

[0113] Exemplary diagnostic agents include paramagnetic molecules, fluorescent compounds, magnetic molecules, and radionuclides, x-ray imaging agents, and contrast agents. Diagnostic agents include commercially available imaging agents used in positron emissions tomography (PET), computer assisted tomography (CAT), single photon emission computerized tomography, x-ray, fluoroscopy, and magnetic resonance imaging (MRI); anti-emetics; and contrast agents. Examples of suitable materials for use as contrast agents in MRI include gadolinium chelates, as well as iron, magnesium, manganese, copper, and chromium. Examples of materials useful for CAT and x-ray imaging include iodine-based materials.

[0114] In some embodiments, a diagnostic and / or therapeutic agent may be a radionuclide. Among the radionuclides used, gamma-emitters, positron-emitters, and X-ray emitters are suitable for diagnostic and / or therapeutic purposes, while beta emitters and alpha-emitters may also be used for therapy. Suitable radionuclides are known in the art.

[0115] In some embodiments, a diagnostic agent may be a fluorescent, luminescent, or magnetic moiety. Fluorescent and luminescent moieties include a variety of different organic or inorganic small molecules commonly referred to as "‘dyes,” “labels / ’ or “indicators.” Examples include fluorescein, rhodamine, acridine dyes, Alexa dyes, cyanine dyes, etc. Fluorescent and luminescent moieties may include a variety of naturally occurring proteins and derivatives thereof, e.g., genetically engineered variants. For example, fluorescent proteins include green fluorescent protein (GFP), enhanced GFP. red, blue, yellow, cyan, and sapphire fluorescent proteins, reef coral fluorescent protein, etc. Luminescent proteins include luciferase, aequorin and derivatives thereof. Numerous fluorescent and luminescent dyes and proteins are known in the art

[0116] In some embodiments, nanoparticles produced using the methods described herein contain less than 80%. less than 75%, less than 70%, less than 60%, less than 50% by weight, less than 40% by weight, less than 30% by weight, less than 20% by weight, less than 15% by weight, less than 10% by weight, less than 5% by weight, less than 1% by weight, less than 0.5% by weight, or less than 0.1% by weight of the active agent. In some embodiments, the active agent may be a mixture of pharmaceutically active agents.

[0117] Targeting Moieties

[0118] In some embodiments, the method further comprises covalently functionalizing the red blood cell lipid nanoparticles with a targeting moiety7.

[0119] In some embodiments, the particles can be functionalized with one or more targeting moieties. such as cell-type or cell-state specific targeting domain or targeting signal. Examples of moieties which may be linked or unlinked to the particles include, for example, targeting moieties which provide for the delivery of molecules to specific cells. The targeting signal or sequence can be specific for a host, tissue, organ, cell, organelle, non-nuclear organelle, or cellular compartment. For example, the nanoparticles disclosed herein can be modified with galactosyl-terminating macromolecules to target the compositions to the liver or to liver cells. The modified compositions selectively enter hepatocytes after interaction of the carrier galactose residues with the asialoglycoprotein receptor present in large amounts and high affinity7only on these cells. Moreover, the compositions disclosed here can be targeted to other specific intercellular regions, compartments, or cell types.

[0120] In one embodiment, the targeting moiety7binds to its ligand or receptor which is located on the surface of a target cell such as to bring the vector and cell membranes sufficiently close to each other to allow penetration of the vector into the cell. Additional embodiments of the present disclosure are directed to specifically delivering polynucleotides to specific tissue or cell types, wherein the polynucleotides can encode a polypeptide or interfere with the expression of a different polynucleotide. The polynucleotides delivered to the cell can encode polypeptides that can enhance or contribute to the functioning of the cell.

[0121] The targeting moiety can be an antibody or antigen binding fragment thereof, an antibody domain, an antigen, a T-cell receptor, a cell surface receptor, a cell surface adhesion molecule, a major histocompatibility locus protein, a viral envelope protein and a peptide selected by phage display that binds specifically to a defined cell.

[0122] One skilled in the art will appreciate that the tropism of the particles described can be altered by merely changing the targeting moiety7. It is know n in the art that nearly every cell type in a tissue in a mammalian organism possesses some unique cell surface receptor or antigen. Thus, it is possible to incorporate nearly any ligand for the cell surface receptor or antigen as a targeting moiety. For example, peptidyl hormones can be used a targeting moieties to target delivery to those cells which possess receptors for such hormones. Chemokines and cytokines can similarly be employed as targeting moieties to target delivery of the complex to their target cells. A variety of technologies have been developed to identify genes that are preferentially expressed in certain cells or cell states and one of skill in the art can employ such technology7to identity7targeting moieties which are preferentially or uniquely expressed on the target tissue of interest.

[0123] Nucleic Acid Targeting Moieties

[0124] As used herein, a “nucleic acid targeting moiety” is a nucleic acid that binds selectively to a target. In some embodiments, a nucleic acid targeting moiety is a nucleic acid aptamer. An aptamer is usually a polynucleotide that binds to a specific target structure that is associated with a particular organ, tissue, cell, extracellular matrix component, and / or intracellular compartment. In general, the targeting function of the aptamer is based on the three-dimensional structure of the aptamer. In some embodiments, binding of an aptamer to a target is typically mediated by the interaction between the two- and / or three-dimensional structures of both the aptamer and the target. In some embodiments, binding of an aptamer to a target is not solely based on the primary sequence of the aptamer, but depends on the three-dimensional structure(s) of the aptamer and / or target. In some embodiments, aptamers bind to their targets via complementary^ Watson-Crick base pairing which is interrupted by structures (e.g. hairpin loops) that disrupt base pairing. In some embodiments, a nucleotide sequence that is homologous to a nucleic acid targeting moiety may be used. In some embodiments, a nucleotide sequence is considered to be “homologous” to a nucleic acid targeting moiety if it contains fewer than 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 nucleic acid substitutions relative to the aptamer. In some embodiments, a nucleotide sequence is considered to be “homologous” to a nucleic acid targeting moiety if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. In some embodiments, a nucleic acid sequence is considered to be “homologous” to a nucleic acid targeting moiety if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%. 65%. 70%. 75%. 80%. 85%. 90%. 95%. or 99% similar.

[0125] Nucleic acids may be prepared according to any available technique including, but not limited to chemical synthesis, enzymatic synthesis, enzymatic or chemical cleavage of a longer precursor, etc. Methods of synthesizing RNAs are known in the art (see, e.g. Gait, M. J. (ed.) Oligonucleotide synthesis: a practical approach. Oxford [Oxfordshire], Washington, D C.: IRL Press, 1984; and Herdewijn. P. (ed.) Oligonucleotide synthesis: methods and applications. Methods in molecular biology, v. 288 (Clifton, N.J.) Totowa, N.J.: Humana Press, 2005).

[0126] A nucleic acid that forms the nucleic acid targeting moiety may contain naturally occurring nucleosides, modified nucleosides, naturally occurring nucleosides with hydrocarbon linkers (e.g. an alkylene) or a polyether linker (e.g., a PEG linker) inserted between one or more nucleosides, modified nucleosides with hydrocarbon or PEG linkers inserted between one or more nucleosides, or a combination of thereof. In some embodiments, nucleotides or modified nucleotides of the nucleic acid targeting moiety can be replaced with a hydrocarbon linker or a polyether linker provided that the binding affinity and selectivity of the nucleic acid targeting moiety is not substantially reduced by the substitution (e.g. the dissociation constant of the nucleic acid targeting moiety for the target should not be greater than about 1. times. 10. sup.-3 M).

[0127] Nucleic acids containing a variety of different nucleotide analogs, modified backbones, or non-naturally occurring intemucleoside linkages can be utilized in accordance with the present invention. Nucleic acids of the present invention may include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxy cytidine) or modified nucleosides. Examples of modified nucleotides include base modified nucleoside (e.g. aracytidine, inosine, isoguanosine, nebularine, pseudouridine, 2,6-diaminopurine, 2-aminopurine, 2-thiothyrmdine, 3-deaza-5-azacytidine, 2'- deoxyuridine, 3-nitropyrrole, 4-methylindole, 4-thiouridine, 4-thiothymidine, 2-aminoadenosine, 2 -thiothymidine, 2-thiouridine, 5 -bromocytidine, 5 -iodouridine, inosine, 6-azauridine, 6- chloropurine, 7-deazaadenosine. 7-deazaguanosine. 8 -azaadenosine, 8-azidoadenosine, benzimidazole, Ml -methyladenosine, pyrrolo-pyrimidine, 2-amino-6-chloropurine, 3-methyl adenosine, 5-propynylcytidine, 5-propynyluridine, 5-bromouridine, 5-fluorouridine, 5- methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)- methylguanine, and 2-thiocytidine), chemically or biologically modified bases (e.g., methylated bases), modified sugars (e.g., 2'-fluororibose, 2'-aminoribose, 2'-azidoribose, 2'-O-methylribose, L-enantiomeric nucleosides arabinose, and hexose), modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages), and combinations thereof. Natural and modified nucleotide monomers for the chemical synthesis of nucleic acids are readily available. In some cases, nucleic acids containing such modifications display improved properties relative to nucleic acids consisting only of naturally occurring nucleotides. In some embodiments, nucleic acid modifications described herein are utilized to reduce and / or prevent digestion by nucleases (e.g. exonucleases, endonucleases, etc.). For example, the structure of a nucleic acid may be stabilized by including nucleotide analogs at the 3' end of one or both strands order to reduce digestion.

[0128] Modified nucleic acids need not be uniformly modified along the entire length of the molecule. Different nucleotide modifications and / or backbone structures may exist at various positions in the nucleic acid. Nucleic acids may, for example, contain a modification to a sugar, nucleoside, or intemucleoside linkage.

[0129] Small Molecule Targeting Moieties

[0130] In some embodiments, a targeting moiety7may be a small molecule. In certain embodiments, small molecules are less than about 2000 g / mol in size. In some embodiments, small molecules are less than about 1500 g / mol or less than about 1000 g / mol. In some embodiments, small molecules are less than about 800 g / mol or less than about 500 g / mol. Any small molecule that specifically binds to a desired target can be used. One exemplary small molecule targeting moiety is folic acid. Folic acid (i.e., pteroylglutamic acid, Vitamin B9) specifically binds to the folate receptor (FR), which is preferentially expressed in tumor tissues relative to healthy tissues (Low et al., 2004, Adv. Drug Deliv. Rev., 56: 1055).

[0131] In some embodiments, small molecule targeting moieties that may be used to target cells associated with prostate cancer tumors include PSMA peptidase inhibitors, such as 2-PMPA, GPI5232, VA-033. phenylalkylphosphonamidates, and / or analogs and derivatives thereof. In some embodiments, small molecule targeting moieties that may be used to target cells associated with prostate cancer tumors include thiol and indole thiol derivatives, such as 2-MPPA and 3-(2- mercaptoethyl)-lH-indole-2-carboxylic acid derivatives. In some embodiments, small molecule targeting moieties that may be used to target cells associated with prostate cancer tumors include hydroxamate derivatives. In some embodiments, small molecule targeting moieties that may be used to target cells associated with prostate cancer tumors include PBDA- and urea-based inhibitors, such as ZJ 43. ZJ 11, ZJ 17, ZJ 38 (Nan et al., 2000, J. Med. Chem., 43:772; and Kozikowski et al., 2004. J. Med. Chem.. 47: 1729), and / or and analogs and derivatives thereof In some embodiments, small molecule targeting moieties that may be used to target cells associated with prostate cancer tumors include androgen receptor targeting agents (ARTAs). In some embodiments, small molecule targeting moieties that may be used to target cells associated with prostate cancer tumors include polyamines, such as putrescine, spermine, and spermidine.

[0132] Tumor Targeting

[0133] In one embodiment, the targeting signal is used to selectively target tumor cells. Tumor cells express cell surface markers which may only be expressed in the tumor or present in nontumor cells but preferentially presented in tumor cells. Such markers can be targeted to increase delivery of the particles to cancer cells.

[0134] For example, in some embodiments, the targeting moiety is a polypeptide including an arginine-glycine-aspartic acid sequence. For example, the targeting moiety can be an arginineglycine-aspartic acid-lysine (RGDK, mRGD) other polypeptide that includes the RGD sequence and is capable of binding to tumor endothelium through the interaction of RGD with a. [T and a fT. In some embodiments, a targeting moiety includes the polypeptide sequence R / KxxR / K, where “x” is any amino acid, and which allow-s binding to neuropilin- 1. Binding with integrins or neuropilin- 1 are two approaches for improving tumor-targeted and tissue-penetrating delivery to tumors in vivo. Similar approaches have been reported to facilitate ligand-specific gene delivery in vitro and targeted gene delivery to liver, spleen, and bone marrow in vivo.

[0135] Other, exemplary7tumor specific cell surface markers include, but are not limited to, alfa- fetoprotein (AFP), C-reactive protein (CRP), cancer antigen-50 (CA-50), cancer antigen-125 (CA-125) associated with ovarian cancer, cancer antigen 15-3 (CA15-3) associated with breast cancer, cancer antigen- 19 (CA-19) and cancer antigen-242 associated with gastrointestinal cancers, carcinoembryonic antigen (CEA), carcinoma associated antigen (CAA), chromogranin A, epithelial mucin antigen (MC5), human epithelium specific antigen (HEA), Lewds(a)antigen, melanoma antigen, melanoma associated antigens 100, 25, and 150, mucin-like carcinoma- associated antigen, multidrug resistance related protein (MRPm6), multi drug resistance related protein (MRP41), Neu oncogene protein (C-erbB-2), neuron specific enolase (NSE), P- gly coprotein (mdrl gene product), multi drug-resistance-related antigen, p!70, multidrugresistance-related antigen, prostate specific antigen (PSA), CD56, NCAM, EGFR, CD44, and folate receptor. In one embodiment, the targeting signal consists of antibodies which are specific to the tumor cell surface markers.

