PH-induced structure-converted lipid nano-carrier, semi-synthetic extracellular vesicle, and preparation method and application of pH-induced structure-converted lipid nano-carrier and semi-synthetic extracellular vesicle

Abstract

The present disclosure provides a pH-induced structure-converted non-layered lipid nanocarrier (LNV) comprising: (a) at least one ionizable cationic lipid; (b) at least one phospholipid having a critical stacking parameter value (CPP) greater than 1; and (c) at least one nonionic surfactant having a CPP of less than 1 at a molar concentration of from 20% to 50%, a method of preparing the LNV, a semi-synthetic extracellular vesicle (ssEV) produced by fusing the lipid nanocarrier with the extracellular vesicle at a pH of greater than 6 and up to about 10, a kit and the use of the ssEV as a medicament or diagnostic agent.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to pH-induced structural-switching lipid nanocarriers, semi-synthetic extracellular vesicles, methods of making and uses thereof. More specifically, the present disclosure relates to lipid nanocarriers with spontaneous fusion ability with extracellular vesicles. BACKGROUND

[0002] Extracellular vesicles (EVs) are vesicular structures derived from cells and are important intercellular communication mediators that can transport messenger ribonucleic acid (mRNA) or proteins and other macromolecules through the extracellular space (O'Brien et al., 2020). Encapsulating large amounts of therapeutic nucleic acids, such as small interfering RNA (siRNA) and mRNA, into extracellular vesicles remains a challenging task, and there is currently a lack of universal methods to achieve this goal (De Jong et al., 2019). Active loading strategies usually involve perturbing the vesicle bilayer to achieve drug encapsulation (De Jong et al., 2019). This method is particularly important for loading extracellular vesicles with relatively high molecular weight drugs. For example, in nucleic acid-based therapies, electroporation has become a commonly used method (Alvarez-Erviti, et al., 2011). However, the extent of vesicle disruption and whether the biological load is lost during the loading process is not fully understood. Similarly, sonication has been used as a means to temporarily disrupt membrane stability and load biological agents into extracellular vesicles (Lamichhane et al., 2016).

[0003] There is still a need for a simpler, passive loading method that can load macromolecules (e.g., therapeutic macromolecules) into extracellular vesicles without compromising the functionality of the extracellular vesicles.

[0004] This specification refers to several documents, the contents of which are incorporated herein in their entirety by reference. SUMMARY

[0006] The present disclosure provides a lipid nanostucture (i.e., lipid nanocarrier (LNV)) that is capable of spontaneous hybridization / fusion with EVs. This passive process does not require additional external stimuli (e.g., sonication, electroporation, specific buffer components) and can be completed within minutes under very mild conditions (e.g., physiological conditions of pH 7.4, 37°C). The LNV can facilitate the formation of fusion stalks in EVs, thereby generating semi-synthetic EV particles (ssEVs) without compromising the biological activity of EVs. These universal particles can load a variety of molecules and / or macromolecular therapeutic agents.

[0007] The present disclosure also provides methods of making the LNV and ssEV.

[0008] Without being limited thereto, it is speculated that the intrinsic fusogenic properties of the LNVs of the present disclosure are based on their non-lamellar lyotropic liquid crystalline structure (e.g. sponge-like structure).

[0009] More specifically, according to the present disclosure, the following items are provided:

[0010] Item 1. A pH-induced structural transition non-lamellar lipid nanocarrier (LNV) comprising:

[0011] (a) at least one ionizable cationic lipid;

[0012] (b) at least one phospholipid having a critical packing parameter value (CPP) greater than 1; and

[0013] (c) at least one non-ionic surfactant having a CPP less than 1, at a molar concentration of 20% to 50%.

[0014] Item 2. The LNV of item 1, wherein:

[0015] (a) the at least one ionizable cationic lipid has an apparent acid dissociation constant (pKa) of 5 to 7.5 prior to incorporation into the LNV;

[0016] (b) the at least one phospholipid is a phosphatidylethanolamine (PE) lipid;

[0017] (c) the at least one surfactant is a fatty acid polyethylene glycol ester or a polysorbate; or

[0018] (d) a combination of at least two of (a) to (c).

[0019] Item 3. The LNV of item 1 or 2, wherein:

[0020] (a') the at least one ionizable cationic lipid is D-Lin-MC3-DMA (MC3);

[0021] (b') the at least one phospholipid is dioleoylphosphatidylethanolamine (DOPE);

[0022] (c') the at least one non-ionic surfactant is a fatty acid polyethylene glycol ester, such as polyethylene glycol 12-hydroxystearate, preferably Kolliphor ® HS15 (KLP); or

[0023] (d') a combination of at least two of (a') to (c').

[0024] Item 4. The LNV of any one of items 1-3, loaded with a drug or diagnostic agent, preferably wherein the drug or diagnostic agent is a nucleic acid molecule.

[0025] Item 5. A composition comprising the LNV as defined in any one of items 1 to 4 and at least one pharmaceutically acceptable excipient.

[0026] Item 6. A method of preparing the LNV as defined in any one of items 1 to 3, comprising mixing: (a) an organic phase comprising at least one ionizable cationic lipid, at least one phospholipid and at least one surfactant, and dissolved in an organic solvent; and (b) an aqueous medium.

[0027] Item 7. The method of item 6, wherein at least one of the following conditions is met:

[0028] (i) the organic phase further comprises a lipophilic drug or diagnostic agent; and

[0029] (ii) the aqueous phase further comprises a hydrophilic drug or diagnostic agent, preferably wherein the hydrophilic drug or diagnostic agent is a nucleic acid molecule.

[0030] Item 8. The method of item 7, wherein (ii) the aqueous phase further comprises a hydrophilic drug or diagnostic agent, and the aqueous medium has a pH of about 4 to about 6.

[0031] Item 9. The method of any one of items 6-8, wherein the mixing is performed using a microfluidic device.

[0032] Item 10. A method of preparing a semi-synthetic extracellular vesicle (ssEV), comprising mixing the LNV as defined in any one of items 1-4 or the composition as defined in item 5 with an extracellular vesicle (EV) at a pH higher than 6 and up to about 10, thereby generating the ssEV.

[0033] Item 11. A semi-synthetic extracellular vesicle (ssEV) prepared by the method as defined in item 10.

[0034] Item 12. A semi-synthetic extracellular vesicle (ssEV) comprising substantially all or a portion of the mixture of:

[0035] (a) the lipid nanocarriers as defined in any one of items 1-4; and

[0036] (b) an extracellular vesicle (EV).

[0037] Item 13. A composition comprising the ssEV as defined in item 11 or 12 and at least one pharmaceutically acceptable excipient.

[0038] Item 14. A kit comprising:

[0039] (A) (a) the LNP as defined in any one of items 1-4 or the composition as defined in item 5; and

[0040] (b) (i) a solution for hydrating (a);

[0041] (ii) extracellular vesicles (EVs) or a composition comprising said EVs and at least one pharmaceutically acceptable excipient;

[0042] (iii) instructions for using (a) and (i) and / or (ii); or,

[0043] (iv) a combination of at least two of (i) to (iii);

[0044] or,

[0045] (B) (a') the ssEV as defined in item 11 or 12 or the composition as defined in item 13; and

[0046] (b') (i') a solution for hydrating (a');

[0047] (ii') instructions for using (a') and (i'); or,

[0048] (iii') a combination of (i') and (ii').

[0049] Item 15. The ssEV as defined in item 11 or 12 or the composition as defined in item 13 for use as a medicament or diagnostic agent.

[0050] Other objects, advantages and features of the present disclosure will become more apparent from the following non-limiting description of specific embodiments thereof, taken in conjunction with the annexed drawings. BRIEF DESCRIPTION OF DRAWINGS

[0051] In the drawings:

[0052] FIGS. 1A-1R : Composition, preparation process and physicochemical characterization of unloaded and siRNA-loaded LNVs prepared using a microfluidic device, wherein LNVs have different molar concentrations of ionizable cationic lipids (40, 50 and 60 mol % MC3) and N / P ratios (ratio of positively charged lipid amine (N) groups to negatively charged nucleic acid phosphate (P) groups). FIG. 1A ) illustrates the preparation materials and flow of siRNA-loaded LNVs used in the examples herein. Created using BioRender.com. FIG. 1B ) and ( FIG. 1C ) illustrate the hydrodynamic diameters (d h) with polydispersity index (PDI); and FIG. 1D ) and ( FIG. 1E ) show the corresponding d h with PDI. ( FIG. 1F ) shows the zeta potential values of LNVs and siRNA-loaded LNVs at pH 7.4 and 5 (60 mol % MC3; N / P = 25). ( FIG. 1G ) and ( FIG. 1H ) show the cryo-TEM images of LNVs and siRNA-loaded LNVs (60% MC3) at pH 7.4, respectively, while ( FIG. 1I ) and ( FIG. 1J ) show the corresponding images at pH 5. ( FIG. 1K ) and ( FIG. 1L ) show the small-angle X-ray scattering analysis of LNVs and siRNA-loaded LNVs (N / P = 25, 60 mol % MC3) at pH 5 and 7.4, respectively. ( FIG. 1M ) and ( FIG. 1N ) show the particle size and PDI of siRNA-loaded LNVs with 60 mol % MC3 at different N / P ratios (i.e., 6, 12, 25, and 50) at pH 7.4, respectively. ( FIG. 1O ) shows the electrophoretic mobility shift assay (EMSA) of siRNA-loaded LNVs with 60 mol % MC3 at different N / P ratios (i.e., 6, 12, 25, and 50) with or without Triton™ X-100. ( FIG. 1P ) and ( FIG. 1Q ) show the loading efficiency of siRNA in LNVs at different N / P ratios (i.e., 6, 12, 25, and 50) with 60 mol % MC3, and at different lipid ratios (40, 50, and 60 mol % MC3), respectively, as detected by RiboGreen™. ( FIG. 1R ) shows the integrity of siRNA loaded in LNVs (N / P = 25, 60 mol % MC3) after RNase treatment at 37 °C by EMSA. Statistical significance was assessed by two-way ANOVA, Tukey’s multiple comparison test (**P≤0.01, *P≤0.05). All experiments were performed in triplicate (n=3).

[0053] FIGS. 2A-2N : siRNA-loaded LNVs (60 mol % MC3, N / P 25) ( FIG. 2AMSC-derived EVs (EVs) and ssEVs formed after incubation at 37 °C, pH 7.4 for 5 min (A), 30 min (B) and 4 h (C) and pH 5 for 30 min (D) and 4 h (E) (scale bar, 100 nm). FIG. 2B ) and ssEVs formed after incubation at 37 °C, pH 7.4 for 5 min (A), 30 min (B) and 4 h (C) and pH 5 for 30 min (D) and 4 h (E) (scale bar, 100 nm). FIG. 2C ) and ssEVs formed after incubation at 37 °C, pH 7.4 for 5 min (A), 30 min (B) and 4 h (C) and pH 5 for 30 min (D) and 4 h (E) (scale bar, 100 nm). FIG. 2D ) and ssEVs formed after incubation at 37 °C, pH 7.4 for 5 min (A), 30 min (B) and 4 h (C) and pH 5 for 30 min (D) and 4 h (E) (scale bar, 100 nm). FIG. 2E ) and ssEVs formed after incubation at 37 °C, pH 7.4 for 5 min (A), 30 min (B) and 4 h (C) and pH 5 for 30 min (D) and 4 h (E) (scale bar, 100 nm). FIG. 2F ) and ssEVs formed after incubation at 37 °C, pH 7.4 for 5 min (A), 30 min (B) and 4 h (C) and pH 5 for 30 min (D) and 4 h (E) (scale bar, 100 nm). FIG. 2G ) and ssEVs formed after incubation at 37 °C, pH 7.4 for 5 min (A), 30 min (B) and 4 h (C) and pH 5 for 30 min (D) and 4 h (E) (scale bar, 100 nm). FIG. 2G ) and ssEVs formed after incubation at 37 °C, pH 7.4 for 5 min (A), 30 min (B) and 4 h (C) and pH 5 for 30 min (D) and 4 h (E) (scale bar, 100 nm). FIG. 2H ) and ssEVs formed after incubation at 37 °C, pH 7.4 for 5 min (A), 30 min (B) and 4 h (C) and pH 5 for 30 min (D) and 4 h (E) (scale bar, 100 nm). FIG. 2I ) and ssEVs formed after incubation at 37 °C, pH 7.4 for 5 min (A), 30 min (B) and 4 h (C) and pH 5 for 30 min (D) and 4 h (E) (scale bar, 100 nm). FIG. 2J ) and ssEVs formed after incubation at 37 °C, pH 7.4 for 5 min (A), 30 min (B) and 4 h (C) and pH 5 for 30 min (D) and 4 h (E) (scale bar, 100 nm). FIG. 2K ) and ssEVs formed after incubation at 37 °C, pH 7.4 for 5 min (A), 30 min (B) and 4 h (C) and pH 5 for 30 min (D) and 4 h (E) (scale bar, 100 nm). 50 ) and ssEVs formed after incubation at 37 °C, pH 7.4 for 5 min (A), 30 min (B) and 4 h (C) and pH 5 for 30 min (D) and 4 h (E) (scale bar, 100 nm). FIG. 2L ) and ssEVs formed after incubation at 37 °C, pH 7.4 for 5 min (A), 30 min (B) and 4 h (C) and pH 5 for 30 min (D) and 4 h (E) (scale bar, 100 nm). FIG. 2L ) and ssEVs formed after incubation at 37 °C, pH 7.4 for 5 min (A), 30 min (B) and 4 h (C) and pH 5 for 30 min (D) and 4 h (E) (scale bar, 100 nm). FIG. 2M ) and ssEVs formed after incubation at 37 °C, pH 7.4 for 5 min (A), 30 min (B) and 4 h (C) and pH 5 for 30 min (D) and 4 h (E) (scale bar, 100 nm). FIG. 2N ) and ssEVs formed after incubation at 37 °C, pH 7.4 for 5 min (A), 30 min (B) and 4 h (C) and pH 5 for 30 min (D) and 4 h (E) (scale bar, 100 nm).

[0054] FIGS. 3A-3G :( FIG. 3A The hydrodynamic diameters (d) of LNVs and mRNA-loaded LNVs, as determined by DLS, under pH 7.4, N / P 0 (unloaded LNVs) and 25 and 60 mol% MC3 conditions. h )and( FIG. 3B Polydispersion index (PDI). FIG. 3C The zeta potential (ζ-pot) of LNVs and mRNA-loaded LNVs under pH 5 and 7.4, N / P 0 (unloaded LNVs), and 25 and 60 mol% MC3 conditions. FIG. 3D The loading efficiency (LE%) of mRNA in LNVs was analyzed by RiboGreen assay (N / P 25, 60 mol% MC3). FIG. 3E -F) shows cryo-transmission electron microscopy images of mRNA-loaded LNVs ( FIG. 3E ), and the ability of mRNA-loaded LNVs to fuse with EVs to form ssEVs under the conditions of pH 7.4, 37°C, incubation time of 5 min, N / P 25 and 60 mol % MC3 ( FIG. 3F ). FIG. 3G The diagram shows bivariate dot plots of CFSE-labeled EVs alone, Cy5-mRNA-loaded LNVs alone, CFSE-labeled EVs with naked Cy5-mRNA, and CFSE-labeled EVs with Cy5-mRNA-loaded LNVs at pH 7.4. A double-positive population (double-positive population in the upper right box) was observed when CFSE-labeled EVs were mixed with Cy5-mRNA-loaded LNVs, confirming the formation of ssEVs. CFSE: carboxyfluorescein succinimide. Mean ± standard deviation (n=3). All experiments were performed in triplicate (n=3).

[0055] FIGS. 4A-4B HeLa cells were incubated with LNV and siRNA-loaded LNV for 4 hours using MTS assay (a colorimetric method for sensitive quantitative analysis of live cells). FIG. 4A ) and 24 hours ( FIG. 4BCell viability after treatment (60 mol% MC3; N / P 25), lipid concentration ranged from 12.5 to 200 μg / mL. Mean ± standard deviation (n=3). All samples were compared with the negative control (culture medium). Statistical significance was assessed by one-way ANOVA and Tukey's post-hoc test (*P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001). All experiments were performed in triplicate (n=3).

[0056] FIGS. 5A-5B Two EV-specific membrane proteins, CD73, isolated from the bone marrow mesenchymal stem cell (MSC) line HS-5, were identified. FIG. 5A ) and CD26 ( FIG. 5B Enzyme activities of CD73 and CD26 were measured by absorbance-based and luminescence-based assays, respectively, after ssEV formation (incubation in HEPES-buffered saline (HEBS) for 30 min, 4°C and 37°C, pH 7.4) or after heterozygote formation (in CD73 assay only, incubation in HEPES-buffered saline (HEBS) for 30 min, 37°C, pH 5), and compared with results obtained using Exo-Fect™ (incubation in HEBS buffer, pH 7.4, 37°C for 10 min). Adenosine 5'-(α,β-methylene)bisphosphate (APCP, 40 μM) was used in CD73 assay, and linagliptin (1 μM) was used as an inhibitor control in CD26 assay. Results were normalized to enzyme activities of EVs subjected to the same fusion conditions without LNV addition. Mean ± standard deviation (n=4–6). Statistical significance was assessed by unpaired two-tailed Student t-test (Welch corrected) (***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05) (n=4–6). All experiments were performed in at least four replicates (n=4–6).

[0057] FIG. 6Comparison of siRNA transfection efficiency in HeLa cells expressing green fluorescent protein. LNVs loaded with GFP-siRNA (N / P 25, 60 mol% MC3) (LNVs (w siGFP)) and LNVs loaded with scrambled control siRNA (LNVs (w siCTRL)) (60 mol% MC3; N / P 25) were compared to ssEVs loaded with GFP-siRNA (ssEVs (w siGFP)), ssEVs loaded with control siRNA (ssEVs (w siCTRL)), Lipofectamine™ RNAiMAX loaded with GFP-siRNA (RNAiMAX (w siGFP)), PBS, and naked GFP-siRNA (naked siGFP). Transfection efficiency was assessed by measuring the fluorescent signal by flow cytometry after 48 h incubation in 10% serum. 5 pmol of GFP-siRNA in each formulation was added per well (corresponding to a concentration of 10 nM). Mean ± s.d. (n = 3). Statistical significance was assessed by one-way ANOVA, Tukey’s post-hoc test (****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05) (n = 3). All experiments were performed in triplicate (n = 3). Please confirm or delete.

[0058] FIG. 7 Comparison of firefly luciferase (FLuc)-mRNA transfection efficiency in HeLa cells. LNVs loaded with FLuc-mRNA (LNVs) (N / P 25 with 60 mol% MC3) and ssEVs loaded with FLuc-mRNA (ssEVs) were compared to PBS, naked FLuc-mRNA, and Lipofectamine™ MessengerMAX™ (MessengerMAX™) loaded with FLuc-mRNA (w FLuc-mRNA). Transfection efficiency was analyzed by measuring the expression of luciferase in cell lysates after 24 h incubation in ~90% serum. HeLa cells were treated with 4 different mRNA doses (100 ng, 10 ng, 1 ng, and 0.1 ng per well). Mean ± s.d. Statistical significance was assessed by unpaired two-tailed Student’s t-test (Welch’s correction) (**P < 0.01, *P < 0.05). All experiments were performed in triplicate (n = 3).

[0059] FIG. 8Representative cryo-transmission electron microscopy images of particles prepared using a microfluidic device, which are composed of phospholipids with a CPP > 1 and nonionic surfactants with a CPP < 1 (DOPE and KLP, molar ratio 48:52, respectively), but do not contain ionizable cationic lipids.

[0060] FIGS. 9A-9H :( FIG. 9A The particle size (d) of particles prepared using microfluidic devices and siRNA-loaded particles h )and( FIG. 9B PDI, the particles of which are prepared from ionizable cationic lipids, phospholipids with a CPP value of ~1, and nonionic surfactants with a CPP value <1 (MC3:DOPC:KLP, molar ratio 60:18:22, N / P 0 (unloaded LNV) and 25). FIG. 9C and 9E )DOPC-NPs, ( FIG. 9D and 9F Representative cryo-transmission electron microscopy images of siRNA-loaded DOPC-NPs (60% DOPC, N / P 25) at pH 5 and pH 7.4, respectively, and images of them mixed with EV at pH 7.4. FIG. 9G ). FIG. 9H DOPC-NPs were analyzed by small-angle X-ray scattering at pH 5 and 7.4, with or without siRNA (N / P 25). All experiments were performed in triplicate (n=3).

[0061] FIG. 10A -10I: ( FIG. 10A The particle size (d) of unloaded nanoparticles and siRNA-loaded nanoparticles prepared using microfluidic devices h )and( FIG. 10B PDI particles were prepared at pH 5 and 7.4 from phospholipids with a CPP > 1 and nonionic surfactants with a CPP < 1, but using fixed cationic lipids instead of ionizable cationic lipids (fixed cationic lipid (DOTMA): DOPE: KLP, molar ratio 60:18:22, with N / P 0 and 25). FIG. 10C and E)DOTMA-NPs, ( FIG. 10D and 10F Representative cryo-transmission electron microscopy images of siRNA-loaded DOTMA-NPs (60% DOTMA, N / P 25) at pH 5 and pH 7.4, respectively, and images of them mixed with EV at pH 7.4. FIG. 10G ). FIG. 10H) Small angle X-ray scattering analysis of DOTMA-NPs at pH 5 and 7.4 without or with siRNA (N / P 25). All experiments were performed in triplicate (n=3).

[0062] FIG. 11 : Particle size distribution of nanoparticles prepared using a microfluidic device, prepared from ionizable cationic lipid, phospholipid with CPP > 1, and a lysophospholipid with CPP < 1 (MC3, DOPE, and 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (MPPC), molar ratio 60:18:22) in place of a non-ionic surfactant with CPP value < 1. All experiments were performed in triplicate (n=3).

[0063] FIG. 12 : Effect of non-ionic surfactant concentration showing CPP value < 1 on the nanoparticles formed. (1) Nanoparticles formed with 5 mol% surfactant (MC3:DOPE:HS15 molar ratio 60:35:5; N / P = 25); (2) 10 mol% surfactant (MC3:DOPE:HS15 molar ratio 60:30:10; N / P = 25); and (3) 15 mol% surfactant (MC3:DOPE:HS15 molar ratio 60:25:15; N / P = 25). All experiments were performed in duplicate (n=2). DETAILED DESCRIPTION

[0064] DEFINITIONS

[0065] LNV component

[0066] The present disclosure provides pH-induced structural transition non-lamellar LNVs. The LNVs of the present disclosure have pH-induced structural transition properties, i.e., they are capable of transitioning from a hexagonal phase at acidic pH (e.g., below about 6) to a sponge phase at neutral pH (e.g., about 6.5 or higher), and vice versa. The LNVs of the present disclosure comprise at least one ionizable cationic lipid, at least one phospholipid with CPP > 1, and at least one non-ionic surfactant with CPP value < 1, which components impart the LNVs with a non-lamellar structure and their pH-induced structural transition properties. The sponge-based structure of the LNVs enables them to successfully fuse with EVs, forming ssEVs that retain biological activity. The LNVs of the present disclosure are not vesicles.