[0136] Antibodies

[0137] Another embodiment provides an antibody or antigen binding fragment thereof bound to the disclosed particles acts as the targeting signal.

[0138] The antibodies or antigen binding fragment thereof are useful for directing the particle to a cell type or cell state. In one embodiment, the particles are coated with a polypeptide that is an antibody binding domain, for example from a protein known to bind antibodies such as Protein A and Protein G from Staphylococcus aureus. Other domains known to bind antibodies are known in the art and can be substituted. The antibody binding domain links the antibody, or antigen binding fragment thereof, to the particle.

[0139] In certain embodiments, the antibody that serves as the targeting signal is polyclonal, monoclonal, linear, humanized, chimeric or a fragment thereof. Representative antibody fragments are those fragments that bind the antibody binding portion of the non- viral vector and include Fab, Fab', F(ab'), Fv diabodies, linear antibodies, single chain antibodies and bispecific antibodies known in the art.

[0140] In some embodiments, the targeting signal includes all or part of an antibody that directs the particle to the desired target cell type or cell state. Antibodies can be monoclonal or polyclonal, but are preferably monoclonal. For human gene therapy purposes, antibodies can be derived from human genes and are specific for cell surface markers, and are produced to reduce potential immunogenicity to a human host as is known in the art. For example, transgenic mice which contain the entire human immunoglobulin gene cluster are capable of producing "human" antibodies can be utilized. In one embodiment, fragments of such human antibodies are employed as targeting signals. In a preferred embodiment, single chain antibodies modeled on human antibodies are prepared in prokaryotic culture.

[0141] Methods of Attaching Targeting Moieties

[0142] In certain embodiments, the red blood cell lipid nanoparticles are covalently functionalized with the targeting moiety using a click chemi s tty reaction. Click chemistry refers to a class chemical reaction (referred to as a “click reaction”) between two click groups that exhibit good yields, wide functional group tolerance, and are highly selective even in the presence of a complex mixture of biological molecules. These characteristics allow the click reactions to proceed even in vivo. Thus, a nanoparticle functionalized with a first click motif (e.g., an azide) can be contacted with targeting moieties bearing a complementary second click motif (e.g., an alkyne) under conditions that allow for the efficient formation of a covalent bond between the nanoparticle and the targeting moiety, thus covalently attaching the targeting moiety to the nanoparticles. Example click motif pairs used as the first click motif and the second click motif include, but not limited to, azide with phosphine; azide with cyclooctyne; nitrone with cyclooctyne; nitrile oxide with norbomene; oxanorbomadiene with azide; trans-cyclooctene with s-tetrazine; quadricyclane with bis(dithiobenzil)nickel(II).

[0143] In some embodiments, the second click motif comprises an alkene, e.g., a cyclooctene, e.g., a transcyclooctene (TCO) or norbomene (NOR), and the first click motif comprises a tetrazine (Tz). In other embodiments, the second click motif comprises an alkyne, e.g., a cyclooclyne such as dibenzocyclooctyne (DBCO), and the first click motif comprises an azide (Az). In some embodiments, the second click motif comprises a Tz, and the first click motif comprises an alkene such as transcyclooctene (TCO) or norbomene (NOR). Alternatively or in addition, the first click motif comprises an Az, and the second click motif comprises a cyclooctyne such as dibenzocyclooctyne (DBCO). TCO reacts specifically in a click chemistry reaction with a tetrazine (Tz) moiety. DBCO reacts specifically in a click chemistry reaction with an azide (Az) moiety. Norbomene reacts specifically in a click chemistry reaction with a tetrazine (Tz) moiety.

[0144] Exemplary click chemistry reactions (and by extension click motifs) are shown below. For example. copper(I)-catalyzed Azide-Alkyne Cycloaddition (CuAAC) comprises using a Copper (Cu) catalyst at room temperature. The Azide-Alkyne Cycloaddition is a 1,3-dipolar cycloaddition between an azide and a terminal or internal alkyne to give a 1,2,3-triazole.

[0145] Another example of click chemistry includes Staudinger ligation, which is a reaction that is based on the classic Staudinger reaction of azides with triarylphosphines. It launched the field of bioorthogonal chemistry as the first reaction with completely abiotic functional. The azide acts as a soft electrophile that prefers soft nucleophiles such as phosphines. This is in contrast to most biological nucleophiles which are typically hard nucleophiles. The reaction proceeds selectively under water-tolerant conditions to produce a stable product. Phosphines are completely absent from living systems and do not reduce disulfide bonds despite mild reduction potential. Azides had been shown to be biocompatible in FDA-approved drugs such as azidothymidine and through other uses as cross linkers. Additionally, their small size allows them to be easily incorporated into biomolecules through cellular metabolic pathways.

[0146] Copper-free click chemistry is a bioorthogonal reaction first developed by Carolyn Bertozzi as an activated variant of an azide alkyne cycloaddition. Unlike CuAAC, Cu-free click chemistry has been modified to be bioorthogonal by eliminating a cytotoxic copper catalyst, allowing reaction to proceed quickly and without live cell toxicity7. Instead of copper, the reaction is a strain- promoted alkyne-azide cycloaddition (SPAAC). It was developed as a faster alternative to the Staudinger ligation, with the first generations reacting over sixty times faster. The incredible bioorthogonality of the reaction has allowed the Cu-free click reaction to be applied within cultured cells, live zebrafish, and mice. Cyclooctynes were selected as the smallest stable alkyne ring which increases reactivity through ring strain which has calculated to be 19.9 kcal / mol.

[0147] Copper-free click chemistry also includes nitrone dipole cycloaddition. Copper-free click chemistry has been adapted to use nitrones as the 1,3-dipole rather than azides and has been used in the modification of peptides.

[0148] This cycloaddition between a nitrone and a cyclooctyne forms N-alkylated isoxazolines. The reaction rate is enhanced by water and is extremely fast with second order rate constants ranging from 12 to 32 M-1-s-1, depending on the substitution of the nitrone. Although the reaction is extremely fast, incorporating the nitrone into biomolecules through metabolic labeling has only been achieved through post-translational peptide modification.

[0149] Another example of click chemistry includes norbomene cycloaddition. 1,3 dipolar cycloadditions have been developed as a bioorthogonal reaction using a nitrile oxide as a 1,3- dipole and a norbomene as a dipolarophile. Its primary use has been in labeling DNA and RNA in automated oligonucleotide synthesizers.

[0150] Norbomenes were selected as dipolarophiles due to their balance between strain- promoted reactivity and stability. The drawbacks of this reaction include the cross-reactivity of the nitrile oxide due to strong electrophilicity and slow reaction kinetics.

[0151] Another example of click chemistry includes oxanorbomadiene cycloaddition. The oxanorbomadiene cycloaddition is a 1,3-dipolar cycloaddition followed by a retro-Diels Alder reaction to generate a triazole-linked conjugate with the elimination of a furan molecule. This reaction is useful in peptide labeling experiments, and it has also been used in the generation of SPECT imaging compounds.

[0152] Ring strain and electron deficiency in the oxanorbomadiene increase reactivity towards the cycloaddition rate-limiting step. The retro-Diels Alder reaction occurs quickly afterwards to form the stable 1,2,3 triazole. Limitations of this reaction include poor tolerance for substituents which may change electronics of the oxanorbomadiene and low rates (second order rate constants on the order of 10 ').

[0153] Another example of click chemistry includes tetrazine ligation. The tetrazine ligation is the reaction of a trans-cyclooctene and an s-tetrazine in an inverse-demand Diels Alder reaction followed by a retro-Diels Alder reaction to eliminate nitrogen gas. The reaction is extremely rapid with a second order rate constant of 2000 M '-S1(in 9: 1 methanol / water) allowing modifications of biomolecules at extremely low concentrations.

[0154] The highly strained trans-cyclooctene is used as a reactive dienophile. The diene is a 3,6- diaryl-s-tetrazine which has been substituted in order to resist immediate reaction with water. The reaction proceeds through an initial cycloaddition followed by a reverse Diels Alder to eliminate N2 and prevent reversibility of the reaction.

[0155] Not only is the reaction tolerant of water, but it has been found that the rate increases in aqueous media. Reactions have also been performed using norbomenes as dienophiles at second order rates on the order of 1 M1-s1in aqueous media. The reaction has been applied in labeling live cells and polymer coupling.

[0156] Another example of click chemistry includes is [4+1] cycloaddition. This isocyanide click reaction is a [4+1] cycloaddition followed by a retro-Diels Alder elimination of N2.

[0157] The reaction proceeds with an initial [4+1] cycloaddition follow ed by a reversion to eliminate a thermodynamic sink and prevent reversibility. This product is stable if a tertiary amine or isocyanopropanoate is used. If a secondary or primary isocyanide is used, the produce will form an imine which is quickly hydrolyzed.

[0158] Isocyanide is a favored chemical reporter due to its small size, stability, non-toxicity, and absence in mammalian systems. However, the reaction is slow, with second order rate constants on the order of lO ^ M ^s1.

[0159] Another example of click chemistry includes quadricyclane ligation. The quadricyclane ligation utilizes a highly strained quadricyclane to undergo [2+2+2] cycloaddition with n systems.

[0160] Quadncyclane is abiotic, unreactive with biomolecules (due to complete saturation), relatively small, and highly strained (~80 kcal / mol). However, it is highly stable at room temperature and in aqueous conditions at physiological pH. It is selectively able to react with electron-poor 71 systems but not simple alkenes, alkynes, or cyclooctynes.

[0161] Bis(dithiobenzil)nickel(II) was chosen as a reaction partner out of a candidate screen based on reactivity. To prevent light-induced reversion to norbomadiene, diethyldithiocarbamate is added to chelate the nickel in the product.

[0162] These reactions are enhanced by aqueous conditions with a second order rate constant of 0.25 M-1-s-1. Of particular interest is that it has been proven to be bioorthogonal to both oxime formation and copper-free click chemistry.

[0163] The exemplary click chemistry' reactions have high specificity, efficient kinetics, and occur in vivo under physiological conditions. See, e.g., Baskin et al. Proc. Natl. Acad. Sci. USA 104(2007): 16793; Oneto et al. Acta biomaterilia (2014); Neves el al. Bioconjugate chemistry 24(2013):934; Koo et al. Angewandte Chemie 51(2012): 11836; and Rossin et al. Angewandte Chemie 49(2010):3375. For a review of a wide variety of click chemistry' reactions and their methodologies, see e g., Nwe K and Brechbiel M W, 2009 Cancer Biotherapy and Radiopharmaceuticals, 24(3): 289-302; Kolb H C et al., 2001 Angew. Chem. Int. Ed. 40: 2004-2021. The entire contents of each of the foregoing references are incorporated herein by reference.

[0164] Exemplary click motif pairs are shown in the table below. Functional groups fonned by reaction of click motif pairs are well known in the art.

[0165] Other suitable include the motifs can be found, for example, in Patterson, D.M., et al. "‘Finding the Right (Bioorthogonal) Chemistry.” ACS Chem. Biol., 2014, 9(3): 592-605; Akgun. B., et al. “Synergic "Click" Boronate / Thiosemicarbazone System for Fast and Irreversible Bioorthogonal Conjugation in Live Cells,” . Am. Chem. Soc., 2017, 139(40): 14285-14291; and Akgun. B. and Hall, D.G. “Fast and Tight Boronate Formation for Click Bioorthogonal Conjugation.” Angew. Chem., Int. Ed. 2016. 55(12): 3909-3913, each of which is hereby incorporated by reference in its entirety.

[0166] In some embodiments the nanoparticles can contain a linker which binds the targeting moiety to the particle. In some embodiments, the linker can be a polypeptide, or any other suitable linker that is known in the art. for example, polyethylene glycol (PEG).

[0167] Polymeric Conjugation

[0168] In certain embodiments, the method can further comprise conjugating one or more polymers to the red blood cell lipid nanoparticles. The polymers can comprise any suitable polymers. In some examples, the polymer can comprise a hydrophilic polymer or a zw itterionic polymer.

[0169] Examples of suitable polymers can include poly(alkylene oxides), such as polyethylene glycol (PEG), polyesters, including polymers and copolymers comprising lactic acid and / or glycolic acid units, such as polylactic acid, polygly colic acid, poly(lactic)-co-poly(gly colic) acid, poly(lactic acid-co-glycolic acid), and poly(lactide-co-glycolide). polyhydroxyacids, and / or polyanhydrides, poly(ortho ester), PEGylated poly(ortho ester), polylysine, PEGylated polylysine, polyethylene imine), PEGylated poly(ethylene imine), poly(L-lactide-co-L-lysine), poly(serine ester), poly(4-hydroxy-L-proline ester), poly [a-(4-aminobut l)-L-gly colic acid], polyesters bearing cationic side chains, such as poly(L-lactide-co-L-lysine), poly(serine ester), poly(4-hydroxy-L-proline ester), acrylic polymers, such as acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacry late, amino alkyd methacrylate copolymer, poly(acry lie acid), poly(methacrylic acid), methacrylic acid alky lamide copolymer, poly(methyl methacrylate), poly(methacry lie acid polyacrylamide, amino alkyl methacrylate copolymer, glycidyl methacrylate copolymers, poly cyanoacrylates, copolymers thereof, and blends thereof.

[0170] Pharmaceutical Compositions

[0171] Also provided are compositions comprising red blood cell lipid nanoparticles prepared by the methods described herein. These compositions can be used, for example, for drug delivery or diagnostics or combination of the two (e.g., depending on the active agent(s) present in the red blood cell lipid nanoparticles).