[0067] The average particle size (dh) of the LNVs is between about 20 nm and about 200 nm.

[0068] Ionizable cationic lipid

[0069] The function of the ionizable cationic lipid (i.e., a lipid whose charge varies with pH) in the LNV of the present disclosure is: 1) to complex with a negatively charged drug (e.g., a nucleic acid such as RNA) at acidic pH; 2) to co-create a pH-induced structural transition non-lamellar LNV with a phospholipid having a CPP > 1 (e.g., dioleoyl phosphatidyl ethanolamine) and a non-ionic surfactant having a CPP value < 1 (e.g., KLP); and 3) to support the fusogenic tine process with EVs. In particular embodiments, the ionizable cationic lipid of the present disclosure (prior to incorporation into the LNV) has an apparent acid dissociation constant (pKa) between 5 and 7.5 (or between 5.3 and 7.4; 5.4 and 7.4; 5.5 and 7.4; or 5.6 and 7.4, or 5.7 and 7.4, or 5.8 and 7.4, or 5.9 and 7.4, or 6 and 7.4; or between 5.3 and 7.3; 5.4 and 7.3; 5.5 and 7.3; or 5.6 and 7.3, or 5.3 and 7.3, or 5.8 and 7.3, or 5.9 and 7.3, or 6 and 7.3; or between 5.3 and 7.2; 5.4 and 7.2; 5.5 and 7.2; or 5.6 and 7.2, or 5.3 and 7.2, or 5.8 and 7.2, or 5.9 and 7.2, or 6 and 7.2; or between 5.3 and 7.1; 5.4 and 7.1; 5.5 and 7.1; or 5.6 and 7.1, or 5.3 and 7.1, or 5.8 and 7.1, or 5.9 and 7.1, or 6 and 7.1; or between 5.3 and 7; 5.4 and 7; 5.5 and 7; or 5.6 and 7, or 5.3 and 7, or 5.8 and 7, or 5.9 and 7, or 6 and 7); and / or is a dimethylamino ionizable cationic lipid derivative; and / or is a conical ionizable cationic lipid. In particular embodiments, the ionizable lipid of the present disclosure is a (dimethylamino)butanoate ionizable cationic lipid derivative.Without being limited thereto, but in particular embodiments, the ionizable cationic lipid of the present disclosure is 4-(dimethylamino)-butyric acid, (10Z,13Z)-1-(9Z,12Z)-9,12- octadecadien-1-yl-10,13-nonadecadien-1-yl ester (also known as DLin-MC3-DMA, pKa = 6.44), N,N-dimethyl-2-(2-((10Z,13Z)-octadeca-10,13-dien-1-yl)-2-((9Z,12Z)-octadeca-9,12- dien-1-yl)-1,3-dioxolan-4-yl)ethan-1-amine (also known as DLin-KC2-DMA, pKa 6.68), 1,2-dilinoleyl-oxy-N,N-dimethyl-3-aminopropane (also known as DLin-DMA, pKa = 6.8), 3-(dimethylamino)propane-1,2-diyl (9Z,9'Z,12Z,12'Z)-bis(octadeca-9,12-dienoate) (also known as DLin-DAP, pKa = 6.2), N,N-dimethyl-1-(2-((10Z,13Z)-octadeca-10,13-dien-1-yl)-2-((9Z,12Z)- octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)methanamine (also known as D-Lin-K- DMA, pKa = 5.9), N,N-dimethyl-3-(2-((10Z,13Z)-octadeca-10,13-dien-1-yl)-2-((9Z,12Z)- octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)propan-1-amine (also known as DLin- KC3-DMA, pKa = 7.2), N,N-dimethyl-4-(2-((10Z,13Z)-octadeca-10,13-dien-1-yl)-2-((9Z,12Z)- octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)butan-1-amine (also known as DLin- KC4-DMA, pKa = 7.3), SM-102 (pKa = 6.68), ALC-0315 (pKa = 6.09) (see, e.g., US2017151333A1), L319 (pKa = 6.38), and YSK13-C3 (pKa = 6.45).

[0070] The pKa value of the ionizable cationic lipid can be measured by commercial software, e.g., software provided by ACD / Labs.

[0071] Phospholipid with a CPP > 1

[0072] The function of the CPP > 1 phospholipid (e.g. DOPE) in the LNV of the present invention is to promote the formation of a non-lamellar structure in the LNV and to enhance the stability and delivery efficiency of the nanoparticle. The phospholipids of the present disclosure are those with a critical packing parameter value > 1 (i.e. an intrinsic curvature < 0, lipids that are inverted cone shaped) that generally promote the formation of a non-lamellar phase (i.e. a pH dependent hexagonal phase (HII) and / or a sponge (L3) phase) in the LNV and in turn promote the formation of a fusion stalk with EVs. Without being limited thereto, but in accordance with the present disclosure, the following CPP > 1 phospholipids can be used: a CPP > 1 phosphatidylethanolamine (PE) class lipid, a CPP > 1 monogalactosyldiacylglycerol (MGDG) class lipid, a CPP > 1 monoglucosyldiacylglycerol (MGIcDG) class lipid, a CPP > 1 diphosphatidylglycerol (DPG) class lipid, a CPP > 1 phosphatidylserine (PS) class and a phosphatidic acid (PA) class lipid.

[0073] In a particular embodiment, the at least one CPP > 1 phospholipid used in the LNV of the present disclosure is a CPP > 1 phosphatidylethanolamine (PE) class lipid, which in a more particular embodiment is DOPE.

[0074] Non-ionic surfactant

[0075] The function of the non-ionic surfactant with a CPP value < 1 (e.g. KLP) in the LNV of the present invention is to stabilize / promote the formation of a non-lamellar structure in the LNV. In a particular embodiment, the non-ionic surfactant acts as a membrane permeation enhancer. Non-ionic surfactants with an intrinsic curvature > 0 and a CPP value < 1 can be used in the LNV of the present disclosure. Without being limited thereto, but in accordance with the present disclosure, the following surfactants can be used: fatty acid polyethylene glycol esters (e.g. polyethylene glycol 12-hydroxystearate, such as a mixture of polyethylene glycol monoesters and 12-hydroxystearic acid diesters, e.g. Kolliphor® HS15 (KLP)), polysorbates (e.g. Tween ® 20 and 80), polyethylene glycol fatty alcohol ethers (those commonly known as Brij 35 and 58), polyethylene glycol fatty acid esters (e.g. PEG stearate) and sorbitan esters (e.g. Span 60).

[0076] In a particular embodiment, a fatty acid polyethylene glycol ester, e.g. the non-ionic surfactant KLP, is used.

[0077] Spontaneous lipid curvature (CPP value)

[0078] Lipid geometry can be expressed in terms of CPP. CPP is related to the hydrocarbon chain volume (v) and chain length (l) of the lipid and the interfacial area (a) occupied by the polar head group. Specifically, CPP is calculated by the following equation:

[0079]

[0080] The volume difference between the head group and tail segment can be considered as a measure of the spontaneous curvature of the lipid. The spontaneous curvature of a monolayer is determined by the way the lipids pack in the membrane, i.e. the interactions of the lipids with their surrounding molecules. Thus, the spontaneous curvature of a monolayer can be slightly different in a bilayer membrane and at an oil-water interface, as the way the oil chains penetrate into the hydrophobic part of the monolayer can be different from the way the penetration occurs in the case of a bilayer. Lipids with a large head group and a small tail segment (prolate, positive curvature, CPP<1) tend to self-assemble into convex structures, such as micelles; while lipids with a head group and tail segment of comparable volume (cylindrical lipids, CPP ~ 1) form planar bilayers or large vesicles; lipids with a small head group and a large tail segment (oblate, negative curvature, CPP>1) give rise to inverted aggregates, such as the inverse hexagonal phase. Different methods, i.e. X-ray diffraction and osmotic pressure, can be used to measure the spontaneous curvature of lipids and their membranes. These methods and techniques are disclosed in, for example, Sodt et al., 2013; Barragan Vidal et al., 2017; Rand et al., 1990; and Hamai et al., 2006.

[0081] LNV component ratio

[0082] The LNVs of the present disclosure comprise the following molar concentrations: the at least one ionizable cationic lipid: about 30% to about 70% (e.g., about 35% to about 70%, about 35% to about 65%, about 35% to about 60%, about 40% to about 60%, about 40% to about 65%, about 40% to about 70%); the at least one CPP > 1 phospholipid: about 10% to about 50% (e.g., about 10% to about 45%, about 10% to about 40%, about 10% to about 38%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%; about 15% to about 45%, about 15% to about 40%, about 15% to about 38%, about 15% to about 35%, about 15% to about 30%, about 15% to about 25%, about 15% to about 20%; about 18% to about 45%, about 18% to about 40%, about 18% to about 38%, about 18% to about 35%, about 18% to about 30%, about 18% to about 25%, about 18% to about 20%); the at least one CPP value < 1 non-ionic surfactant: about 20% to about 50% (e.g., about 20% to about 49%; about 20% to about 48%; about 20% to about 47%; about 20% to about 46%; about 20% to about 45%; about 20% to about 44%; preferably about 20% to about 43%; about 20% to about 42%; about 21% to about 50%; about 21% to about 49%; about 21% to about 48%; about 21% to about 47%; about 21% to about 46%; about 21% to about 45%; about 21% to about 44%; preferably about 21% to about 43%; about 21% to about 42%; about 22% to about 50%; about 22% to about 49%; about 22% to about 48%; about 22% to about 47%; about 22% to about 46%; about 22% to about 45%; about 22% to about 44%; about 22% to about 43%; about 22% to about 42%; about 20% to about 35%; about 20% to about 30%; about 20% to about 25%; about 21% to about 35%; about 21% to about 30%; about 21% to about 25%; about 22% to about 35%; about 22% to about 30%; about 22% to about 25%). The above molar concentrations are selected to achieve useful drug or diagnostic agent (e.g., nucleic acid) loading efficiency in the LNV. When the LNV of the present disclosure consists only of the at least one ionizable cationic lipid, the at least one CPP > 1 phospholipid, and the at least one CPP value < 1 non-ionic surfactant, the sum of the molar concentrations of each of these components is 100%.

[0083] Drug

[0084] As used herein, the term "drug" refers to any molecule or mixture of molecules that is pharmacologically active (including unapproved active pharmaceutical ingredients). As used herein, a mixture of molecules is pharmacologically active. Not limited thereto, biological extracts (e.g., plant or animal extracts) are included in the term "molecular mixture" and therefore also in the term "drug." Drugs that can be incorporated into the carriers of this disclosure can be low molecular weight active pharmaceutical ingredients (< 1000 Da) or large molecules (> 1000 Da), such as polysaccharides, peptides, proteins, nucleic acids (e.g., RNA (such as siRNA, mRNA, etc.), DNA), targeted proteolytic chimeras (PROTACs), antibody-drug conjugates, and vaccine antigens.

[0085] Diagnostic agent

[0086] As used herein, "diagnostic agent" refers to any pharmaceutical product used as a component of a diagnostic test, such as radiopharmaceuticals, contrast agents for imaging techniques (including X-rays, magnetic resonance imaging, and ultrasound), compounds for diagnostic tests that do not involve radioisotopes or imaging techniques, various stains / labels (i.e., stains for detecting malignant cells), antibodies (i.e., monoclonal antibodies), and nucleic acid-based probes. In specific embodiments, a diagnostic agent is a negatively charged molecule (e.g., a nucleic acid-containing diagnostic agent) or a hydrophobic molecule.

[0087] Concentration of drug and diagnostic agent

[0088] The LNV disclosed herein can be used to encapsulate drugs or diagnostic reagents. When the drug or diagnostic reagent is a nucleic acid, the ratio of ionizable cationic lipid to nucleic acid drug or diagnostic reagent (i.e., N / P ratio) in the nucleic acid-loaded LNV of this disclosure is ≥ about 1 (e.g., ≥ 2, ≥ 3, ≥ 4, ≥ 5, between 6 and 300, or between 6 and 200, or between 6 and 150, or between 6 and 100, or between 6 and 50).

[0089] Formulation of drug or diagnostic

[0090] LNVs are prepared by mixing an aqueous phase (e.g., brine, pure water) and an organic medium (e.g., ethanol) at an acidic pH. Hydrophilic drugs are dissolved in the aqueous phase before being encapsulated into the LNV, while hydrophobic drugs are dissolved in the organic phase before being loaded into the LNV.

[0091] Subject

[0092] As used herein, the term "subject" refers to any animal, such as a mammal, including humans, dogs, cats, pigs, cows, monkeys, cattle, horses, etc. In one particular implementation, it refers to a human, and in a specific implementation, it refers to an adult subject.

[0093] Method of making drug or diagnostic loaded LNVs

[0094] The LNV of the present disclosure is prepared by mixing (i) an organic phase comprising at least one ionizable cationic lipid, at least one phospholipid with a CPP > 1, at least one non-ionic surfactant with a CPP value < 1, dissolved in a solvent; and (ii) an aqueous phase. During mixing, the volume flow ratio of the aqueous phase to the organic solvent is between 0.5:1 and 5:1. In a more particular embodiment, the ratio is 1.5:1. When preparing LNVs loaded with a hydrophobic drug (or diagnostic agent), the hydrophobic drug (or diagnostic agent) is added to the organic phase; when preparing LNVs loaded with a hydrophilic drug (or diagnostic agent) such as a nucleic acid, the hydrophilic drug (or diagnostic agent) is added to the aqueous phase.

[0095] The mixing method used to prepare the LNVs of the present disclosure includes any microfluidic device. Without being limited thereto, it includes any microfluidic chip design, including micro-mixers, hydrodynamically focused chips, T-junction chips, chaotic micro-mixers, split-and-recombine mixers and baffle mixers. In a particular embodiment, a chip formed from a Y-shaped inlet design is used. In a particular embodiment, the mixing device used is a microfluidic device using high-precision syringe pumps. In a particular embodiment, the microfluidic device has a total flow rate of between 0.001 mL / min and 100 mL / min, preferably a flow rate of about 0.05 mL / min; and / or a volume flow ratio of the microfluidic device of between 0.5:1 and 5:1; and / or a microchannel depth of the microfluidic device of between 1 pm and 500 pm, preferably about 100 pm, a channel inlet width of between 1 pm and 500 pm, preferably about 100 / 200 pm, a channel mixer width of between 1 pm and 500 pm, preferably about 200 pm, and a channel outlet width of between 1 pm and 500 pm, preferably about 200 pm.

[0096] The mixing method also includes ethanol injection, thin film evaporation, reverse phase evaporation, high pressure homogenization (HPH), high shear homogenization, sonication, microemulsion formation, and any other method that can produce lipid-based particles.

[0097] The non-lamellar LNVs thus prepared, having a pH-induced structural transition property, have a particle size of between 20 and 200 nm, in a particular embodiment of about 100 nm, and form a hexagonal phase structure (HII) at acidic pH and a sponge (L3) phase structure at neutral pH. FIG. 1A , 1G-1H, 2A)

[0098] Organic phase solvent

[0099] The organic solvent used in the present disclosure is any biocompatible solvent in which the ionizable cationic lipid and the phospholipid having a CPP > 1 are soluble. Without being limited thereto, it includes alcohols (e.g., ethanol), oils (e.g., fluorinated oils), and chloroform. In a particular embodiment, the organic phase solvent is ethanol. The amount of organic solvent used is sufficient to solubilize the lipids. In a particular embodiment, the concentration of lipids in the solvent is between 10 micrograms of lipids per milliliter of solvent and 1 gram of lipids per milliliter of solvent. In a particular embodiment, the concentration of lipids in the solvent is between 1 milligram of lipids per milliliter of solvent and 20 milligrams of lipids per milliliter of solvent, or between 5 milligrams of lipids per milliliter of solvent and 15 milligrams of lipids per milliliter of solvent, or between 10 milligrams of lipids per milliliter of solvent and 15 milligrams of lipids per milliliter of solvent.

[0100] Aqueous medium

[0101] The aqueous medium is used for the production of the LNV (the aqueous phase) as well as the hybridization step of the LNV with the EV. The aqueous medium used in the present disclosure is any aqueous medium, including pure water or saline. The aqueous medium can have different compositions, pH, and osmolarity.

[0102] When the drug or diagnostic agent to be loaded into the LNV is negatively charged (e.g., a nucleic acid such as RNA), the pH of the aqueous medium used for the production of the LNV (e.g., nucleic acid-loaded LNV) can range from about 4 to about 6, e.g., 4, 4.5, 5, 5.5, 6, preferably between about 4 and 6. In a particular embodiment, a sodium acetate buffer (pH 4) is used. The pH can be adjusted to reach the desired pH value by adding buffer components (e.g., acetate buffer, sodium acetate, citrate buffer, sodium citrate, or citrate phosphate buffer) and / or acids or bases (e.g., hydrochloric acid and / or sodium hydroxide). When the drug or diagnostic agent to be loaded into the LNV is neutral, the pH of the aqueous medium can also be higher than 6 and up to 10. After the LNV is formed, the aqueous solution can be neutralized to pH 7.4 with, e.g., a phosphate buffer (e.g., PBS) or HEPES-buffered saline (HEBS) or Tris buffer.

[0103] The aqueous solution containing the LNV can be formulated for storage for future use by increasing the pH to about 7 or higher, up to about 10, or adjusted for the hybridization step with the EV. If the aqueous solution containing the LNV has been neutralized (e.g., with PBS), a more precise adjustment can be made with an acid or base (e.g., hydrochloric acid and / or sodium hydroxide).

[0104] The aqueous medium used for the hybridization step can further comprise an osmotic agent to achieve an isotonic solution with the EV. The osmolarity of the EV is typically 200-500 mOsm / kg. Osmotic agents can be used, such as sodium chloride, dextrose, gelatin, xylitol, sorbitol, mannitol, glucose polymers (e.g., icodextrin), or amino acids.

[0105] The aqueous medium used in the hybridization step has a pH higher than 6 and up to about 10, higher than 7, higher than 7.1, higher than 7.2, higher than 7.3, or between about 7.4 and about 10.

[0106] EVs

[0107] The general term “EVs” describes herein both natural EVs as well as synthetic or bioengineered EVs prepared to mimic natural EVs (see e.g. exoIL-12™, exoSTING™, exoASO™-STAT6, AGLE-102™, ExoAAV, OmniSome, ILB-202). “Natural EVs” can be defined as a heterogeneous population of membrane vesicular structures derived from any eukaryotic or prokaryotic cell or tissue. They can be classified according to their properties (such as size, density and composition) or biogenetic pathways, for example, small EVs (≤ 200 nm) from mammalian cells are mainly derived from endosomes, commonly known as exosomes. Medium or large EVs (> 200 nm) are usually formed by budding through the plasma membrane, known as exosomes / microvesicles, or by shedding through apoptotic bodies upon cell death (Théry et al., 2018).

[0108] The term EVs also refers to a portion of EVs as described above and / or to extruded / homogenized forms of natural EVs, as well as to extruded forms of whole cells. Extrusion / homogenization is performed by any known method, for example, extrusion through nano / micrometric filter pores using a high pressure extruder or a centrifugation apparatus, as well as mechanical homogenization / breaking of cells / EVs by means of sonication, nitrogen cavitation, freeze-thaw cycles or Dounce homogenizer.

[0109] The ssEVs of the present disclosure retain at least a portion of the EV surface / membrane protein functionality, as well as the biological activities inherent to EVs (e.g. renotic activity, anti-inflammatory activity, angiogenic, cardioprotective and homing / targeting properties, i.e. the ability to recognize specific tissues / cells).

[0110] Method of isolating EVs

[0111] EVs can be isolated from biological samples using known methods, such as centrifuge-based methods (e.g., ultracentrifugation and density gradient centrifugation), affinity-based methods (e.g., protein-based affinity capture (e.g., antibody capture or MagCapture™ kit) or membrane-based affinity capture (e.g., ExoEasy™ kit)), precipitation-based methods (e.g., ExoQuick™ kit), microfluidic-based methods, filtration-based methods (e.g., tangential flow filtration and ultrafiltration), and size exclusion chromatography as described in Wu et al., 2023. In a particular embodiment, EVs are isolated using a method combining differential centrifugation, microfiltration, and ultracentrifugation.

[0112] The term “biological sample” (from which EVs for the present disclosure can be isolated) as used herein includes any cells and tissues derived from eukaryotic and prokaryotic organisms. It includes cultured animal (mammalian, such as human) cell lines or primary cells belonging to any tissue / organ (e.g., lung, brain, pancreas, tumor / cancer, liver, bone marrow, blood, etc.); any human / mammalian biological fluid (e.g., blood, cerebrospinal fluid, serum, urine, (breast milk) milk, saliva, synovial fluid, bile, ascites fluid, amniotic fluid, semen, feces); any cultured bacterial strain (gram-positive and gram-negative); any EVs from fungi and plants.

[0113] Method of making semi-synthetic EV particles (ssEVs)

[0114] LNVs (e.g., drug-loaded such as nucleic acids, diagnostic agent-loaded) are mixed (e.g., incubated) with EVs in an aqueous medium (see above, e.g., PBS) at a temperature between 4 and 60 °C, at a pH value between greater than 6 and 10, e.g., 6.1 to 10, 6.2 to 10, 6.3 to 10, 6.4 to 10, 6.5 to 10, 6.6 to 10, 6.7 to 10, 6.8 to 10, 6.9 to 10, 7 to 10, 7.1 to 10, 7.2 to 10, 7.3 to 10, 7.4 to 10, 7.5 to 10, 6.1 to 9, 6.2 to 9, 6.3 to 9, 6.4 to 9, 6.5 to 9, 6.6 to 9, 6.7 to 9, 6.8 to 9, 6.9 to 9, 7 to 9, 7.1 to 9, 7.2 to 9, 7.3 to 9, 7.4 to 9, 7.5 to 9. The duration of mixing is 1 second to 48 hours (e.g., 5, 10, 15, 20, 30, 40, 50, 55 seconds; 1, 2, 3, 4, 5, 6, 10, 15, 20, 25, 30, 55 minutes; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 24, 30, 36, or 42 hours, and any range therebetween).