[0172] The compositions described herein can include red blood cell lipid nanoparticles in combination with pharmaceutically acceptable carriers. As would be appreciated by one of skill in this art, the carriers may be chosen based on the route of administration as described below, the location of the target issue, the drug being delivered, the time course of delivery of the drug, etc.

[0173] The pharmaceutical compositions of this disclosure can be administered to a patient by any means known in the art including oral and parenteral routes. The term “patient,” as used herein, refers to humans as well as non-humans, including, for example, mammals, birds, reptiles, amphibians, and fish. For instance, the non-humans may be mammals (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a primate, or a pig). In certain embodiments parenteral routes are desirable since they avoid contact with the digestive enzymes that are found in the alimentary canal. According to such embodiments, inventive compositions may be administered by injection (e.g., intravenous, subcutaneous or intramuscular, intraperitoneal injection), rectally, vaginally, topically (as by powders, creams, ointments, or drops), or byinhalation (as by sprays).

[0174] In a particular embodiment, the red blood cell lipid nanoparticles of the present disclosure are administered to a subject in need thereof systemically, e.g., parenterally, or by IV infusion or injection.

[0175] The present invention may be understood more readily by reference to the following detailed description of various aspects of the invention and the examples included therein and to the Figures and their previous and following description.

[0176] EXAMPLES

[0177] Example 1: Microfluidic Nano-Assembly of Red-Blood-Cell (RBC) Lipids and Components for Engineering Therapeutic Extracellular Vesicles and Nanoparticles

[0178] Summary Lipid nanoparticles and engineered vesicles for advanced therapeutic applications have yielded significant benefits recently. Red blood cells (RBCs) are a unique type of oxygencarrying cell, with lipids — specifically phospholipids — on their cell membrane. Traditionally, RBC nanoparticles are synthesized using a top-down approach through hemolysis to create erythrocyte ghosts, which undergo several physical processes to form nanoparticles. However, this approach hinders reproducibility, batch-to-batch consistency, and scaling up. Microfluidics allows scalable, high-throughput, and controlled synthesis. Our calibrated bottom-up approach produces highly homogeneous and tunable RBC lipid-based engineered red blood cell extracellular vesicles (eRBCEVs). New generation multi-physics simulation was incorporated to optimize flow dynamics in real-time by considenng, channel geometry, phase concentrations, and flow rate ratios to reinforce homogeneity. The versatility of loading components into eRBCEVs was explored by nano-encapsulating small-to-large protein complexes, metal nanoparticles, and nucleic acid. Furthermore, eRBCEVs stand out as distinctive drug-carrying shuttles with the potential to circumvent immune responses, marking them as promising candidates for personalized nanomedicine.

[0179] Introduction

[0180] Lipid nanoparticles (LNPs) prevent the degradation of mRNA in vivo and thus were utilized in several COVID-19 vaccines, significantly contributing to our ability to combat the pandemic, and effectively restore normalcy to our lives. Furthermore, LNPs excel in therapeutic formulations by ensuring improved bi oavai 1 abi 1 i ty . efficient cellular uptake, targeted delivery, and versatile formulations. Achieving homogeneity, actuation of size, consistent loading, and large-scale purification are significant challenges. LNP size influences pharmacokinetics, whereby heterogeneous populations poorly distribute throughout the body, impacting delivery efficiencies of therapies. Traditional LNP synthesis techniques like sonication carry the risk of particle aggregation and contamination, the ethanol-inj ection method faces challenges in precise solvent controllability and removal, affecting the stability of particles, bulk mixing lacks scalability and consistency, and non-solvent emulsion techniques are complex labor intensive, and have poor batch-to-batch consistency. Microfluidics have evolved into a cutting-edge platform for manipulating milliliters, nanoliters, and pico-liters of fluids to enhance mixing and particle synthesis with highly homogeneous sizes ranging from several microns to nanometers. Additionally, automated pressure-based microfluidics have demonstrated highly controlled mixing, scalability, enhanced encapsulation efficiency, real-time monitoring, complex particle design capabilities, and size and composition control. Therefore, microfluidics is the appropriate technology for complex therapeutic design and it allows precise control of synthesis and reproducible delivery of therapeutics.

[0181] The composition of lipids significantly impacts various aspects of LNP, including their size, stability, encapsulation capacity, and release kinetics, affecting the potential for targeted delivery. The surface modifications of LNPs including their charge, surface proteins, and composition, influence their interaction with immune cells. For example, positively charged nanoparticles interact strongly with negatively charged cell membranes due to electrostatic forces. Smaller particles exhibit extended circulation half-lives by evading mononuclear phagocyte system clearance and can readily traverse endothelium, facilitating entry’ into target tissues. Furthermore, in many LNP formulations, the use of poly -(ethylene glycol) (PEG) and ionizable lipids are required, since PEGylated lipids enhance circulation and, ionizable lipids reduce cytotoxic effects. However, large quantities of PEGylated lipids and cationic lipids incorporated in LNPs induce accelerated blood clearance, which in turn leads to PEG-induced opsonization. On the other hand, high-purity ionizable lipids like popular ALC-0315 are expensive, and continuous flow large-scale production is process-intensive. Therefore, there is an unmet need to consider natural lipid membranous structures that are capable of transferring cargo, such as extracellular vesicles (EVs).

[0182] Recently. EVs have emerged as promising candidates for therapeutic applications due to their unique surface properties, and potential advantages over LNP and cell-based therapies. EVs are cell-derived, membrane-bound structures that are secreted by cells under various phy siological and pathological conditions with native cargo, including proteins, nucleic acids, and lipids. EVs naturally traffic cargo between cells, making them a versatile delivery system for therapeutic agents, targeted drug delivery, and regenerative medicine. EVs are cell-derived, EV- based therapeutics are less immunogenic and tumorigenic, and reduce the risk of embolisms compared to cell-based therapies. Among the EVs generated from all ty pes of cells, red blood cell-derived extracellular vesicles (RBCEVs) have gained significant attention as potential therapeutic agents due to their abundance in the bloodstream, facile isolation, and ability to cross biological barriers. RBCEV-based therapeutics are limited to translational clinical settings, due to their immense heterogeneity' and an inability to control the components of molecular cargo. Furthermore, RBCs undergo oxidation and, eventually degrade after 6 weeks of storage, whereby EVs from such stock contain large amounts of proteins making it complex to purify. On the other hand, stored RBCEVs upregulate reactive oxygen species (ROS), leading to endothelial dysfunction limiting the potential for translation and scale-up. Therefore, it is necessary to develop engineered EVs through non-biosynthetic methods to control particle size and cargo flexibility.

[0183] In this Example, a tunable bottom-up approach to synthesize engineered RBCEVs (eRBCEVs) by assembling RBC lipid components through microfluidic approaches is presented. Microfluidic multi-physics simulations were performed to model flow dynamics and estimate mixing tendencies to optimize the influence of channel design, flow rate, and diffusion parameters. Highly homogeneous eRBCEVs of size ranging from 80 to 200 nm were obtained by tuning the flow rate linearly and optimizing the concentration of starting materials. Encapsulation flexibility was explored with different types of molecular cargos, including hemoglobin (Hb), ultra-high-molecular-weight Hb, nanoparticles, and oligonucleotides. Encapsulated molecular cargos were characterized with elemental mapping and high-resolution optical techniques, demonstrating the breadth of characterization techniques across encapsulates. High-purity eRBCEVs were obtained by incorporating process-intensified soft buffer exchange, concentrating, and purifying using tangential flow filtration (TFF). Furthermore, the purified eRBCEVs possess minimal activation with human peripheral blood neutrophils. Altogether, the tunable parametric configurations, scalable optimized technology, and flexibility of encapsulation coupled with RBC lipids realize the development of a variety of personalized nano-medicine designs.

[0184] Materials and Methods

[0185] RBC lipid extraction. Total lipid extraction from red blood cells (RBCs) was performed using a procedure schematically illustrated in Figure 9A. Briefly, 800 pL of washed-packed RBCs were lysed using 800 pL of deionized (DI) water in glass centrifuge tubes (Sigma- Aldrich, MO, USA). 6 mL of methanol and chloroform mixture in 2: 1 volume ratio, 2 mL of chloroform, and 2 mL of deionized (DI) w ater were added, with vigorous vortex mixing in between each step. After 15 minutes of centrifugation at 1750 x g at 4 °C, the organic phase was isolated and dried under a nitrogen blanket. The resulting lipid films were then reconstituted in a small volume of chloroform, and pooled, and the final solution was filtered through a 0.45 pm polytetrafluoroethylene (PTFE) filter to ensure the retention of any suspended proteins. The final filtrate w as dried under a nitrogen blanket once more, and the resulting dried lipids were weighed and stored at -20 °C.

[0186] RBC lipid formulation. A batch of extracted RBC lipids, weighing precisely 24 mg, was dissolved in a 200-proof ethanol solution (Decon Laboratories Inc., PA, USA) within a 5 mL low-binding Eppendorf tube to achieve final concentrations of 2, 4, 6, and 12 mg / mL. For instance, to attain a final concentration of 6 mg / mL, the lipid was dissolved in 4 mL of the ethanol solution. The lipid solution then underwent sonication for 10 minutes to ensure no lipid aggregation during dissolution. Subsequently, the obtained lipid solution was filtered using a 0.22 pm syringe filter (Minisart® NML, GmbH, Germany) pre-wetted with ethanol to eliminate any potential remaining aggregation, even after sonication. The filtered lipid solution was stored at 4 °C.

[0187] Buffer preparation. A 100 mL CB with a final concentration of 100 mM was prepared by dissolving 2.426 grams of sodium citrate dihydrate (EMD Millipore Corp., MA, USA) and 336 mg of citric acid (Sigma- Aldrich, MO, USA) in 80 mL of water for injection (WFI) (Life Technologies Corp., NY, USA) in a 200 mL borosilicate conical flask, vortexed (VWR™, GA, USA) for better mixing. The solution's pH was measured using the digital pH meter (Oakton® Instruments, Cole-Parmer, IL, USA) and accurately adjusted to 6.0 by carefully adding either 0.1N HC1 or 0.1N NaOH (Thermo-Fisher Scientific, PA, USA) solutions. Subsequently, DI water was added to bring the volume to 100 mL. Furthermore, a 500 mL vacuum filter system equipped with a 0.22 pm pore filter (Coming Inc. NY, USA), was utilized, boasting a 33.2 cm2filter area and a polyether sulfone membrane to filter the CB.

[0188] Microfluidic device. A flow-focusing microfluidic device (SKU# 10000158, LabSmith, CA, USA) was used. In this configuration, the RBC lipids flow in the primary' microchannel, with dimensions of 400 pm, and two secondary channels, each measuring 100 pm deliver CB. A herringbone design microfluidic device (SKU# 10000076, LabSmith, CA, USA) made of durable Zenor material was adopted. These microchannels within the chip possess channel dimensions of 400 pm in width and 200 pm in height and w ere characterized by a series of precisely engineered grooves and ridges measuring 50 pm. The CB and lipid phases were fed in 50 pm separately.

[0189] Multiphysics simulation. All computational calculations were performed on COMSOL Multiphysics version 6.0.0.405 (Stockholm, Sweden) licensed through Ohio Supercomputer Center (OH, USA). The geometric design was developed and imported from Solid-Works CAD version 5.0 licensed through The Ohio State University. Columbus. USA. The study was focused on understanding the dynamics of mixing using volume of fluid (VOF) and level set configuration integrated w ith the transport of dilute species (TDS) module. The time-dependent Lagrangian study was chosen along with the TDS and VOF equations module. The user-defined 1 pm element size at a growth rate of 1 and a curvature factor of 0.5 was utilized for meshing. Boundary layer refinement was incorporated with 8 iterations and 16 element depth for developing structured mesh (Figure 9B). The diffusion across the length of the channel was computed for a total time of 1 minute with 0.001s as the step size. The computation w as across 112 nodes boosting from 6 cores of the graphics processing unit. It took about 6 hours to compute each flowrate ratio (FRR) on each microfluidic design.

[0190] Nanoparticle synthesis setup. A pressure-based microfluidic control system (Elveflow Microfluidics, Paris, France) was utilized to synthesize eRBCEVs. The system creates a pressure differential along a fluidic conduit that extends from the pressure controller outlet to the inlet of the microfluidic channel (expressed as AP = Pout - Pin). Typically, the outlet pressure (Pout) was maintained at atmospheric pressure, thereby making the pressure controller responsible for regulating the pressure difference (AP). This pressure variance propels the movement of liquid within the system, and the flow rate was quantified using a digital flow sensor with a capacity of 200 pL / min - 5000 pL / min. The polyether ether ketone capillary resistor with small internal diameters was employed to gain precise control of flow in the system. To this end, the polytetrafluoroethylene (PTFE) tube with an inner diameter of 0.3 mm, with a length of 12 inches connecting to the microfluidic channel from each of the flow resistors was utilized. Two inlets were connected to deliver distinct components of the reaction mixture, such as RBC lipids dissolved in ethanol and CB of pH 6.0 solutions. The build-up of pressure was facilitated through nitrogen pressure lines with an air filter, and variable operating pressure was utilized, maxing out at 2000 mbar, to each inlet. An integrated Elveflow smart interface (ESI) software was used to command pressure values and flow rates to the fluidic configuration.