[0115] ssEVs

[0116] ssEVs are the result of fusion of LNVs with EVs (see, e.g. FIG. 2C - E, 2G-I, FIG. 3E and 3G ). In particular embodiments, they are prepared by the methods of the present disclosure. In other particular embodiments, they comprise essentially all components of the LNVs and EVs of the present disclosure (partially or completely mixed). The term "essentially all components" used in this context means that while a substantial portion (at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, 96%, 97%, 98%, or 99%) of the components of each of the LNVs and EVs are present in the ssEVs, they do not necessarily comprise all components (e.g., due to the method of preparation). The term "partially or completely mixed" used in this context is intended to encompass ssEVs present at all stages of fusion (see, e.g. FIG. 2C-2E and 3F).

[0117] The average particle size of ssEVs according to the present disclosure is between about 20 nm to 2000 nm (depending on, for example, EV source, extrusion, etc.). In particular embodiments, the average particle size is about 20 nm ~ 500 nm, 20 nm ~ 450 nm, 20 nm ~ 400 nm, 20 nm ~ 350 nm, 20 nm ~ 300 nm, 20 nm ~ 250 nm, about 50 nm ~ 250 nm, about 75 nm ~ 250 nm, about 100 nm ~ 250 nm, about 125 nm ~ 250 nm, about 130 nm ~ 250 nm, about 140 nm ~ 250 nm, about 150 nm ~ 250 nm, about 160 nm ~ 250 nm, about 20 nm ~ 225 nm, about 50 nm ~ 225 nm, about 75 nm ~ 225 nm, about 100 nm ~ 225 nm, about 125 nm ~ 225 nm, about 130 nm ~ 225 nm, about 140 nm ~ 225 nm, about 150 nm ~ 225 nm, about 160 nm ~ 225 nm, about 20 nm ~ 215 nm, about 50 nm ~ 225 nm, about 75 nm ~ 215 nm, about 100 nm ~ 215 nm, about 125 nm ~ 215 nm, about 130 nm ~ 215 nm, about 140 nm ~ 215 nm, about 150 nm ~ 215 nm, about 160 nm ~ 215 nm, about 20 nm ~ 200 nm, about 50 nm ~ 200 nm, about 75 nm ~ 200 nm, about 100 nm ~ 200 nm, about 125 nm ~ 200 nm, about 130 nm ~ 200 nm, about 140 nm ~ 200 nm, about 150 nm ~ 200 nm, about 160 nm ~ 200 nm.

[0118] The present method allows for the in situ production of ssEVs at the point of care (i.e., point of treatment) prior to administration to a subject in need thereof.

[0119] Composition / formulation

[0120] Pharmaceutically acceptable excipients include one or more of the following: cryoprotectants (e.g., sucrose, glucose, trehalose, or synthetic polymers such as poloxamers and polyvinylpyrrolidone, etc.), osmotic agents (e.g., glucose, lactulose, sorbitol, magnesium hydroxide, magnesium citrate, sodium chloride, potassium chloride, polyethylene glycol, etc.), salts (e.g., sodium phosphate, potassium phosphate, tris-hydroxymethyl aminomethane, sodium citrate, etc.).

[0121] LNV composition / formulation

[0122] LNVs can be mixed with one or more pharmaceutically acceptable excipients to form a composition (liquid or solid) and stored, for example, for future use. For example, they can be freeze-dried in the presence of (i) one or more cryoprotectants (e.g., sucrose, glucose, trehalose, or synthetic polymers such as poloxamer and PVP), (ii) one or more osmotic agents (e.g., glucose, PEG, lactulose, sorbitol, magnesium hydroxide, magnesium citrate, sodium chloride, and potassium chloride), and (iii) one or more salts (e.g., sodium phosphate, potassium phosphate, Tris, sodium citrate), then resuspended with an aqueous medium, or stored as an aqueous suspension in the temperature range of -80 to +8 °C.

[0123] In particular embodiments, at least one cholesterol and PEG lipid (polyethyleneglycolated) can be added to the LNVs (e.g., DSPE-PEG, molecular weight = 2 kDa; DMG-PEG, molecular weight = 2 kDa).

[0124] ssEV composition / formulation

[0125] ssEVs can be mixed with one or more pharmaceutically acceptable excipients to form a composition (liquid or solid) and stored, for example, for future use. For example, they can be freeze-dried in the presence of cryoprotectants (e.g., sucrose, glucose, trehalose, or synthetic polymers such as poloxamer and PVP) and salt compositions (e.g., sodium phosphate, potassium phosphate, Tris, sodium citrate, sodium chloride, and potassium chloride), then resuspended with an aqueous medium, or stored as an aqueous suspension in the temperature range of -80 to +8 °C.

[0126] They can also be embedded in a polymeric matrix (e.g., as a gel, implant, or device) or made into classic pharmaceutical preparations (e.g., ointments, capsules, pre-filled syringes, etc.).

[0127] In particular embodiments, cholesterol and / or PEG lipids (polyethyleneglycolated) can be added to the ssEVs, for example, to improve their stability (e.g., DSPE-PEG, molecular weight = 2 kDa; DMG-PEG, molecular weight = 2 kDa).

[0128] Alternatively, ssEVs can be generated in situ (i.e., at the site of treatment) prior to injection into a subject in need thereof.

[0129] Kit

[0130] The scope of this invention also includes kits comprising (a) at least one LNV of this disclosure or a composition (liquid or solid, such as LNV powder (e.g., lyophilized powder)) comprising at least one LNV and at least one pharmaceutically acceptable excipient; and (b) (i) a solution for hydrating said at least one LNV or a composition thereof (e.g., prior to its use), optionally comprising at least one pharmaceutically acceptable excipient; (ii) a pharmaceutical or diagnostic agent (e.g., for loading into an LNV or as a control); (iii) a composition (liquid or solid) comprising EVs and at least one pharmaceutically acceptable excipient; (iv) instructions for using said at least one LNV or a composition thereof (e.g., instructions for producing ssEVs from LNVs and / or instructions for administering ssEVs); or (v) a combination of at least two of (i) to (iv). In one specific embodiment, the kit comprises (a) at least one LNV (unloaded or loaded with a drug or diagnostic agent) in solid form (e.g., lyophilized) or a composition in solid form (e.g., lyophilized) containing said at least one LNV and at least one pharmaceutically acceptable excipient (e.g., at least one cryoprotectant); and (b) (i) a solution for hydrating said at least one LNV or composition, the solution containing at least one pharmaceutically acceptable excipient (e.g., at least one permeabilizer and salt to provide an isotonic environment for the LNVs). In another specific embodiment, the kit further comprises (b) (ii) a drug or diagnostic agent (e.g., for loading into the LNV or as a control).

[0131] The scope of this invention also includes kits comprising (a) at least one ssEV of this disclosure, or a composition (liquid or solid) comprising said at least one ssEV and at least one pharmaceutically acceptable excipient; and (b) (i) a solution for hydrating said at least one ssEV or a composition thereof (before its use); (ii) instructions for using these ingredients (e.g., instructions for administering at least one ssEV or a composition thereof); or (iii) a combination of (i) and (ii).

[0132] A kit will typically include a label indicating that the contents are intended for a particular use. The term "label" is used herein in the broadest sense to refer to any written, recorded, or printed matter accompanying a kit or supplied therewith, or to any other means of communication accompanying the kit or supplied therewith. The kit can further include one or more containers, reagents, administration devices. One example of such a kit includes (i) a first composition (liquid or solid) comprising LNVs, contained in a first container or in a first compartment of a first container; and (ii) a second composition (liquid or solid) comprising EVs and at least one pharmaceutically acceptable excipient, contained in a second container or in a second compartment of the first container. Prior to use, the contents of the first and second compartments can be mixed by, for example, breaking a wall separating the compartments, thereby mixing the first and second compositions. After the ssEVs are formed in the mixture, the resulting solution can be administered to a subject in need thereof.

[0133] Use

[0134] LNVs of the present disclosure (e.g., loaded with a drug or diagnostic agent) can be included in commercial kits for the production of ssEVs. Such ssEVs can be used for in vitro and in vivo studies, or for clinical (e.g., therapeutic or diagnostic) purposes.

[0135] Route of administration

[0136] The ssEVs of the present disclosure can be administered by parenteral, topical, or oral routes.

[0137] Subject

[0138] The term "subject" or "subject in need thereof" as used herein refers to a subject who would benefit from receiving an effective amount of the ssEVs of the present disclosure or a composition thereof. In particular embodiments, it refers to animals, mammals, and humans. The ssEVs and compositions of the present disclosure can also be used for veterinary applications, and for use in pets or other animals (e.g., cats, dogs, horses, etc.; as well as cows, fish, pigs, poultry, etc.).

[0139] In the description of the present disclosure, and particularly in the context of the claims below, the use of the terms "one" and "an" and "the" and similar referents is to be interpreted as including both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0140] The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise noted.

[0141] The description of ranges of values should be considered as a shorthand for describing each separate value falling within the range, unless the context clearly dictates otherwise. All subsets of values within a range are implicitly described, and each separate value is incorporated in the specification as if it were individually recited herein.

[0142] All methods described herein can be performed in any suitable order unless otherwise specified herein or otherwise clearly contradicted by context.

[0143] The term "about" has its ordinary meaning. In embodiments, it can mean ±10% of the defined value. Herein, the term "approximately" has its ordinary meaning. In embodiments, it can mean ±10% of the defined value.

[0144] The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the application and does not pose a limitation on the scope of the application unless otherwise claimed.

[0145] No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the application.

[0146] The following non-limiting examples further illustrate the disclosure.

[0147] Example 1 : Materials and Methods

[0148] Preparation of LNVs

[0149] Unloaded LNVs

[0150] LNVs and nanoparticles were prepared by mixing the organic phase with the aqueous phase. The organic phase contained the components listed in Table I below (molar concentrations are shown in Table I), dissolved in ethanol solvent; the aqueous phase contained acetate buffer at pH 4 (see FIG. 1A ). The nanoparticles used in Examples 10-13 were prepared as shown in FIG. 1A , except that the organic phase contained the components listed in the respective columns of Table I.

[0151] Table I: Components of LNVs and nanoparticles used in the examples herein

[0152]

[0153] Mixing was performed by introducing each phase separately into the microfluidic device using separate syringes (see FIG. 1A). A chip based on a Y-inlet design enabling diffusive mixing was used, with a channel depth of 100 pm, an inlet channel width of 100 / 200 pm, a mixer channel width of 200 pm, and an outlet channel width of 200 pm (Fluidic™ 186 micro mixer, purchased from Microfluidic ChipShop, Germany). During mixing, the volume flow ratio of the aqueous phase to the organic phase was set to 1.5:1, and a high-precision syringe pump was used with a total flow rate of 0.05 mL / min.

[0154] Subsequently, the prepared LNVs were purified from the organic solvent by dialysis using a Slide-A-Lyzer™ Mini Dialysis Device (MWCO = 20 kDa) against PBS at pH 7.4 or HEBS (20 mM HEPES, 150 mM NaCl) at pH 7.4, with a 1000-fold excess of dialysis liquid volume. This procedure resulted in LNVs at pH 7.4. Hydrochloric acid was added to the sample to prepare LNVs at pH 5. The pH of all preparations was carefully checked using a pH meter.

[0155] siRNA loaded LNVs

[0156] The preparation of siRNA-loaded LNVs was identical to the preparation of unloaded LNVs, except that siRNA was added after adjusting the pH of the aqueous phase.

[0157] The nucleotide sequence of the siRNA against green fluorescent protein (GFP-siRNA) was 5'-GAACUUCAGGGUCAGCUUGGG-3' (SEQ ID NO: 1) (3' overhang: sense strand (5'-3') dGdG; antisense strand (5'-3') dTdT); molecular weight = 13365.1 g / mol). The siRNA concentration used was 5.7, 11.4, 22.8, and 45.6 pM, respectively, to obtain N / P ratios of 50, 25, 12, and 6, respectively. The GFP-siRNA was custom synthesized by Microsynth AG (Switzerland).

[0158] The siRNA used for the nanoparticle flow cytometry experiments was FAM-labeled siRNA (MISSION® siRNA Fluorescent Universal Negative Control, 6-FAM, Sigma-Aldrich, USA); molecular weight = 13854 g / mol) with an N / P ratio of 25; or, GFP-siRNA-loaded LNVs were labeled by replacing 1 mol% of DOPE with Atto 488-labeled DOPE.

[0159] In the in vitro knockdown experiments, the LNVs were additionally loaded with a scrambled control siRNA sequence as a negative control (CTRL-siRNA, nucleotide sequence 5'-UAAGGCUAUGAAGAGAUACTT-3' (SEQ ID NO: 2), 3' overhang: sense strand (5'-3') dTdT; antisense strand (5'-3') dTdT; molecular weight = 13270.0) at an N / P ratio of 25. The control siRNA was custom synthesized by Microsynth AG (Switzerland).

[0160] mRNA loaded LNVs

[0161] The LNVs were prepared as described before, with the difference that the mRNA was added after adjusting the pH of the aqueous phase.

[0162] The mRNA was either Firefly luciferase mRNA (FLuc-mRNA, TriLink BioTechnologies, USA; molecular weight ~ 637715.0 g / mol) or Cy-5 labeled Firefly luciferase mRNA (Cy5-FLuc-mRNA, Cellerna Bioscience, Germany; molecular weight ~ 785980.0 g / mol) at a concentration of 0.25 µM and an N / P ratio of 25.

[0163] Nanoparticles without MC3

[0164] The nanoparticles were prepared using the same protocol as for the LNVs, as described before, with the difference that the organic phase contained DOPE and KLP at a molar concentration of 48% and 52%, respectively.

[0165] Gel electrophoresis mobility shift assay (EMSA)

[0166] For the EMSA experiments, 0.1 µg of GFP-siRNA loaded in LNVs or free GFP-siRNA were mixed with or without 1% (v / v) Triton™ X-100 and loaded onto a 0.5% (w / v) agarose gel. The gel was run in 0.5 x TAE buffer (Tris-base 20 mM, 10 mM acetic acid, 0.5 mM EDTA) at 70 V for 30 min, then stained with SYBR™ Gold nucleic acid gel stain (Thermo Fischer Scientific, USA) for 45 min and imaged using a ChemiDoc™ MP imaging system (Bio-Rad, USA). Similarly, EMSA experiments were performed after pre-incubation with 5 µg / mL RNase A for 1 h at 37°C (with / without Triton™ X-100) to assess the resistance of the siRNA cargo to nuclease-mediated degradation.

[0167] Cell culture

[0168] EVs were prepared from a bone marrow-derived mesenchymal stem cell (MSC) line, HS-5, cultured in a three-dimensional culture system on microcarriers. Specifically, 118 x 10 6 HS-5 cells were seeded on 4.5 g of Corning™ Enhanced Attachment microcarriers (Corning, USA) in a 500 mL Corning™ reusable glass stirred flask (Corning, USA) containing a total volume of 300 mL of serum-containing DEME medium. An intermittent stirring program (30 rpm for 5 minutes, off for 60 minutes) was used initially, followed by setting the stirrer paddle speed to continuous mode at 35 rpm. After 48 hours, the microcarriers were washed twice with PBS and 400 mL of fresh serum-free phenol red-free medium was added to the stirred flask. The cell culture supernatant was collected after 48 hours of incubation in serum-free medium.

[0169] Isolation of EVs from cell culture supernatant

[0170] EVs were isolated from the cell culture supernatant using a combination of centrifugation, microfiltration and ultracentrifugation. First, the supernatant was sequentially centrifuged at 500 x g, 2,000 x g for 5 minutes each, and 10,000 x g for 20 minutes using a Sorvall™ LYNX 6000 centrifuge (both Thermo Fisher Scientific, USA) equipped with a Fiberlite™ F12-6 x 500 LEX fixed-angle rotor. The clarified cell culture supernatant was then filtered through a 0.2-µm filter membrane, followed by ultracentrifugation (4°C, 100,000 x g, 70 minutes) using an Optima™ XE-90 ultracentrifuge (UC) (Beckman Coulter, USA) and a Type 45 Ti fixed-angle titanium rotor. After the first ultracentrifugation, the EV pellet was washed with PBS or HEBS and pooled. The EV pellet was re-centrifuged under the same conditions, resuspended in ice-cold PBS / HEBS and stored at -20°C.

[0171] Formation of nucleic acid (NA) loaded LNVs and EV hybrids - ssEVs

[0172] Different RNA-loaded LNVs (i.e. siRNA and mRNA described above) were mixed with MSC-derived EVs in aqueous media (PBS at pH 5, 6, 7.4 or 9 or HEBS) using different EV:LNV particle number ratios (4:1, 1:1 and 1:4), time points (5 min, 30 min, 1 h and 4 h), temperatures (4°C, 25°C and 37°C) and pH values (5, 6, 7.4 and 9). To achieve the specific pH values used, the pH of the LNVs was carefully adjusted, if necessary, by the addition of hydrochloric acid and / or sodium hydroxide and confirmed with a pH meter. More specifically, 62.5 mM solutions of hydrochloric acid or sodium hydroxide were used to lower or raise the pH, respectively. The pH of the samples was carefully checked each time using an InLab Micro™ pH electrode (Mettler Toledo, USA).

[0173] HeLa cell viability

[0174] CellTiter 96 ® Aqueous One Solution Cell Proliferation Assay (MTS) kit (Promega, Switzerland) was used to determine cell viability. Briefly, HeLa cells (2500 cells per well, total volume 200 µL complete DMEM medium) were seeded in 96-well plates and incubated at 37°C for 24 h. Then, 2 µL of LNVs or GFP-siRNA-loaded LNVs were added to the wells to achieve specific final lipid concentrations (ranging from 12.5 to 200 µg mL -1 , dilution factor 1:2) and incubated with the cells for 4 and 24 h. Negative control cells were added with 2 µL PBS. Positive control cells were added with 0.5% (v / v) Triton™ X-100. Subsequently, the medium was removed and the cells were gently washed with PBS and 100 µL complete DMEM medium was added. 20 µL MTS reagent was added and the HeLa cells were incubated at 37°C for 1 h. The absorbance was recorded at 490 nm using a Tecan Spark ® and Infinite™ M200 microplate reader (both Tecan, Switzerland). Results were processed using i-control™ 2.0 software. All formulations were tested in three independent batches (n=3).

[0175] Cryo transmission electron microscopy

[0176] Three microlitres of PBS suspension of ssEV, LNV or EV were added to glow-discharge treated (30 seconds treatment in an Emitech™ K100X glow-discharge system, Quorum Technologies Ltd., UK) 300-mesh lacey carbon supported film copper grids. Samples were flash-frozen in a mixture of liquid ethane and propane using a Vitrobot™ Mark II (Thermo Fisher Scientific, USA). Excess sample was removed by controlled suction filtration and the grid was then transferred to a Gatan™ cryo-sample holder and imaged using a Tecnai™ F20 cryo-EM (Field Electron and Ion Company, USA) in bright-field mode at an acceleration voltage of 200 kV, the sample being held at -180°C during observation. Micrographs were recorded using a Falcon II™ 4K direct electron detector (Field Electron and Ion Company, USA) under low-dose conditions (<500 electrons nm -2 -2).

[0177] Small angle X-ray scattering (SAXS)

[0178] Twenty microlitres of sample were carefully added to borosilicate glass capillaries. Capillary length was 80 mm with a wall thickness of 0.01 mm and were loaded using a hypodermic stainless steel needle. To ensure sealing, the upper part of each capillary was precision sealed using ultrahigh vacuum 2-K epoxy resin glue (1 part resin to 1 part hardener, v / v, UHU plus, Germany) in two successive steps, one hour each. Subsequently, the capillaries were inserted into a dedicated sample holder and then loaded into the instrument for further analysis. SAXS measurements were performed using an in-house SAXS system (Xeuss™ 3.0, Xenocs, Grenoble, France) equipped with a Genix™ 3D light source (Xenocs). The X-ray energy was 50 kV (l = 1.54 A, Cu K α ), with a photon flux > 10 -7 counts per second at the sample position. The beam size on the sample was approximately 0.5 x 0.5 mm2(FWHM). The sample-to-detector distance was calibrated using a silver behenate standard and the scattered signal was collected by a Dectris Eiger 1 M detector (1028 x 1062 pixels, pixel size 75 x 75 pm 2 , Dectris Ltd., Baden, Switzerland). The total signal recording time was > 8400 seconds. SAXS data are shown in the q range 0.016 to 0.69 A -1In-house, the corresponding actual length scale is 1.0466 to 39.25 nm. Detector data were masked, azimuthally integrated, normalized to absolute intensity (cm -1 ) and background subtracted using the XSACT (Xenocs) 2.6 software package.

[0179] The integrated intensity I(q) was plotted against the magnitude of the scattering vector q, which is calculated as:

[0180]

[0181] Dynamic and static light scattering (DLS / SLS) experiments

[0182] The hydrodynamic size (d h , in nm) and zeta potential (ζ-pot, in mV) of LNVs were measured by DLS at 25 °C, 179° scattering angle using a Malvern Zetasizer™ Advance Pro instrument (Malvern Panalytical, UK). DLS and SLS time course experiments were performed in a sealed 384-well microplate (Aurora Microplates, USA) using a DynaPro™ Plate Reader III (Wyatt Technology, USA) under temperature-controlled conditions (4 °C, 25 °C and 37 °C). The microplate was pre-incubated with 30 µL of LNV suspension (~8 x 10 8 particles / mL) for about 30 minutes until a stable particle concentration reading was achieved. Equal number of particles (except for the scaling experiments) of EVs were then added and the measurements (particle concentration and d h ) were continued for 1 hour.

[0183] Evaluation of CD73 and CD26 enzyme activity

[0184] Prior to measuring enzyme activity, EVs were either treated with LNVs to form ssEVs of the disclosure or transfected with the Exo-Fect™ exosome transfection kit (System Biosciences, USA). For measuring the activity of the EV membrane protein CD73, an absorbance-based malachite green assay was employed. Specifically, 0.1 pg EV protein from different test conditions (in HEBS, pH 7.4) were resuspended in 60 pL 10 mM Tris buffer (pH 7.4) and 30 pL of 800 pM AMP stock (dissolved in 10 mM Tris buffer) was added to a final concentration of 240 pM. The mixture was then incubated for 10 min at 25 °C and the enzymatic reaction was stopped by adding 40 pL of color reagent (0.034% (w / v) malachite green, 1.55% (w / v) ammonium molybdate tetrahydrate, 0.0625% (v / v) polysorbate 20) to the well. The plate was incubated for 1 h at 25 °C and the absorbance was measured at 620 nm using an Infinite™ M200 plate reader. The absorbance values after background subtraction were normalized to the values obtained for EVs subjected to the same hybridization conditions.