[0191] Pressure pumping system calibration. A digital universal serial bus cytoSMART™ lux2 (Don Whitley Scientific Limited, England, UK) camera was employed to visualize the mixing interface and captured an image every 10 seconds. The flow sensors were linked to the pressure controller, forming a feedback loop that auto-adjusts flow rates to maintain the setpoint values. The tubes were primed with WFI at 500 mbar pressure for 5 minutes. CB and lipid solutions were then pumped at 300 mbar for 15 seconds to ensure a smooth transition of fluids within the channels. Furthermore, activated the feedback proportional integral derivative (PDI) control loop by switching to flow rate mode on the software. The P calibration factor was set to 0.010 and 0.08 for the target flow rate for the dispersed and continuous phases respectively, and the integral factor was set to 0.035 and 0.05 for the dispersed and continuous phases respectively, this tuning allowed precise adjustments to achieve the target flow rate by minimizing errors that occurred while adjusting the pressure difference between channel and the reservoir. The derivative was auto-tuned to reduce error bands during the P and I fine-tuning process.

[0192] Mass spectroscopy of RBC lipids. Mass spectroscopy of extracted RBC membrane lipids was performed by the Kansas State Lipidomics Research Center (KS, USA) using an Applied Biosystems API 4000 and an Applied Biosystems Q-TRAP in tandem. Extensive lipid profiles were compiled for three biological replicates of RBC membrane lipids. In addition to mass spectroscopy data, the Lipidomics Research Center ran a secondary procedure to quantify the cholesterol present in each sample, which was necessary to normalize the data sets to allow for direct comparison of the lipid species distribution across all three replicates. GC / MS was performed on an Agilent 6890N GC coupled to an Agilent 5975N quadrupole mass selective detector (with El). The GC was fitted with a VF-5MS capillary column (inert 5% phenylmethyl column, length: 30 mm, internal diameter: 250 pm, film thickness: 0.25 pm) with a 10 m EZ guard column. Helium was used as the carrier gas at a column flow rate of 1 mL / min. The front inlet was operating at 250 °C. The Agilent 7683 autosampler was used to inject 1 pL of the sample in the split-less mode. The GC oven temperature program was set to an initial temperature of 150 °C, held for 1 min, 30 °C / min to 300 °C, and 3 °C / min to a final temperature of 315 °C, held for 1 min. Total run time 12 min. The mass spectrometer was operated in the electron impact mode at 70 eV ionization energy. The MS quad temperature was at 150 °C and the MS source temperature was at 230 °C. The data acquisition was set to scan mode with a scan mass of 50 to 650. The data were processed with Agilent Chem-station software.

[0193] Nanoparticle tracking analysis (NTA) of eRBCEVs. The eRBCEVs size distribution and particle concentration were investigated on nanoparticle tracking analysis (NTA) NS300 (Malvern Panalytical, UK). Initially, the microfluidic NTA cell was thoroughly rinsed three times with 1 mL of DI w ater. The system w as operated at 25 °C, with a sample flow rate of 50 pL / min, and 500 pL of the sample w as introduced. To match the NTA's w orking concentration, the sample was diluted 1000-fold with phosphate-buffered saline (PBS). Five videos, each lasting 30 seconds, were recorded to ensure accurate distribution measurements. The screen gain was set to 15, and the detect threshold was adjusted to 5. A setup featuring a scientific complementary' metal-oxide-semiconductor (sCMOS) camera was employed to capture the moving particles, and data post-processing was performed on NTA 3.3.301 version software.

[0194] Zeta potential of eRBCEVs. The zeta-sizer (Malvern Panalytical, UK) was used to analyze the zeta potential of eRBCEVs. The zeta cell was rinsed with 1 mL of PBS twice, and then the sample was loaded onto it. The zeta potential setting was preset to appropriate solvent properties.

[0195] Human hemoglobin (hHb) isolation and purification. The hHb isolation and TFF purification were conducted by using established procedures. Briefly, the RBC was lysed with DI water and centrifuged at 1750 x g to remove large debris. Furthermore, multistage tangential flow' filtration was swift and practically purified hemoglobin, yielding high-purity samples suitable for subsequent processing. Moreover. TFF-purified hemoglobin maintained oxy genbinding characteristics and cooperativity coefficients consistent with established literature values. The isolated hHb of concentration 225 mg / mL was stored at -80 °C.

[0196] Human hemoglobin encapsulation and functionality assessment. 50 pL of stock hHb (~ 5 nm) concentration in PBS was utilized to obtain 2 mg / mL hHb by dissolving in 5 mL CB and filtering with a 0.22 pm Teflon syringe filter. The FF and HB microfluidic devices were utilized with 4 mM RBC lipid concentration, at FRR of 20 pL / min lipid flow for 400 pL / min CB flow at a 1 :20 ratio. CO binding assay w as performed as described in published literature. Briefly, the eRBCEVs encapsulating hHb was sealed in glass vial and CO gas was supplied for 10 minutes, then it was analyzed by using spectra deconvolution technique on UV-Vis spectrometer. Furthermore, experimental protocol was scaled to the working concentration of 0.3 mg / mL.

[0197] Lutnbricus terrestris erythrocruorin (LtEc) isolation and purification. The isolation and purification of erythrocruorin from these organisms is performed by following the published research methods. Briefly, erythrocruorin (Ec) is a large, multi-subunit protein of size ~3.6 MDa found in the blood of certain segmented worms, such as Lumbricus terrestris (Lt), worms were collected and their blood was extracted through physical processes. The blood was then centrifuged to separate the erythrocruorin from the other components of the blood. Furthermore. LtEc was purified using tangential flow filtration (TFF), concentrated to 83 mg / mL, and stored at -80 °C.

[0198] LtEc encapsulation. To encapsulate larger proteins, the herringbone device was utilized with 4 mM RBC lipid concentration at FRR 1 :5 pL / min. The LtEc concentrations of 2, 4, 8, and 12 mg / mL were considered for optimization, LtEc was dissolved in CB, and the mixture was vortexed for 30 seconds and filtered with a 0.22 pm Teflon syringe filter before loading to the reservoirs.

[0199] Gold nanoparticle encapsulation. A 100 pL AuNPs of 10 nm size, 0.01 % Au at 5.7 x 1012parti cles / mL (Nanocs Inc., MA. USA) were dissolved in 5 mL citrate buffer and filtered with 0.22 pm Teflon syringe filter. To encapsulate gold nanoparticles (AuNPs), the herringbone device was utilized with 2 mM RBC lipid concentration, FRR 1:20 for 10 nm AuNPs, and FRR 1:5 for 30 nm AuNPs.

[0200] Oligonucleotide encapsulation. FAM-0 referring to oligonucleotides that are labeled with the fluorophore FAM (6-carboxyfluorescein) with sequence 5’TACCGCGTGCGACCC (SEQ. ID 1) (Alpha DNA, Quebec, Canada) was utilized to encapsulate in eRBCEVs. Green fluorescence was detected using total internal reflection fluorescence microscopy (TIRFM) for each well with a 100 * oil objective. The loading concentration was initially optimized considering FAM-0 concentrations 50 nM, 200 nM, 7 pM, and 10 pM in CB.

[0201] Cationic lipid nanoparticle (cLNP) synthesis. The nanoparticle synthesis pack (Elveflow Microfluidics. Paris, France) was employed as described in previous sections at a flow rate ratio of 1:20 pL / min. Commercial lipids, including l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene gly col-2000 (DMG-PEG2000), dioleoyl-3-trimethylammonium propane (DOTAP), and 1,2-dioleoyl-sn- glycero-3 -phosphocholine (DOPC) (Avanti Polar Lipids AL, USA), were dissolved at w / v ratio 10:20:50:20 in ethanol solution. The lipid mixture was sonicated for 10 minutes and filtered using a 0.22 pm syringe filter (Minisart® NML, Sartorius). eRBCEVs purification by dialysis. A 20kDa molecular weight cut-off (MWCO) Slide- A-Lyzer MINI® Dialysis device (Thermo-Fisher Scientific), was employed for purification using dialysis. 2 mL of sample was introduced onto the membrane, and PBS, 20 times the volume of the sample was used. The membrane was immersed in the PBS solution and left for 4 hours. Over 48 hours, the PBS solution was changed every 6 hours. This strategy allowed the concentrations to reach equilibrium, enhancing the purification process. Subsequently, the sample w as recovered from the top of the membrane and stored in a low-binding Eppendorf tube. eRBCEVs purification by centrifugation. An ultracel regenerated cellulose lOOkDa NMWL Amicon Spin column (Merck Millipore, MA, USA) was utilized for purification via centrifugation (Eppendorf Centrifuge 5810R, Thomas Scientific, NJ, USA). To wet the membrane. 1 mL of buffer was spun at 1000 x g for 5 minutes. The sample was then carefully introduced onto the membrane and spun at 3000 x g for 20 minutes. This step was repeated thrice by adding 500 pL PBS each cycle. eRBCEVs purification by tangential flow filtration (TFF). TFF was operated with lower shear stress without back pressure, which allowed for maintaining the structural integrity', minimized the risk of particle aggregation during the purification process. The eRBCEV sample was purified using a 28 cm2surface area 500 kDa and 0.05 pm pore size poly ethersulfone membrane (Repligen, MA, USA). The flexi tubes were flushed with 70% ethanol and the membrane was flushed with 45 mL of DI water and PBS solutions. Subsequently , the eRBCEVs sample was introduced into the membrane inlet using a peristaltic pump, with a flow rate set at 35 mL / min. No back pressure was applied, allowing the particles to circulate freely without inducing any squeezing action. The retentate line was supplied with PBS solution, facilitating a gentle exchange of the buffer. The permeate, containing unwanted components, was collected from the outlet. This purification process was repeated for several Dia-cycles, with each Dia- cycle maintained at a constant volume of 5 mL, ensuring comprehensive purification.

[0202] Bicinchoninic acid protein assay (BCA). The BCA is performed to validate the purification of the eRBCEV sample by analyzing free proteins in the sample. The BCA was performed following the well-established protocol for protein detection45. Briefly, the calibration standard was prepared by serial diluting the cocktail of 25 pL of Pierce™ RIPA buffer 10x (Thermo-Fisher Scientific), 2.5 pL of protease inhibitor (Thermo-Fisher Scientific), and 225 pL DI water. A working solution (WS) was prepared by adding a 50:1 volume ratio of rapid gold BCA reagents A and B from the BCA protein assay kit (Thermo-Fisher Scientific). 20 pL of each standard and sample was added on 96-well plate (Greiner Bio-One CellstarK. Fisher Scientific, NH, USA) in triplicate. 200 pL of WS was added to each well, mixed, and incubated at room temperature for 20 minutes. Absorbance was measured at 480 nm on a VERS Amax microplate reader (Molecular Devices LLC., CA, USA).

[0203] Cloning of Proteins and Purification. 5 pL of Kanamycin (50 mg / mL) was added to a culture tube with 5 mL of LB media and protein cloned into NEB T7 Shuffle Express cell line. The culture was incubated at 30 °C, shaking at 225 RPM for 16 hours. The culture was transferred to a 2 L baffled flask containing 500 mL of autoclaved 2*YT media and 500 pL of Kanamycin (50 mg / mL). and shaking proceeded for 5 hours at 30 °C. The cultures were cooled to 16 °C and induced with IPTG (2.5 mM final concentration). The induced cultures were shaken at 16 °C for 24 hours. The cells were harvested the next day using centrifugation (4000 RPM for 10 min) and the pellet was resuspended in 1 x PBS with Dnase-I and a protease inhibitor cocktail (EDTA free) tablet. The cells were lysed by sonication in an ice bath for 5 second increments over 15 minutes. The lysate was centrifuged at 11000 RPM for 20 min to separate cellular debris. The resulting supernatant was purified by affinity chromatography using 5 mL of Nickel- NTA histidine binding resin, which was preequilibrated with 1 x PBS. The column was left to shake for 1-2 hours at room temperature on a benchtop shaker. The column w as drained out and washed with 1 x PBS. A low concentration of imidazole of 10 mL at 10 mM was used to detach any weakly bound proteins from the resin. Elution buffer volume of 15 mL of 150 mM imidazole was then added to the column and collected in a lOkDa spin column where it was concentrated to 0.5 mL before further purification by size exclusion chromatography (superdex column) on Akta FPLC. Fractions were determined with SDS page and concentrated together in 1 xPBS and stored in 4 °C.

[0204] Azide Conjugation. The reaction occurred in a buffer (10 mM CaCh, 150 mM NaCl, 20 mM HEPES, with a pH 7.5-8), between eSrtA (100 pM), nanobody with a ligation tag (75 pM), and an amine group (20 mM). The reaction proceeded for 24 hours and then quenched with 1 : 1 volume of stop buffer (10 mM EDTA, 300 mM NaCl, 20 mM HEPES, pH 7.5-8) and left to shake for 1-2 hours. The reaction was concentrated three times via centrifugation in a lOkDa spin column and 1 x PBS before the reaction was added to 2 mL of Nickel-NTA resin and left to shake for 2 hours. Unbound proteins were collected with 1 x PBS. For proteins with histidine residues, an elution buffer of 5 mM imidazole (10 mL) followed by 10 mL of 10 mM imidazole was used to collect weakly bound proteins. Collected proteins were buffer exchanged with 1 x PBS and verified by SDS Page.

[0205] Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). Protein samples were diluted in 1 x PBS for a concentration of 10 pM in 10 pL. To the sample 10 pL of Laemmli sample buffer was added before the sample was boiled at 95 °C for 5 minutes. The samples were briefly spun using a microcentrifuge. 15 pL sample was pipetted into a Bio-Rad precast 10-well gel and left to run at a constant 200 V for 30 mins. The gel was stained with Coomassie-B.