[0185] Hybridization was performed using the commercially available DPPIV-Glo™ Protease Assay Kit (Promega, Switzerland) and the CD26 enzyme activity after hybridization was assessed according to the manufacturer’s instructions. Briefly, 0.08 pg EV protein in 25 pL HEBS (pH 7.4) was mixed with an equal amount of DPPIV-Glo™ reagent and incubated for 30 min at 25 °C. Luminescence values were measured using an Infinite™ M200 Pro plate reader (Tecan, Switzerland) and normalized to the values obtained for EVs subjected to the same hybridization conditions.

[0186] Nanoparticle flow cytometry (Nano FCM) analysis

[0187] EVs were labelled with CellTrace™ Far Red (Thermo Fisher Scientific, USA) or 5(6)-Carboxyfluorescein diacetate N-succinimidyl ester (CFDA-SE) (Chemodex AG, Switzerland) according to the manufacturer’s instructions with slight modifications. Briefly, EVs were mixed with 5 mM of the respective dye and incubated overnight at 4°C. Subsequently, the mixture was incubated at 37°C for 15 min to allow for acetate hydrolysis of the dye. Afterwards, unbound dye was removed by dialysis against 1000-fold volume of PBS overnight at 4°C using Pur-A-Lyzer™ Mini 6000 Dialysis Kit (Sigma-Aldrich, USA). Fluorescently labelled LNVs (FAM-siRNA, Cy5-FLuc-mRNA and Atto 488 DOPE) and labelled EVs were then mixed at a 1 : 1 particle ratio for 30 min at 4°C or 37°C and the formation of ssEVs was assessed using the Flow NanoAnalyzer™ N30 (NanoFCM Co., Ltd, UK) instrument.

[0188] For control experiments using the Exo-Fect™ Exosome Transfection Kit, an equal amount of FAM-siRNA (~ 12 mM) was mixed with the Exo-Fect™ reagent according to the manufacturer’s instructions. Events were recorded for 1 min on three different channels 488 / 10 (side scatter), 525 / 40 and 670 / 30 (bandpass filters) and collected using a single photon counting avalanche photodiode detector (SPC MAPD).

[0189] For FAM-siRNA and Atto 488 DOPE experiments, the blue 488 nm laser was set to 10 / 50 mW and the red 638 nm laser to 40 / 100 mW with a SS attenuation of 0.2%. For Cy5-FLuc-mRNA experiments, the 488 nm laser was changed to 20 / 50 mW and the 638 nm laser to 20 / 100 mW. Most samples were diluted 1 :50 (v / v) with PBS prior to measurement, except for Exo-Fect™ transfected samples which were diluted 1 :80 (v / v) with PBS. Samples were processed using the NanoFCM™ Professional Suite software (version 1.8, NanoFCM Co., Ltd, UK) and data analysed using FlowJo™ v10 software (BD Biosciences, USA).

[0190] GFP knockdown experiments

[0191] For GFP knockdown experiments, 30,000 HeLa-GFP cells (Cell Biolabs, Inc., USA) were seeded per well in a 24-well plate. After incubation at 37 °C and 5% CO2for 24 h, cells were treated with LNVs or ssEVs loaded with GFP-siRNA (siGFP) or scrambled control siRNA (siCTRL). Lipofectamine™ RNAiMAX (Thermo Fisher Scientific, USA) loaded with GFP-siRNA and naked GFP-siRNA were used as positive and negative controls, respectively. All samples were prepared in 100 µL OptiMEM™ and added to 400 µL DMEM containing 10% (v / v) serum, resulting in a final siRNA concentration of 10 nM for each condition. After an additional 48 h of incubation, GFP expression was analyzed using a CytoFLEX™ flow cytometer (Beckman Coulter, USA). Cells were then washed twice with PBS and trypsinized. Cells were collected in serum-containing DMEM and centrifuged at 300 g for 5 min. The cell pellet was washed once with PBS and treated with a Zombie NIR™ fixable viability kit (Biolegend, USA) according to the manufacturer’s instructions. After 15 min of incubation, cells were pelleted by centrifugation at 300 g for 10 min and resuspended in 120 µL ice-cold flow cytometry buffer (PBS containing 0.5% (w / v) BSA and 2 mM EDTA) for analysis. Data were processed using FlowJo™ v10 software.

[0192] Luciferase expression

[0193] For luciferase experiments, 5,000 HeLa cells were seeded per well in 96-well plates the day before transfection. After incubation at 37 °C and 5% CO2for 24 h, the culture medium was replaced with 90 µL FBS. Cells were then treated with different concentrations of FLuc-mRNA-loaded LNVs and ssEVs. Lipofectamine™ MessengerMAX™ (Thermo Fisher Scientific, USA) loaded with FLuc-mRNA and naked FLuc-mRNA were used as positive and negative controls, respectively. For each condition, 12 µL of sample in PBS was added to the wells, so that the final mRNA dose added to the cells was 100, 10, 1, and 0.1 ng per well, and the final serum concentration was approximately 90% (v / v). After 24 h of incubation, cells were washed twice with DPBS, then lysed, and luciferase expression was determined using the Pierce™ Luciferase Glow Assay Kit (Thermo Fisher Scientific, USA). Luminescence measurements were performed using a Tecan Spark® ® Microplate Reader and normalized according to the protein loading amount determined by the Micro BCA™ Protein Assay Kit (Thermo Fisher Scientific, USA).

[0194] Example 2: Effect of lipid ratio, pH, and N / P ratio on physicochemical properties of unloaded and siRNA-loaded LNVs

[0195] Unloaded LNVs and siRNA-loaded LNVs were prepared using different molar concentrations of MC3 (40%, 50%, and 60%) at pH 5 and 7.4, as described in Example 1.

[0196] The hydrodynamic diameters (d h ) of unloaded LNVs and siRNA-loaded LNVs increased with increasing molar concentration of MC3 or more alkaline pH at which they were prepared ( FIG. 1B (unloaded LNVs), FIG. 1D (siRNA-loaded LNVs)). The zeta potentials of unloaded LNVs and siRNA-loaded LNVs decreased with more alkaline pH at which they were prepared ( FIG. 1F ). The polydispersity coefficients did not significantly change with the percentage of MC3 or pH in unloaded LNVs ( FIG. 1C ) or siRNA-loaded LNVs ( FIG. 1E ). Different N / P ratios did not significantly affect the particle size and PDI of siRNA-loaded LNVs ( FIG. 1M-N). The encapsulation of siRNA in LNVs was confirmed by EMSA in the presence or absence of Triton™ X-100 FIG. 1O ). siRNA was only detected when LNVs were solubilized with Triton™ X-100.

[0197] The loading efficiency of siRNA was not affected by different N / P ratios and MC3 molar concentrations tested FIG. 1P -Q). The integrity of siRNA loaded in LNVs (N / P 25, 60% MC3, pH 7.4) was confirmed FIG. 1R .

[0198] Cryo-transmission electron microscopy (cryo-TEM) images showed that LNVs and siRNA-loaded LNVs had a particle size of about 100 nm and a non-lamellar structure at pH 7.4 and 5 conditions FIG. 1G -J).

[0199] Small-angle X-ray scattering (SAXS) analysis of LNVs and siRNA-loaded LNVs (N / P 25, 60 mol% MC3) at pH 5 and 7.4 conditions revealed more detailed information about the particle structure FIG. 1K -L, pH 5 and pH 7.4, respectively).

[0200] More specifically, the SAXS scan of unloaded LNVs at pH 5 conditions FIG. 1K ) showed the formation of a clear hexagonal phase (HII phase) with a q-value position ratio of 1 : V3 : 2. Its characteristic repeat distance (d) and center-to-center distance (a) were estimated to be 6.93 nm and 8.01 nm, respectively. Upon addition of siRNA at N / P ratio of 25, an additional peak appeared at q-value of 1.2 nm -1 , indicating the formation of a second phase with a smaller characteristic spacing. This can be due to the presence of a population of particles containing complexed siRNA. At pH 7.4 conditions, the high-order peak disappeared, but the first structural peak was still present, although much broader FIG. 1L . This was observed with and without siRNA (i.e., LNVs and siRNA-loaded LNVs), indicating a transition of the HII phase to a more disordered non-lamellar phase, more specifically a sponge-like structure (L3) phase. Once siRNA was added, the structure based on the HII phase became more disordered and a fourth peak appeared FIG. 1K . The L3 phase at neutral pH helps LNVs to acquire proper fusogenic capacity.

[0201] Example 3: Physico-chemical characterization of ssEVs prepared using siRNA-loaded LNVs

[0202] In two different experimental setups, CellTrace™ Far Red (CT) labeled EVs were mixed with FAM-siRNA loaded LNVs ( FIG. 2G ) or Atto 488 DOPE labeled LNVs ( FIG. 2H ) according to the method described in Example 1, using LNVs (60% MC3; N / P 25) incubated at 37°C, pH 5 and pH 7.4 for 30 min. FIG. 2G The appearance of a double positive population (boxed) after mixing LNVs with EVs at pH 7.4 confirms the formation of ssEVs (column 5, row 1). This population does not appear when only naked FAM-siRNA is added to EVs ( FIG. 2G column 4, rows 1 and 2), or when using the Exo-Fect™ exosome transfection kit ( FIG. 2G column 4 and 5, row 3), or in all other control samples tested. At pH 5, large hybrids are produced ( FIG. 2G column 5, row 2). FIG. 2I shows that the efficiency of loading FAM-siRNA into ssEVs using LNVs at pH 7.4, or Hyb at pH 5, is significantly higher than that achieved using the commercial Exo-Fect™ kit. FIG. 2H shows that a double positive population (boxed) also appears when Atto 488 DOPE labeled LNVs are mixed with CellTrace™ labeled EVs at pH 7.4, confirming the formation of ssEVs ( FIG. 2H column 3, row 1). At pH 5, large hybrids (Hyb) are formed ( FIG. 2H column 3, row 2). FIG. 2J shows the percentage of the double positive ssEVs (pH 7.4) or Hyb (pH 5) population out of the total particle population, i.e. the proportion of Atto 488 DOPE transferred from LNVs to EVs, confirming the occurrence of the hybridization process. FIG. 2K shows that ssEVs are generated at pH 7.4 (d50 < 200 nm), while large hybrids are formed at pH 5, confirming that the double positive population obtained at pH 5 is an aggregate, while ssEVs are obtained at pH 7.4.

[0203] At pH 7.4, when LNVs are present as a sponge ( FIG. 1H and 1L), LNVs spontaneously and transiently generate a fusion handle in EVs in the hybridization step, generating a hybrid system, herein referred to as semi-synthetic extracellular vesicles (ssEVs), such as FIG. 2A NanoFCM measurements also confirmed this interaction (Fig. 2J). In contrast, at pH 5, a high amount of aggregation between LNVs and EVs occurred (Fig. 2K). FIG. 2G , FIG. 2F , 2G and 2H), failing to form ssEVs.