[0206] Human neutrophil isolation. Human peripheral blood was collected with the explicit consent of informed volunteers according to IRB&2018H0268 at The Ohio State University7in BD Vacutainer K2-EDTA tubes (Thermo-Fisher Scientific). The freshly collected blood was then added at a 1:20 volume ratio to an RBC lysis buffer consisting of 150 mM ammonium chloride, 10 mM sodium bicarbonate, and 0. 1 mM EDTA at 7.4 pH for 5 minutes. The solution was then centrifuged at 350 x g for 5 minutes to concentrate the leukocytes. With the EasySep™ Human Neutrophil Isolation Kit (STEMCELL Technologies, Vancouver, Canada) neutrophils were negatively selected through an immunomagnetic separation following the manufacturer’s instructions and subsequentially resuspended in Iscove’s Modified Dulbecco’s Medium (Thermo-Fisher Scientific) with 20 % fetal bovine serum (Gibco, Thermo-Fisher Scientific) and 1 % penicillin-streptomysin (Gibco, Thermo-Fisher Scientific) at a concentration of 1 x 106cells / mL.

[0207] Lipophilic staining of eRBCEVs. 1,1 '-Dioctadecyl-3, 3, 3', 3'- tetramethylindotricarbocyanine iodide (DiR), a lipophilic near-infrared fluorescent dye was employed to stain lipids in eRBCEVs. A 10 pM DiR dye was added to the eRBCEVs sample of concentration 5 x 1011particles / mL and incubated at 37 °C for 20 minutes. A three-time quick wash with 500 pL PBS was performed through centrifugation at 1750 x g to remove excess dye. free FAM-O, and free RBC lipids if any exist.

[0208] LtEc-FITC labelling. The hemoglobin of the earthworm, LtEc was isolated and purified by following a published procedure and dissolved in PBS at 1.2 mg / mL concentration. Fluorescein isothiocyanate (F1TC) (Thermo-Fisher Scientific) was dissolved in N, N- Dimethylformamide (DMF) at a concentration of 20 mg / mL and thoroughly mixed to ensure complete dissolution of the FITC. The FITC solution was added to the LtEc solution at a molar ratio of 20: 1 and promptly mixed. The mixture was incubated for 1 hour at room temperature in the dark. The excess FITC dye was removed by Zeba™ (Thermo-Fisher Scientific) 7kDa MWCO spin desalting columns (Thermo-Fisher Scientific) and subsequently dialyzed using 20kDa MWCO Slide-A-Lyzer™ MINI dialysis device (Thermo-Fisher Scientific). The modified LtEc-FITC was stored in PBS at 4 °C before proceeding to the next step.

[0209] Characterization of eRBCEVs and encapsulates by TIRFM. Total internal reflection fluorescence microscopy (TIRFM) (Nikon Eclipse Ti2 Inverted Microscope System, NY, USA) was used to detect co-localization of FAM-0 and FITC labeled LtEc inside a DiR labeled eRBCEV at 80 microns region of interest. A 100-fold dilution of 10 pL of FAM-O-eRBCEVs and FITC-LtEc-eRBCEVs sample was drop cast on a 25 x 75 mm cleaned high precision glass coverslip (Paul Marienfeld GmbH & Co., KG, Germany). For the FAM-O-eRBCEVs sample. 45% laser intensity was used for both channels 657 nm and 488 nm at perfect focus mode. Similarly, for the FITC-LtEc sample, 20% laser intensity was used for the channel 488 nm and 60% laser intensity was used for the channel 657 nm at perfect focus mode. The DiR being far red, emits red at 657 nm wavelength while FAM being green emits at 488 nm, similarly FITC being fluorescent green emits at 488 nm. The program was set to auto to capture a hundred images at perfect focus on distinct locations in the sample.

[0210] Functionalization of eRBCEVs with biotin and detection by TIRFM. 2% w / v of DSPE-PEG-2000Biotin (Avanti Polar Lipids, AL. USA) of concentration 20 mg / mL was dissolved in ethanol and mixed with RBC lipids, sonicated for 8 minutes, and filtered with 0.22 pm syringe filter. The lipid solution was then used for eRBCEVs production like the previously described method. The selective capture on gold biochip technique was utilized for detection, and the fabrication and functionalization of the biochip were followed as described in a published protocol. Briefly, the high precision cover glass was cleaned thrice in ethanol and water, then it was subjected to UV treatment for 10 minutes. Furthermore, the cover glass was coated with 2 nm titanium (Ti) and 12 nm gold (Au) using electron gun-evaporator at Nanotech West Laboratories (The Ohio State University, OH, USA). The eRBCEVs of concentration 5 x 108were linked to the NeutrAvidin (NA; Thermo-Fisher Scientific) via 30 minutes of incubation and three times of washing with l x PBS. Later, NA 488 was added, incubated for 15 minutes, and washed as earlier. After the incubation, 45% laser intensity was used for 488 nm channel at perfect focus mode to detect Biotin- NeutrAvidin signal from eRBCEVs surface. Transmission electron microscopy (TEM) of eRBCEVs. Two 20 pL droplets of DI water and two 20 pL droplets of Urany-Less EM contrast stain from Electron Microscopy Science (EMS, PA, USA) were placed on a parafilm surface. A 300 mesh nickel grid (Ted Pella Inc., Redding, CA) featuring lacy carbon film was employed. Subsequently, 10 pL of eRBCEVs were drop-cast onto the grid and incubated for 60 seconds, followed by blotting with filter paper (Whatman™ 1, Cytiva, MA, USA) to remove excess liquid. The TEM grids were promptly washed by dipping into a DI water droplet, blotted with filter paper, and the process was repeated with another DI water droplet. A parallel procedure was carried out for the contrast stain, with a shorter incubation time of 22 seconds. The treated TEM grids were then left in the grid box overnight to ensure complete drying before imaging. TEM imaging was executed on a Tecnai TF-20 microscope (Center for Electron Microscopy and Analysis, OH, USA) operating at 300 kV.

[0211] Energy dispersive X-ray spectroscopy (EDS) of eRBCEVs encapsulating AuNPs. TEM with energy dispersive X-ray spectroscopic analysis (TEM-EDS) technique was utilized to achieve high-resolution imaging capabilities of TEM with the elemental analysis. The eRBCEVs encapsulating 10 nm gold nanoparticles were subjected to EDS to determine the presence and distribution of elements, including gold (Au), phosphorous (P), and oxygen (O). The sample preparation and TEM grid incubation were followed exactly as mentioned earlier. High- Angle Annular Dark-Field (HAADF) imaging technique w as incorporated within scanning transmission electron microscopy (STEM) mode to gain high-resolution images of the atomic structure of materials. The sample w as scanned for 45 minutes.

[0212] Cryogenic electronic microscopy (cryo-EM) of eRBCEVs. A 3 Lil. aliquot of eRBCEVs was dropped onto lacy 300-mesh copper specimen grids (Product #01883; Ted Pella Inc., Redding, CA). After allowing the excess liquid to be blotted away for 4 seconds using Whatman™ grade 1 filter papers, the grid was promptly immersed into liquid ethane using the Vitrobot Mark IV system (Thermo-Fisher Scientific) to rapidly induce the formation of a thin layer of amorphous ice. Subsequently, the grid was transferred beneath liquid nitrogen to a Glacios™ Cryo-TEM (Thermo-Fisher Scientific). Finally, images were captured utilizing a Felcon™ direct electron detector (Thermo-Fisher Scientific).

[0213] Nanodrop spectrophotometer of eRBCEVs encapsulating LtEc-FITC. A NanoDrop 2000c spectrophotometer (Thermo-Fisher Scientific) was employed to quantify peaks for unencapsulated proteins and excess free FITC leaking from the labeled LtEc sample after encapsulation into eRBCEVs. Drop casted 1 pL of sample onto the low er optical pedestal further a standard protocol was followed for all experiments and data analysis as published. Human neutrophil activation studies and brightfield imaging. 100 pL of the cell solution was added to each well and the dilutions were adjusted back to 40 pL by adding PBS for each sample. Following the loading of neutrophils onto the microwells, the cells' activity was observed through time-lapse imaging at 20* objective. A fully automated Nikon Eclipse Ti2 microscope (Nikon Instruments Inc., NY, USA) equipped with a cage incubator (Oko-lab, NA, Italy), maintained at a temperature of 37 °C and a CO2 concentration of 5%, was employed for this purpose. The program was set to capture an image every 5 minutes for an hour and every 10 minutes for 10 hours. The capturing bright-field channel was acquired sequentially over time, providing comprehensive data on the behavior of neutrophils within the micro well array.

[0214] Image analysis. The intensity, brightness, and contrast adjustments were carried out on open-sourced Java-based image processing software Fiji-ImageJ (GitHub). The cell counter and merge channel function were used to quantity7neutrophil activation and to compile the two channels from TIRFM. Furthermore, MATLAB image processing toolbox (MathWorks, MA, USA) licensed through Ohio Supercomputing Center was utilized to performed image analysis, according to a published algorithm. Briefly, images from TIRFM were binarized, normalized brightness, and normalized channel fluorescence intensities along with eRBCEVs detection area.

[0215] Statistical analysis. Statistical Analysis were performed on JMP Pro 17 software (JMP, Cary. NC). whereby statistical significances were inferred with the satisfaction of p < 0.05. Data expressed as the mean, median, mode and ± SD.

[0216] Results

[0217] Microfluidic nano-assembly of human RBC lipids. The idea of engineering to reassemble human RBC lipids into nanoparticles allows us to develop therapeutics by encapsulating various molecular components, and optionally incorporating accurate surface modifications. In this example, we examine the potential of eRBCEVs by encapsulating variety of molecular cargo namely, human hemoglobin (hHb), Lumbricus terrestris erythrocruorin (LtEc), gold nanoparticles (AuNPs), and oligonucleotides. Furthermore, the eRBCEVs can include surface modification (for example biotinylation) to bind with protein complexes (Figure 1A)

[0218] We use expired and nearly-expired RBCs to extract lipids through the chloroformmethanol phase separation technique, which enabled the extraction of protein-less pure lipid components. However, the cell membrane of RBCs includes a large number of different lipids, including phospholipids that are known to be responsible for the bilayer formation.

[0219] In order to understand the RBC membrane lipid composition, we first performed lipidomics. The mass spectroscopy findings were normalized with respect to cholesterol. Human RBC membranes were found to include 46.59% phosphatidylcholine (PC). 20.4% phosphatidylethanolamine (PE), 0.2% phosphatidylinositol (PI), 2.5% phosphatidylserine (PS), 0.62 % lysophosphatidylcholine (LysoPC), 22.89% sphingomyelin-DSM (SM-DSM), 4.5% ethylphosphatidylcholine (ePC), 0.25% lysophosphatidylethanolamine (LysoPE), 1.7% ethylphosphatidylethanolamine (ePE). 0.04% ethylphosphatidylserine (ePS), 0.2% phosphatidic acid (PA), 0.1% phosphatidylglycerol (PG), and 0.01% phosphatidylethanolamine-ceramide (PE-Cer) (Figure IB). The natural lipids from RBCs show a balance in charge with neutral PC and PE, anionic PA, PS and PI, zwitterionic SM-DSM, cationic ePC, and ePE. The phenomenon of self-assembly ensures cohesive association of the fatty acyl chains that form the inner core, and heads face outwards making it a cargo carrier.

[0220] A focus of the work in this Example is on engineering the RBC lipid self-assembly process through manipulation of influencing factors including FRR, lipid concentration, and loading cargo. The microfluidic device acts as a platform for the mixing of RBC lipids and CB flowing from pressurized reservoirs. FF and HB designs enhance the chaotic advection-inducing transverse flow (Figure 1C). To ensure sterility and large-scale processing capability, industrystandard tangential flow filtration methods for eRBCEVs purification are also incorporated (Figure ID). COMSOL multi-physics w as utilized to compute operating parameters such as FRR. and mixing conditions. The COMSOL simulation predicted the concentration of lipids solution diffusing across the channel. Various FRRs (1 :2, 1 :4, 1 :5, 1 :6, 1:8, 1 : 10, 1 : 16, 1 :20, and 1 :40) w ere studied to optimize the diffusion by quantifying mixing conditions with concentration gradients (Figure 9C). The gradients of diffusion and computational statistics of diffusion across the channel length were studied on the FF device. The gradient studies were performed with lipid concentrations 1 and 4 mM at FRR 1 :20. Streamline plots were used to represent these by cut 2D functions at 10 seconds time-dependent study. The increase in lipid concentration dominates the interface formation, 4 mM solution forms a thicker and narrow er interface which show s lesser surface interexchange of fluid layers (Figure IE). Furthermore, the concentration of lipids at the mixing junction was initialized to 4 mM and the concentration of the aqueous phase was set to 100 mM FRR was maintained at 1 :4 and 1:20. The concentration profile decreased along the path of the channel length, and area of interface have direct proportionality to the concentration of solutes (lipids) and FRR. A gradual diffusion of lipids was observed as it proceeded across the length of the channel, and it was observed that diffusion occurs sideways, the concentration was found to be highest at 3.87 mM at the central part of fluid flow, then gradually decreased to 50 pM and 500 pM as it nears the wall depending on the FRR (Figure IF). These optimized flow' rates were used as input to the pressure system, and the flow of fluid was observed in real-time with a brightfield camera. The VOF flow plots at the lowest FRR 1:4 and higher FFR 1 :20 were compared in real-time in the microfluidic channel at the same flow rate ratios (Figure 1G).

[0221] The FRR 1 :5 and 1: 10 on FF was compared with real-time flow (Figure 9D). The increase in the flow rate of the aqueous phase led to the squeezing of the lipid flow path and then the interface for the diffusion was formed. Therefore, in the case of the FF device, the aqueous phase limits the lipid flows to form a V-shaped neck near the junction, this induces efficient mixing conditions. In reality, this transformation arises from ion-based interactions of the selfassembly lipids in buffer solution. Furthermore, the herringbone design adeptly utilized the fluid dynamics emanating from the secondary flows and complex geometric configuration to enhance interactions among various components within the fluid substantially. Thus, the herringbone design generated a higher level of micro-turbulence and convective mixing. The flow 2D surface w as plotted for diffusion of lipid components at 10 seconds time snap using the X and Y line partition throughout the channel. The transport of concentration gradient equations was incorporated to predict and quantify the diffusion of lipids into the citrate phase (Figures 10A and 10B). The FRR 1 :1, 1:6, 1 : 10, and 1 :40 on HB were compared with the real-time flow (Figure 10C). However, FRR 1:40 pL / min did not show significant mixing, therefore it is neglected for further optimization. Therefore, the multi-parametric simulations reveal that flowrate and diffusion played a role in controlling the mixing of fluids inside a microfluidic channel, flowrate ratio (FRR) from 1:4 pL / min to 1:20 pL / min, and lipid concentration from 1 mM to 4 mM w as considered to ensure the efficient micro-mixing environment within the thin fluid layers. The simulation and visualization of mixing parameters were established based on time dependent mathematical models and logical assumptions to achieve convergence, nonetheless, they are realistic. Therefore, we considered simulated outcomes as the initial parameter to further optimize experimentally with instrument measurements and optical characterizations.

[0222] Optimization and analytical characterization of eRBCEVs. The experimental characterization of eRBCEVs determines the tunable factors for size, concentration, homogeneity dependencies from the type of device, molecular cargo, and lastly optical assessment for topology. The size distribution fromNTA for both FF and HB devices show7variation in mode size starting from 80 nm (SD + / - 4.2 nm) at FRR 1 : 10 on the HB device to 235 nm (SD + / - 7. 1 nm) at FRR 1 :4 on FF device, offering the capability of producing a broad range of size by manipulating FRR and geometric configurations (Figure 2A). These particles w ere produced without cargo. The eRBCEVs produced under various FRRs of 4, 5, 6, 8, 10, 20, and 40 were subjected to statistical analysis using the analysis of variance (AN OVA) test, the average size differed significantly across all FRR conditions (F = 0.0001, p < 0.05). Furthermore, the Tukey -Kramer Test was performed to compare the difference in means and found that few FRRs were not significant including 6 & 10, 5 & 6, and 20 & 40 thus one among each pair of those FRRs was neglected in subsequent optimization. (Figure 11A). Similarly, the eRBCEVs produced on flow focusing and herringbone design microfluidic devices were subjected to statistical analysis (Figure 2B). These results revealed that the average size differed significantly across designs of microfluidic devices (F = 0.0110, p < 0.05) indicating the influence of flow rate and design on the size distribution, suggesting that the manipulation of FRRs and design is a key factor for the size characteristics. The flow-focusing device produced a border size distribution for same synthesis parameters as compared to the herringbone design. Therefore, herringbone design may be preferred for the production of eRBCEVs for consistent and narrower size distribution.

[0223] The effect of loading was studied by comparing differences in size for encapsulated and empty eRBCEVs. The nanoparticle size distributions suggest that both encapsulated and empty nanoparticles were unimodal around 100 nm mean size + / - S.D 23.5 nm and had a similar range of concentration at 8 x 109after 1000-fold dilution (Figure 2C), ( ANOVA and studentized t-test F = 0. 1890, p < 0.05) statistical analysis confirms that there were no significant differences in encapsulated and empty eRBCEVs (Figure 11B). The size of eRBCEVs was majorly influenced by both the concentration of lipids and FRR apart from geometric configurations. Hence, variation of concentration of lipids was considered for flow parameter optimization. In this context, RBC lipids were diluted into 1-, 2- and 4-mM concentrations. The size distribution and statistical analysis were performed (Figure 2D). The FRR was maintained at 1 :20 throughout and a herringbone device was used to estimate the influence of lipid concentration. The particle size versus concentration graph depicts the bimodal distribution for 1 mM and a heterogeneous size pattern for 4 mM concentrations whereas, 2 mM lipid concentration shows a unimodal curve. The concentrations 4 & 2 and 4 & 1 -mM was found to have effects on the eRBCEVs size, therefore the concentration of lipids played key role in size distribution and homogeneity (Tukey and ANOVA tests F= 0.0019, p < 0.05). Furthermore, categorical analysis revealed that there was no association between the FRR and microfluidic design (likelihood ratio = 0.0006, Pearson correlation = 0.0012, and CMH chisq = 0.0047, p < 0.05). Therefore, the effect change in the concentration of lipids shows a significant effect on the size of eRBCEVs (Figure 11C). Thus, the lipid concentrations 2 mM and 4 mM were chosen for the rest of the optimization. The surface charge was analyzed considering the zeta negative standard ZTS1240 as a control and the zeta potential of eRBCEVs was found to be significantly near the neutral value average of - 7.69 mV (S.D + / - -2.34 mV) when compared to standard average -29.0 mV (S.D + / - -0.9 mV), these values were consistent (ANOVA and t-test F = 0.0001, / ? < 0.05) (Figure 11D).

[0224] Next, poly dispersity index (PDI) analysis was essential to confirm the homogeneity of the particles. It was used to quantify the distribution of sizes within a population of eRBCEV particles. The PDI enabled optimization and accurately provided feedback on the impact of changes in flow conditions on the eRBCEV size distribution. This PDI numerical range is from 0 to 1, where PDIs below 0. 1 signify a state of mono-dispersity, indicating a narrow and consistent particle size range of nanoparticles. Conversely, PDIs exceeding 0.3 suggest poly dispersity, indicative of a broader distribution of particle sizes. The PDI was compared in the context of loading and empty eRBCEVs. The PDI variation confirmed that eRBCEVs with loading cargo encouraged particles to stabilize and thus have a lower PDI of 0.05 (Figure 2E). Comparisons confirmed that as FRR was increasing, the PDI was decreasing.

[0225] It was favorable to choose either 2 mM or 4 mM lipid concentration and FRR 10. 20. and 40. At FRR 1 :20 PDI was found to be 0.062 (SD + / -0.003), 0.012 (SD + / -0.002), and 0.038 (SD + / -0.008) for l-,2- and 4 mM lipid concentration. However, FRR 1:20 and 2 mM lipid concentrations showed the most favorable results in terms of PDI (Figure 2F). Furthermore, optimization conditions were different for the different molecular cargos encapsulated. Therefore, PDI analyses were compiled considering five different runs on each loading type. The flow, concentration parameters, and device design conditions were maintained constant throughout. The radar graph showed the average PDI of Oligos and hHb as 0.02 (SD + / -0.005) <0.1 & 0.03 (SD + / - 0.002) <0.1 was found to be homogeneous due to their size and molecular density. The PDI for AuNPs and LtEc was 0.065 (SD + / -0.01) <0.1 and 0.07 (SD + / - 0.015) respectively, though they were still homogeneous (Figure 2G). Furthermore, electron microscopy analysis revealed that the eRBCEVs exhibited a near-circular morphology resembling EVs. The size distribution of RBCEVs was found to be multi-modal with mode 121.4 nm (SD + / - 68.7 nm) (Figures 2H and 12A). The inside of the vesicles appeared as a dark region due to the contrast betw een the lipids and the surrounding void. Since these eRBCEVs were empty, a notable absence of internal structures was observed. This distinguishes them from loaded eRBCEVs.

[0226] Additionally, the size distribution was unimodal with a mean size of 125 nm (SD + / - 12.5 nm, n = 45) (Figure 21). Interestingly, during experiments it was observed that the FF device was more prone to lipid aggregation, it was tested for mixing at different time points including 0, and 15 minutes, and the interface betw een the dispersed and continuous phases w as found to be susceptible to coalescence at 15 minutes of mixing flows. High shear forces at the interface contributed to the formation of larger aggregation towards the later end of the main channel. However, this phenomenon was not significantly observed on the herringbone device due to the presence of ridges arranged on the walls of the microchannel which in turn induced chaotic flow patterns (Figure 4B). Furthermore, the HB device was found to produce highly homogeneous eRBCEVs at 2 mM lipids and FRR 1 :20. Therefore, experimental optimization and critical parameters are essential to produce eRBCEVs for clinical translation. Furthermore, eRBCEVs were set to explore encapsulating various molecular cargo by producing with pre-optimized conditions.

[0227] Characterization of nano-encapsulates in eRBCEVs

[0228] Human hemoglobin (hHb) encapsulation in eRBCEVs. Encapsulation protects hemoglobin from degradation and reduces the risk of renal toxicity associated with unencapsulated hemoglobin. Therefore, encapsulation of hemoglobin w as a versatile approach for overcoming the limitations associated with unmodified hemoglobin. The hHb concentration of 2 mg / mL mixed with aqueous phase to produce hHb encapsulated eRBCEVs and size distribution from NTA showed unimodal with 135 nm mean size (S.D + / - 12.8 nm, n = 3) at FRR 1 :20 (Figure 13A). The eRBCEVs formed the outer shell encapsulating the internal hemoglobin, being a protein, it appeared electron-dense within this core (Figure 3A). The calibrated and optimized eRBCEVs exhibit uniformity in both size and structure. The efficiency of encapsulation was calculated by counting the TEM images across three replicates. The encapsulation efficiency was found to be 77.24 % (S.D + / - 1.55%, n = 138). Furthermore, in RBC, the hemoglobin is responsible for the rate of exchange of gases such as oxygen, carbon monoxide, or nitric oxide through intermembrane diffusion. The CO binding functional assessment shows the mimicking of the natural RBC and makes it suitable for potential blood substitute and advanced therapeutic development. The CO binding ‘konCO IO? was found to be 0. 198, which was not significantly high compared to free hHb at 0.210 however, due to the presence of a lower concentration of hHb inside each eRBCEVs, kOnco>' was considered satisfactory also since mean particle size 135 nm < 500 nm as reported in the literature.

[0229] Gold nanoparticle (AuNPs) encapsulation in eRBCEVs. The streptavidin-coated gold nanoparticles are utilized for biosensors, and for enhancing the specificity of drug release especially in cancer field. However, unencapsulated AuNPs are toxic in circulation and have poor biodistribution. Therefore, eRBCEVs can be utilized to internalize AuNPs for theranostic applications. The eRBCEVs with AuNPs were seen as typically spherical or quasi-spherical structures under TEM as shown in (Figure 3B). The eRBCEVs encapsulating 10 nm and 30 nm AuNPs, appeared as a contrasted boundary. The AuNPs appeared as dark spots compared to the surrounding eRBCEVs. The size distribution showed unimodal with 75 nm (SD + / - 11.3 %) and 215 nm (SD + / - 16.2 %) mean size at FRR 1:20 & 1:5 for 10 nm and 30 nm AuNPs respectively. The encapsulation efficiency was found to be 47.11 % (S.D + / - 4.22%) for 10 nm AuNPs and 37.58% (S.D + / - 2.12%) for 30 nm AuNPs (Figure 13B)

[0230] ED AX was performed on eRBCEVs with AuNP - 10 nm encapsulation, it confirmed the elemental position inside the eRBCEVs. The element mapping depicted the regions with higher gold, oxygen, carbon, and phosphorous overlays, the elements were ty pically represented in warm colors such as yellow, green, turquoise, and dark green respectively along with negative staining inverse contrast. Si, N, CPS mapping, density' mapping and overlay (Figure 13C). In the eRBCEV sample, the oxygen and phosphorous were observed in higher concentrations on lipid layers and Au was seen at higher concentrations inside the eRBCEVs. The intensity' versus energy graph depicted the presence of Au by revealing M and L shell energy band peaks at 2. 1 keV and 8.2-14.5 keV respectively. Elements oxygen and phosphorous were detected at the early energy levels below 1.8-2.1 keV. The elements Ni, C, and Si peaks appeared due to interference of the TEM grid (Figure 3C).

[0231] Lumbricus terrestris erythrocruorin (LtEc ~ 3.6 MDa) encapsulation in eRBCEVs. Like hemoglobin, erythrocruorins function as oxygen earners in the circulatory system of certain annelids but holds the danger of vasodilation and contributing to vasoconstriction. Therefore, shielding LtEc with eRBCEVs leads to the development of a six-fold bigger oxygen carrier than hemoglobin (—28 nm) analyzed from TEM, marking it as a potential candidate for blood substitute. The LtEc concentration of 1.2 x io12at 6 mg / mL was mixed with 5 mL aqueous phase. The size distribution was found to be unimodal with 205 nm mean size (S.D + / - 10.5 nm). The TEM image shows internalization of LtEc. The large protein LtEc appeared as an electron- dense hexagonal bright strip along with a hollow core and eRBCEVs exhibit uniformity' in both size and structure (Figure 3D). The images provide direct visual evidence of the encapsulated LtEc in eRBCEVs. The encapsulation efficiency was found to be 51.55 % (S.D + / - 7.12%. n = 68).

[0232] Lastly, these characterizations were effective to analyze homogeneous nano-assembly of eRBCEVs with nano-encapsulation efficiencies. TEM coupled with ED AX outcomes were excellent for bulk topological quantifications; however, to examine eRBCEVs at the single particle level, high-resolution microscopy techniques like cryo-EM and TIRFM were adopted.

[0233] High-resolution characterization, functional studies, and purification of eRBCEVs. High-resolution cryo-TEM technique was utilized characterize topology' of eRBCEVs. The unloaded eRBCEVs at FRR 1: 10 appeared trapped within the thin layer of ice and holes, and the double-layered membrane was evident, with darker and lighter regions representing different densities of the lipid molecules exhibiting ‘onion-like’ ring structure with uniformity and homogeneity on the cryo-EM (Figure 4A). The mean size was found to be 132 nm (S.D + / - 6. 1% nm, n = 158) (Figure 14A). The biotinylated eRBCEVs selectively captured on gold biochip with Neu were acquired on TIRFM, the bright green spots were observed on the 488 nm channel (5% laser power), and controls PBS and 561 - channel were blank indicating microfluidic approach have abilities to surface modify eRBCEVs (Figure 14B).

[0234] FAM-Oligonucleotide (FAM-O) encapsulation and detection in eRBCEVs. The oligonucleotides were encapsulated in eRBCEVs, considering as a model for nucleic acid therapeutics, encapsulating oligonucleotides provide a more tailored approach based on primary sequence knowledge. The particles with DiR stained appeared red and FAM-0 appeared green under TIRFM (Figure 4B). Cross-interference signal was negative as demonstrated using controls free FAM-Oligos and PBS (Figure 15A). MATLAB intensity mapping program was utilized for mean intensity analysis (Figure 15B) and no crossing over of channels was observed. The optimized particle characteristics were found at 7 pM and size distribution showed unimodal with 145 nm mean size analyzed on NTA with average concentration 4 x 107particles / mL after 1000-fold dilution in PBS at FRR 1 :5 (Figure 15C). The intensity overlapping from MATLAB software was found to be < 3% for controls and 56.03% for Oligos, which means that out of all eRBCEVs, 56% of them contain FAM-Oligos, interestingly the standard cationic LNP (cLNP) encapsulated 33.29% produced with the same parameters (Figure 4D). The control empty eRBCEVs labeled with DiR and free FAM-Oligos were found to have statistically not significant colocalization (F = 0. 1089, / ? < 0.05). Therefore, the bottom-up approach of encapsulating oligos depicted better efficiency with eRBCEVs than cLNPs.

[0235] FITC labelled LtEc and encapsulated in eRBCEVs. Similarly, LtEc was labeled using FITC to precisely correlate encapsulation efficiency at single-EV resolution within DiR-labeled eRBCEVs at a concentration of 4 x 1010particles / mL. (Figures 15D and 15E). The eRBCEVs with DiR stained appeared red and FITC labeled LtEc appeared green under TIRFM (Figure 4C). The co-localization efficiency was found to be 59.86%. The controls w ere statistically significant (F = 0.001 p < 0.05) against the encapsulated eRBCEVs (Figure 4D). The LtEc within eRBCEVs of mean size 205 nm (S.D + / - 10.5 nm) labeled with FITC (Figure 15F) were exposed to a series of wavelengths, no absorbance peaks were observed for controls PBS and WFI at any wavelengths. The unencapsulated LtEc protein showed a significant peak at 400 nm with ~0.4 a.u, whereas a peak for free FITC was observed at 475 nm with - 0.2 a.u. The eRBCEV encapsulated FITC labeled LtEc showed two peaks at 400 nm and 475 nm showing agreement with the dialyzed sample (retentate). Furthermore, there was no peak observed from the permeate solution, therefore there was no significant excess FITC, or free proteins present in the solution after purification (Figure 15G).

[0236] Purification of eRBCEVs. Continuous purification of eRBCEVs on the TFF system was time-efficient providing consistency and scalability. The concentration of free proteins (hHb) in the permeate and retentate solutions was analyzed with the BCA technique. Initially, the protein concentration of permeate exponentially increases until 30 mL cycle volume whereas retentate protein concentration exponentially reduces until about 35 mL. The rest of the graph follows linear co-relation between the concentration of proteins between permeate and retentate volumes. Therefore, at 30 mL most of the free proteins w ere transferred to the permeate side of TFF and the actual required dia-cycle was found to be 18 or until 90 mL volume transfer. Beyond 90 mL it w ould be due to lipid membrane shearing under pressure (Figure 4E). TFF allowed real-time monitoring and control of parameters such as transmembrane pressure and flow rates which facilitates precise control over the purification process to maintain optimal conditions continuously. Visualization and intensity analysis empowered purification strategies. Thus, TFF was chosen for future purifications under these optimized conditions.

[0237] Quantifying human peripheral blood neutrophil activation in eRBCEV triggered immunogenicity. The surface properties of lipids, including their charge, size, and composition, can influence their interaction with immune cells. Understanding neutrophile activation percentage allows us to categorize eRBCEVs as a unique drug-carrying vesicle and a potential candidate for crossing biological barriers. Table 1 contains a comprehensive list of samples used and their concentrations with volume.

[0238] Table 1. Samples for quantifying human peripheral blood neutrophil activation in eRBCEV triggered immunogenicity. The experiment was conducted in triplicate with a total of 45 wells including controls.

[0239]

[0240] Time points 5, 15, 30, 60, 90, 120. 180, and 360 minutes were strategically considered to study activation. The average activation rate was exponential during time 30 minutes to 90 minutes for positive control (BioParticles™) whereas PBS control did not show activation significantly (Figure 16A). It was observed that neutrophils activated after a specific buffer time, where the rate of activation increases and slows down later but activates the surrounding neutrophils due to chemotaxis (Figure 16B). The activation % for free hHb at 0.5 mg / mL was observed constant throughout the time scale; this was the same concentration of hHb that was encapsulated in eRBCEVs. However, once most of the particles were activated, it induces cellular enzymes which activated the rest, which was not true activation induced due to the sample. Therefore, the activation percentage was quantified at 90 90-minute time stamp (Figure 5A). The graph depicted that cLNP by itself activated 42% (S.D + / - 4.8%) neutrophils, thus cLNP at a sufficiently high concentration of 5 x 1012particles / mL was capable of inducing an immunogenic response in humans. The controls PBS and BioParticle™ showed significant activations of 8.22 % (S.D + / - 0.135) and 56.67% (S.D + / - 10.45). The activation % was significantly varying for the samples (ANOVA, F = <0.0001, / ? < 0.05), whereas the samples PBS and eRBCEVs were not significantly different (Tukey-Kramer HSD, F = 0.9705, p < 0.05). Therefore, eRBCEVs did not have a significant influence on neutrophil activation. To estimate the influence of the concentration of hHb on activation %, hHb was diluted as mentioned in Table 1, data was analyzed at timestamps 5, 15. 30. 60. 90. 120, 180. and 360. The rate function of neutrophil activation with respect to time at various dilutions of hHb and eRBCEVs was mapped (Figure 5B). The unencapsulated hemoglobin though induced at a lower concentration of 0.4 pL (0.5 mg / mL) or higher concentration 40 pL (0.5 mg / mL) had no significant difference in activation rate and was as same as BioParticles™. Interestingly eRBCEVs introduced at different concentrations did not show significant differences and were the same as control PBS, however, there was a significant difference among each of the concentrations of unencapsulated hHb and eRBCEVs encapsulating hHb. Therefore, the RBC lipid layer acted as a shield to avoid interaction with neutrophil cells. Thereby, encapsulation was efficient in protecting the hHb from causing the immunogenic response. The unencapsulated hHb upregulated response by 56.19% (S.D + / - 0.85%) and encapsulated showed minimal activation of 11.6% (S.D + / - 1.2%), (ANOVA F = 0.0001, p < 0.05) thus eRBCEVs showed lesser activation than free hHb at the same concentration of 0.5 mg / mL (Figure 5C). Furthermore, the unencapsulated hHb at the same concentration as encapsulated in eRBCEVs showed neutrophil activation after 90 minutes of incubation. Therefore, eRBCEVs did not activate the neutrophil and were less toxic due to no presence of surface major histocompatibility complexes (MHC) although derived from human RBCs.

[0241] Surface modification and nanobody conjugation with eRBCEVs. The conjugation of PD-L1 nanobodies to the surface of eRBCEVs using azide-alkyne click chemistry was found to be precise and stable functionalization which represents a significant advancement in targeted cancer therapy. The chemistry creates a covalent bond between the PD-L1 targeting moiety and the DBCO lipids (Figure 6A). These particles are endowed with the capability to recognize and bind to PD-L1 -expressing tumor cells, like EMT6. The size distribution and structure on the TEM of eRBCEVs after the addition of 1% DSPE-PEG2000-DBCO lipids was found to be consistent with previously reported unmodified eRBCEVs (Figure 6B). A bright yellow colocalized signal was observed om TIRFM with NHSF stained PDL1 nanobodies and DiR stained eRBCEV sample (Figure 6C). whereas it was not observed in the controls (Figure 17). Quantification of percentage colocalization shows that the sample with eRBCEVs to nanobody ratio 1 :700 contains ~58 % successful functionalized eRBCEVs, whereas it is ~68 % for the ratio 1:7000, while the controls PBS and PDLl-Dye remain significantly lower than the colocalized signals from sample (Figure 6D).

[0242] Cell uptake studies with conjugation of PDL1 nanobodies with eRBCEVs. To qualitatively and quantitively assess the internalization of eRBCEVs, the cellular uptake behavior was evaluated using the confocal microscopy technique and flow cytometry. The PDL1 nanobodies with NHSF on eRBCEV’s surface was introduced as a fluorescent probe on for the assessment of cellular internalization. The eRBCEVs stained with DiR were cultured with EMT6 cells for 2 hours, whereas the nuclei were stained using DAPI, and lysosome were stained with RFP. The EMT6 cells were fixed before imaging (Figure 7A). The effects of temperature on cellular membrane fluidity and endocytic activity were observed. At 4°C, the decreased kinetic energy leads to reduced membrane fluidity, which impairs the ability of eRBCEVs to interact with and penetrate the cell membrane. Additionally, the lower temperature slows down the activity of endocytic pathways, such as clathrin-mediated and caveolae-mediated endocytosis. However, while PDL1 is expressed on the cell surface, it is not internalized. Although the protein is present and accessible on the cell membrane, the uptake mechanisms necessary for internalization are not activated or are less effective at lower temperatures (Figure 7B). In contrast, at 37°C, which approximates physiological temperature, cellular membranes appear to be more fluid, and endocytic processes are observed to be more active, facilitating greater uptake of eRBCEVs. Interestingly, there was no significant colocalization was observed within the nucleus, which minimizes the risks of cytotoxicity, genomic instability and unwanted gene expression (Figure 7C). The colocalization of PDL1 green signals overlap on the red signals from eRBCEVs, glows yellow on merging at both the temperatures (Figure 18A). It was evident that the white colocalization signal from NHSF, DiR and RFP was weak at 4 °C condition, whereas the fluorescence colocalization at the lysosome was significantly enhanced at 37 °C temperature during incubation. The eRBCEVs colocalization with lysosomal marker (white signals) indicates that they have been internalized and transported through endocytic vesicles to the lysosomes. Additionally, the controls PBS, eRBCEVs without PDL1 and free PDL1 do not show significant colocalization, whereas the natural RBCEVs show- colocalization at 37 °C (Figure 18B).

[0243] Flow cytometry was also used to investigate the cellular uptake of eRBCEVs. The fluorescence intensity was significantly enhanced at 37 °C. These results demonstrate that eRBCEVs with PDL1 nanobodies could be effectively internalized by cells.

[0244] Furthermore, the box plot effectively illustrates the variation in intensity / area of eRBCEVs, PDL1, and lysosomes with temperature, revealing distinct patterns. For DiR-labelled eRBCEVs, the intensity / area displays a broader range and significantly higher signal at 37°C (Blue) compared to 4°C (Red). In contrast, the RFP lysosome stain maintains a relatively consistent intensity / area across both temperatures and the PBS control, with a slight increase observed at 37°C. This increase is attributed to enhanced RFP uptake by the cell membrane at the higher temperature, as opposed to the uptake of eRBCEVs. Meanwhile, both DiR and GFP in PBS exhibit consistently low intensity / area values across the temperatures, which is true since there is no sample induced. Discussion

[0245] Nano-drug carrier systems have evolved to become versatile platforms to develop from flu vaccines to cancer therapies. The development of personalized nanomedicine with lipid nanoparticles is currently a complex process, it requires precise prediction, design, and formulation. Despite manufacturing difficulties, the personalized nano-drug possesses unique characteristics like being tunable, scalable, efficacious, and safe. The lipid-based drug delivery agents are reliable in treating cancer, overcoming the usage of genetically modified viruses. The T-cell-based therapies with traditional techniques like electroporation are not scalable and toxic to the cell. We proposed interlayer flow dynamics and rapid mixing on microfluidics to produce a homogeneous distribution of particles. In the FF device, the laminar flow forced the RBC lipids to experience constriction at the fluid interface and transform into nanoparticles resembling the EV bi-lipid layer. Whereas the HB device through its geometric design splits the flow into a highly thin layer inducing vortices and eddies. This chaotic mixing ensures continuous and efficient blending of adjacent fluid layers, leading to dean flow and improved mixing efficiency. The advection enables rapid mixing, increasing the interfacial area, and allowing for quicker diffusion of lipids (solutes). Many authors described the flow rate ratio as one of the key factors influencing the size of particles. The inverse trend of variation of size with FRR in our findings is in satisfactory agreement with the trend reported in the literature.

[0246] Engineering EVs from mammalian cells are promising candidates for personalized medicine. Currently, EVs are produced and engineered through biogenesis, which is a small- scale manufacturing technology. Moreover, the smart selection of parent cell and culture parameters is essential to avoid the risk of oncogenicity. The loading of molecular cargo into EVs is performed by two popular methods, such as endogenous (within the parent cell) and exogenous (after isolation from the cells). Healthy RBCs measuring 7.5-8 7 pm diameter and 1.7-2.2 pm thick resemble biconcave shapes and are flexible during their lifespan of ~4 months. Like other cells, RBCs also undergo vesiculation throughout their lifecycle. However, naturally, to escape early clearance from circulation. RBCs accelerate vesicular secretion during the second half of their lifespan shedding large protein molecules. Therefore, the various minute differences in arrangements of phospholipids and surface proteins are expected to give significant membrane uniqueness and allow EVs to have numerous degrees of fluidity for the transmembrane exchange. Unlike other cell-derived EVs, RBCEVs are simply spherical fragments of RBC since RBCs lack lipids and protein-synthesizing capability. Furthermore, approximately a healthy human RBC releases 2.71 EVs per day during their activity. The composition of lipids on RBCEVs resembles closely with RBCs. However, the small EVs and large EVs released from the same batch of stored RBCs induce distinct cellular communication and mostly contain oxidized proteins. EVs though negatively charged are known to be able to up be taken by cells. Therefore, the near-neutral zeta potential of eRBCEVs allowed a border range of encapsulation capability and stability, it showed potential to have cargo to stabilize while encapsulated. Therefore, we isolate lipids from the RBCs to self-assemble on microfluidics, thereby encapsulating variety of molecular cargo while maintaining control over loading, charge and size.

[0247] During the immunogenic studies of RBCEVs, immune cell phagocytosis were observed by upregulation of super-oxides. and CD11 expression. Furthermore, CR3 monocyte-mediated proinflammatory endothelial cell response was observed with the stored RBCEVs. Neutrophils play a crucial role in the innate immune response, serving as the first line of defense against infections. Some of the drug-carrying particles are recognized as foreign and induce inflammation at the injection site while activating neutrophils. Interestingly, there was no significant upregulation of neutrophil activation during eRBCEVs interaction. Therefore, eRBCEVs uphold the advantages of LNPs and EVs together, marking them as unique, tunable, and less toxic nano-drug carriers. Furthermore, the existing human hemoglobin-based oxygen carrier (HBOC) lacks the surface antigens, making it compatible for every' human being, however purification of hHb is tedious process since unpurified can potentially transmit bloodborne diseases. Therefore, encapsulating within eRBCEVs makes it an attractive alternative for artificial oxygen carrier. However, there is a need to explore in vivo mechanics and fine-tune the deliverable strategies for customized eRBCEVs as personalized therapeutics. Furthermore, strategies for scaled-up isolation of RBC lipids, procurement, and characterization of acquired blood units, supply chain management, and legal frameworks for the use of blood components are major challenges for the commercialization of this technology.

[0248] Conclusion

[0249] The development of diverse nano-systems has significantly advanced drug delivery strategies, addressing challenges such as poor drug solubility, limited bioavailability, drug instability, and adverse side effects. Beyond this traditional drug delivery, there is a burgeoning interest in engineering EVs as shuttles for personalized therapeutic drugs. Among these, our nanostructured lipid drug carriers represent a promising avenue by combining benefits from LNPs and EVs to enhance drug loading flexibility and tunability. Traditional RBCEV isolation faces challenges, making microfluidics an attractive alternative to engineer RBCEVs through tunable self-assembly of RBC lipids. Automated pressure-based microfluidics showcases controlled mixing, scalability', and real-time monitoring. Account to the simulation, thinner diffusion boundary layers were identified as crucial for efficient diffusion, prompting optimization of flow rates and concentrations for each phase and microfluidic designs. Furthermore, initial immunogenic response with human peripheral blood neutrophils was found negative. The surface modifications, cell uptake and in vivo studies underscore the versatility of eRBCEVs as drug deli ven’ systems. However, therapeutic efficacy in in vitro and animal models are yet to be explored. This comprehensive analysis provides insights into the adaptability of eRBCEVs for diverse cargo types at optimized conditions and puts forth an idea for the design of therapeutics to cross biological barriers.

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[0366] The materials and devices of the appended claims are not limited in scope by the specific materials and devices described herein, which are intended as illustrations of a few aspects of the claims. Any materials and devices that are functionally equivalent are intended to fall within the scope of the claims. Various modifications of the materials and devices in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative materials and devices disclosed herein are specifically described, other combinations of the materials and devices also are intended to fall within the scope of the appended claims, even if not specifically recited. Thus, a combination of elements, components, or constituents may be explicitly mentioned herein or less, however, other combinations of elements, components, and constituents are included, even though not explicitly stated.

[0367] The term “comprising’' and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of’ and “consisting of’ can be used in place of “comprising” and “including” to provide for more specific embodiments of the invention and are also disclosed. Other than where noted, all numbers expressing geometries, dimensions, and so forth used in the specification and claims are to be understood at the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, to be construed in light of the number of significant digits and ordinary rounding approaches.

[0368] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

Claims

We Claim:

1. A method for generating red blood cell lipid nanoparticles, the method comprising: mixing a first solution comprising a mixture of lipids isolated from red blood cells and a second solution comprising an aqueous buffer within a microfluidic device under conditions effective to form a crude mixture comprising the population of red blood cell lipid nanoparticles dispersed within an aqueous fluid; and filtering the crude mixture comprising the population of red blood cell lipid nanoparticles by microfiltration against a filtration membrane, thereby forming a retentate fraction comprising the population of red blood cell lipid nanoparticles and a permeate fraction comprising low molecular weight contaminants.

2. The method of claim 1, wherein the mixture of lipids isolated from red blood cells comprises a mixture of lipids isolated from membranes of human red blood cells.

3. The method of claim 1, wherein the mixture of lipids isolated from red blood cells comprises a mixture of lipids isolated from membranes of non-human red blood cells.

4. The method of any one of claims 1-3, w herein the mixture of lipids isolated from red blood cells comprises a mixture of lipids isolated from membranes of red blood cells derived from stem cells, "‘recombinant red cells" cultured in bioreactors, or engineered red cells obtained from genetic engineering of stem cells.

5. The method of any one of claims 1-4, wherein the mixture of lipids isolated from red blood cells is substantially free of proteins.

6. The method of any one of claims 1-5, wherein the mixture of lipids isolated from red blood cells comprises one or more phospholipids chosen from cholesterol, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), lysophosphatidylcholine (LysoPC), sphingomyelin-DSM (SM-DSM), ethylphosphatidylcholine (ePC), lysophosphatidylethanolamine (LysoPE), ethylphosphatidylethanolamine (ePE), ethylphosphatidylserine (ePS), phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylethanolamine-ceramide (PE-Cer), or any combination thereof.

7. The method of any one of claims 1-6, wherein the mixture of lipids isolated from red blood cells comprises a mixture of phospholipids, and the mixture of phospholipids comprises from 40% by weight to 50% by weight phosphatidylcholine (PC), from 15% by weight to 25% by weight phosphatidylethanolamine (PE), from greater than 0% by weight to 0.5% by weight phosphatidylinositol (PI), from 1% by weight to 5% by weight phosphatidylserine (PS), from 0.2% by weight to 1% by weight lysophosphatidylcholine (LysoPC), from 20% by weight to 25% by weight sphingomyelin-DSM (SM-DSM), from 3% by weight to 6% by weight ethylphosphatidylcholine (ePC), from greater than 0% by weight to 0.5% by weight lysophosphatidylethanolamine (LysoPE), from 0.5% by weight to 3% by weight ethylphosphatidylethanolamine (ePE), from greater than 0% by weight to 0.2% by weight ethylphosphatidylserine (ePS), from greater than 0% by weight to 0.5% by weight phosphatidic acid (PA), from greater than 0% by weight to 0.5% by weight phosphatidylglycerol (PG), and from greater than 0% by weight to 0.2% by weight phosphatidylethanolamine-ceramide (PE- Cer).

8. The method of any one of claims 1-7, wherein the mixture of lipids isolated from red blood cells comprises a mixture of phospholipids, and the mixture of phospholipids comprises -46.6% by weight phosphatidylcholine (PC), -20.4% phosphatidylethanolamine (PE), -0.2% phosphatidylinositol (PI), -2.5% phosphatidylserine (PS), -0.6 % lysophosphatidylcholine (LysoPC), 22.89% sphingomyelin-DSM (SM-DSM), 4.5% ethylphosphatidylcholine (ePC), 0.25% lysophosphatidylethanolamine (LysoPE), 1.7% ethylphosphatidylethanolamine (ePE), 0.04% ethylphosphatidylserine (ePS), 0.2% phosphatidic acid (PA), 0.1% phosphatidylglycerol (PG), and -0.01% phosphatidylethanolamine-ceramide (PE-Cer).

9. The method of any one of claims 1-8, wherein the mixture of lipids isolated from red blood cells is obtained by a process that comprises extraction of lipids from a population of lysed red blood cells using a chloroform-methanol phase separation technique.

10. The method of any one of claims 1-9, wherein the method further comprises lysing a population of red blood cells; extracting the lysed population of red blood cells with chloroform and methanol to obtain a biphasic mixture comprising an organic phase and an aqueous phase; separating the organic phase from the aqueous phase;filtering the organic phase to remove any suspended proteins; and isolating mixture of lipids isolated from red blood cells from the organic phase.

11. The method of any one of claims 1-10, wherein the first solution comprises the mixture of lipids isolated from red blood cells dissolved or dispersed in water, a water-miscible solvent, or a combination thereof.

12. The method of claim 11, wherein the water-miscible solvent comprises an alcohol, such as ethanol.

13. The method of any one of claims 1-12, wherein the aqueous buffer comprises a citrate buffer.

14. The method of any one of claims 1-13, wherein the aqueous buffer exhibits a pH of from5.5 to 8.5, such as a pH of from 5.5 to 7.5, a pH of from 5.5 to 7.0, a pH of from 5.5 to 6.5, or a pH of about 6.

15. The method of any one of claims 1-14, wherein the microfluidic device comprises a central fluid inlet channel and two outer fluid inlet channels, wherein the central fluid inlet channel and the outer fluid inlet channels converge in a mixing channel upstream of an outlet; and wherein mixing the first solution comprising the mixture of lipids isolated from red blood cells and the second solution comprising the aqueous buffer comprises flowing the first solution through the central fluid inlet and flowing the second solution through the two outer fluid inlet channels.

16. The method of any one of claims 1-14, wherein the microfluidic device comprises a first fluid inlet channel and a second fluid inlet channel that converge in a mixing channel upstream of an outlet; wherein the mixing channel further comprises one or more mixing elements, such as pillars or ridges, that increase chaotic mixing of fluids within the mixing channel; and wherein mixing the first solution comprising the mixture of lipids isolated from red blood cells and the second solution comprising the aqueous buffer comprises flowing the first solutionthrough the first fluid inlet channel and flowing the second solution through the second fluid inlet channel.

17. The method of any one of claims 15-16, wherein the second solution comprising the aqueous buffer and the first solution comprising the mixture of lipids isolated from red blood cells are introduced at a flow rate ratio (FRR) of from 1 : 1 to 1 : 60, such as from 1 :2 to 1 : 20 or from 1:4 to 1:20.

18. The method of any one of claims 15-17, wherein the first solution comprising the mixture of lipids isolated from red blood cells is introduced into the microfluidic device at a flow rate of from 100 pL / min to 1000 pL / min.

19. The method of any one of claims 1-18, wherein the second solution comprising the aqueous buffer is introduced into the microfluidic device at a flow rate of from 10 pL / min to 200 pL / min.

20. The method of any one of claims 1-19, wherein the mixture of lipids isolated from red blood cells are present in the first solution at a concentration of from 0.5 mM to 10 mM, such as from 1 mM to 4 mM.

21. The method of any one of claims 1-20, wherein the filtration membrane is rated for a 500 kDa molecular weight cut-off.

22. The method of any one of claims 1-21, wherein filtering the crude mixture comprising the population of red blood cell lipid nanoparticles by microfiltration comprises filtering for at least 3 diafiltrations cycles, such as at least 4 diafiltration cycles, at least 5 diafiltration cycles, at least 6 diafiltration cycles, at least 7 diafiltrations cycle, at least 8 diafiltration cycles, at least 9 diafiltrations cycle, or at least 10 diafiltration cycles.

23. The method of any one of claims 1-22, wherein the microfiltration comprises tangential flow filtration.

24. The method of any one of claims 1-23, wherein the first solution, the second solution, or a combination thereof further comprises an active agent, and wherein the population of red blood cell lipid nanoparticles comprise the active agent encapsulated therein.

25. The method of claim 24, wherein the active agent comprises a small molecule, an organometallic compound, a nucleic acid (e.g., DNA, RNA), a protein (including multimeric proteins, protein complexes, etc.), a peptide, a lipid, a carbohydrate, a hormone, a metal, a radioactive element or compound, a drug, a vaccine, an immunological agent, a nanoparticle, or a combination thereof.

26. The method of any one of claims 1-25, wherein the method further comprises covalently functionalizing the red blood cell lipid nanoparticles with a targeting moiety.

27. The method of claim 26. wherein the red blood cell lipid nanoparticles are covalently functionalized with the targeting moiety using a click chemistry reaction.

28. The method of any one of claims 26-27, wherein the targeting moiety comprises a nucleic acid, a small molecule, or an antibody.

29. The method of any one of claims 1-28, wherein the population of red blood cell lipid nanoparticles is monodisperse.

30. The method of any one of claims 1-29, wherein the population of red blood cell lipid nanoparticles exhibits a PDI of 0.1 or less.

31. The method of any one of claims 1-30, wherein the population of red blood cell lipid nanoparticles exhibits an average particle size of from 50 nm to 500 nm, such as from 75 nm to 300 nm, as measured by dynamic light scattering (DLS).

32. The method of any one of claims 1-31, wherein the population of red blood cell lipid nanoparticles comprise engineered red blood cell extracellular vesicles (eRBCEVs).

33. The method of any one of claims 1-32, wherein the mixture of lipids isolated from red blood cells further comprises one or more additional lipids added to the mixture of lipids isolated from red blood cells.

34. The method of claim 33, wherein the one or more additional lipids comprise a synthetic PEGylated lipid, such as DSPE-PEG-DBCO.

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