[0204] The effect of LNV:EV number ratio, reaction temperature (4°C, 25°C and 37°C) and pH (5, 6, 7.4 and 9) on the size and number of particles generated was determined in 384-well plates using a DLS / SLS microplate reader. FIG. 2L It was shown that different number ratios did not affect the size population and reaction kinetics of ssEVs by monitoring the increase of hydrodynamic diameter and particle concentration over time (1 hour). FIG. 2M It was shown that the speed of the hybridization process depends on the temperature, being faster at 37°C than at 4°C, while FIG. 2N It was shown that the hybridization of EVs is highly sensitive to pH. pH 7.4 is the optimal value for the formation of ssEVs, while pH 5 generates large hybrid bodies (Hyb). Moreover, at pH 5, no particle concentration could be detected after 5 minutes, probably due to the presence of multiple particle species, including larger aggregates.

[0205] Example 4: Effect of pH on physicochemical properties of unloaded and mRNA-loaded LNVs and physicochemical characterization of ssEVs prepared from mRNA-loaded LNVs at pH 7.4

[0206] Unloaded LNVs and mRNA-loaded LNVs were prepared at pH 5 and 7.4, as described in Example 1, and used to prepare ssEVs at pH 7.4.

[0207] The average hydrodynamic diameter of the mRNA-loaded LNVs (N / P 25, 60 mol % MC3) prepared was about 130 nm (Fig. 3A) FIG. 3A ), with a low polydispersity index value (PDI) of about 0.11 (Fig. 3B) FIG. 3B ). The data also showed a slight decrease in the zeta potential of LNVs once mRNA was loaded into the particles (Fig. 3C) FIG. 3C ), both at acidic and neutral pH conditions. The loading efficiency (LE) of mRNA encapsulated into LNVs was 94% ± 5 (Fig. 3D) FIG. 3D .

[0208] Structure of mRNA-loaded LNVs as seen by cryo-EM micrographs (mRNA-loaded LNVs at a number ratio of 4:1 with EVs, LNVs prepared at N / P ratio 25 and 60 mol% MC3) (scale bar, 50 nm). FIG. 3E ).

[0209] The fusion process of mRNA-loaded LNVs with EVs is also illustrated in cryo-EM micrographs, where it can be seen that, similarly to siRNA-loaded LNVs, mRNA-loaded LNVs spontaneously fuse with EVs at 37°C and pH 7.4 (i.e. very mild conditions) to form ssEVs (scale bar, 50 nm). FIG. 3F The loading of mRNA in ssEVs was confirmed by FIG. 3G -H. As described above, CFSE-labeled EVs were incubated with Cy5-FLuc-mRNA-loaded LNVs or with FLuc-mRNA-loaded LNVs. After mixing Cy5-mRNA-loaded LNVs with CFSE-labeled EVs, a double positive population appeared (boxed), indicating the formation of mRNA-loaded ssEVs at pH 7.4. This population was not present, for example, when naked Cy5-FLuc-mRNA was added to EVs alone, or when unlabelled FLuc-mRNA was encapsulated in LNVs and mixed with unlabelled EVs.

[0210] Example 5: Effect of LNVs and siRNA-loaded LNVs treatment on HeLa cell viability in an in vitro model

[0211] HeLa cells were incubated at 37°C and their viability was tested using the method described in Example 1.

[0212] At all concentrations tested (12.5, 25, 50, 100 and 200 pg / mL) and time points (4 hours and 24 hours), both unloaded LNVs and siRNA-loaded LNVs did not cause a decrease in cell viability (Fig. 5A-B). FIG. 4A -B).

[0213] Example 6: Preservation of EV membrane protein biological activity after hybridization

[0214] The enzymatic activity of EV membrane proteins CD73 and CD26 was determined following the method described in Example 1. The results show that, after hybridization with siRNA-loaded LNVs at pH 7.4, 4°C and 37°C, their activity was not altered (Fig. 6A-B). In contrast, after hybridization with siRNA-loaded LNVs at pH 5, the enzymatic activity of CD73 was significantly reduced (Fig. 6C). FIG. 5A -B). In contrast, after hybridization with siRNA-loaded LNVs at pH 5, the enzymatic activity of CD73 was significantly reduced (Fig. 6C). FIG. 5A). Exo-Fect™ reagent also significantly reduced the enzymatic activity of both enzymes (CD73 and DPP4) after hybridization compared to ssEVs (pH 7.4, 4°C and 37°C) FIG. 5A -B).

[0215] Example 7: Transfection efficiency of siRNA-loaded ssEVs in HeLa cells expressing GFP

[0216] Untreated cells (received only medium), PBS, free GFP-siRNA as negative control, and cells treated with Lipofectamine™ RNAiMAX loaded with GFP-siRNA as positive control. Subsequently, the transfection efficiency of LNVs loaded with GFP-siRNA (LNVs (w siGFP)) and ssEVs loaded with GFP-siRNA (ssEVs (w siGFP)) in HeLa-GFP cells was evaluated in the presence of ~8% (v / v) FBS. The results showed that LNVs loaded with GFP-siRNA reduced the normalized GFP fluorescence intensity by ~59%, while ssEVs significantly enhanced the transfection efficiency of LNVs after hybridization, with a ~75% reduction in GFP fluorescence (ssEVs (w siGFP)), which was comparable to the effect of the positive control Lipofectamine™ RNAiMAX (RNAiMAX (w siGFP)) (Figure 7). FIG. 6 .

[0217] Example 8: Transfection efficiency of mRNA-loaded ssEVs in HeLa cells

[0218] PBS-treated cells and cells receiving naked FLuc-mRNA as negative control, and cells treated with Lipofectamine™ MessengerMAX™ loaded with FLuc-mRNA as positive control. The transfection efficiency of different doses of mRNA (100, 10, 1, and 0.1 ng) encapsulated in LNVs, ssEVs, or Lipofectamine™ MessengerMAX™ in HeLa cells was evaluated in the presence of ~90% (v / v) serum. The results showed that ssEVs significantly improved the expression of luciferase compared to LNVs, with a ~7-fold increase in fluorescence signal at a mRNA dose of 100 ng and a ~10-fold increase at a mRNA dose of 10 ng (Figure 8). FIG. 7 .

[0219] Example 9: Nanostructures prepared with a phospholipid with a CPP > 1 and a non-ionic surfactant with a CPP value < 1 (but without an ionizable cationic lipid)

[0220] Nanoparticles similar to multilamellar liposomes were prepared using DOPE and KLP but without MC3, as shown in FIG. 8 .

[0221] Example 10: Nanoparticles prepared with an ionizable cationic lipid, a phospholipid with CPP ~ 1 and a non-ionic surfactant with CPP < 1

[0222] Nanoparticles were prepared using MC3, DOPC (a phospholipid with CPP ~ 1) and KLP and their d h , PDI, zeta-pot and LE% (see Table II below) were evaluated at pH 5 and 7.4 using Zeta-Sizer and RiboGreen assay. FIG. 9A -B and Table II below.

[0223] Table II: Summary of physico-chemical properties (dh, PDI, zeta-potential and LE%) of DOPC-NPs and siRNA-loaded DOPC-NPs

[0224]

[0225] SAXS analysis showed that DOPC-NPs exhibited a hexagonal phase at pH 5, characterized by three sharp peaks with q value positions ratio of 1 : V3 : 2, especially when siRNA was added in the system ( FIG. 9H ). Cryo-EM images also showed that DOPC-NPs formed well-defined non-lamellar structures at pH 5 ( FIG. 9D ).

[0226] However, at pH 7.4 where fusion can occur (see Figures 2A-E, G-I), multilamellar / vesicular regions were found to appear near the non-lamellar arranged regions upon addition of siRNA ( FIG. 9F ). Cryo-EM images ( FIG. 9C -G) also showed that siRNA / DOPC-based nanoparticles had a multi-vesicular structure.

[0227] As shown in FIG. 9G , siRNA / DOPC-based nanoparticles did not form ssEVs with MSC-derived EVs.

[0228] Example 11: Nanoparticles prepared with a phospholipid with CPP > 1 and a non-ionic surfactant with CPP < 1, but using a fixed cationic lipid instead of an ionizable cationic lipid

[0229] Nanoparticles were prepared using DOTMA (a fixed cationic lipid), DOPE and KLP and their dh , PDI, z-pot and LE% FIG. 10A - B and Table III below.

[0230] Table III: Summary of physicochemical properties (dh, PDI, z-potential and LE%) of DOTMA-NPs and siRNA-loaded DOTMA-NPs

[0231]

[0232] Cryo-EM images show that DOTMA-NPs with or without siRNA cargo form multilamellar and unilamellar liposomes smaller than LNVs and DOPC-NPs (< 100 nm) at pH 5 and pH 7.4 (see Figures 6A and 6B, respectively). FIG. 10C , 10E and FIG. 10D , 10F ). FIG. 10G Cryo-EM images show that DOTMA-NPs with or without siRNA cargo form multilamellar and unilamellar liposomes smaller than LNVs and DOPC-NPs (< 100 nm) at pH 5 and pH 7.4 (see Figures 6A and 6B, respectively). FIG. 10H SAXS plots (Figure 6C) show a series of broad oscillations, indicating the formation of a less multilamellar double-layer structure at pH 5 and pH 7.4, with or without siRNA cargo. Consistent with SAXS and cryo-EM image data, the structure of DOTMA-NPs does not change with buffer pH and presence of siRNA cargo. Moreover, FIG. 10G show that siRNA-loaded DOTMA particles form a large number of aggregates in the presence of MSC-derived EVs at pH 7.4, confirming that only LNVs can spontaneously form ssEVs with appropriate physicochemical properties at neutral pH.

[0233] Example 12: Nanoparticles prepared with an ionizable cationic lipid, a phospholipid with CPP > 1, and a lysophospholipid with CPP < 1 (instead of a non-ionic surfactant with CPP value < 1)

[0234] Nanoparticles were prepared as described in Example 1 using MC3, DOPE and MPPC (a lysophospholipid with CPP < 1). Particle size distribution of the nanoparticles was evaluated at pH 7.4 using a DLS instrument as described in Example 1 (Figure 7A). FIG. 11 A broad particle size distribution was detected, comprising more than one population of particles, which demonstrates the key role of non-ionic surfactants (e.g. KLP) in the formation of particles with appropriate particle size and PDI values.

[0235] Example 13: Nanoparticles prepared with different concentrations of surfactants showing CPP values < 1

[0236] Nanoparticles were prepared using MC3, DOPE and KLP, with the concentration of KLP varying between 5 and 15 mol %. The hydrodynamic size of the nanoparticles was measured at pH 7.4 using a DLS instrument equipped with a Malvern Zetasizer™, as described in Example 1. The particle size obtained at these concentrations was between about 800 and 1050 nm FIG. 12 ), much larger than the size obtained using surfactant concentrations > 20 mol % (see for example FIG. 2C-2E , 2K-2N), confirming that aggregation occurred at these surfactant concentrations.

[0237] The scope of the claims should not be limited to the embodiments set forth in the examples, but should be given the broadest interpretation consistent with the entire specification.

[0238] References

[0239] Alvarez-Erviti, L., Seow, Y., Yin, H., et al. 2011. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat. Biotechnol., 29, pp. 341-345.

[0240] Barragan Vidal, I.A., Müller, M., Generalization of the swelling method to measure the intrinsic curvature of lipids, J. Chem. Phys. 147, 224902, 2017.

[0241] Hamai, C., Yang, T., Kataoka, S., Cremer, P.S., Musser, S. M., Effect of Average Phospholipid Curvature on Supported Bilayer Formation on Glass by Vesicle Fusion. Biophys. J. 90, 1241, 2006.

[0242] Lamichhane, T. N., Jeyaram, A., Patel, D. B., et al., 2016. Oncogene Knockdown via Active Loading of Small RNAs into Extracellular Vesicles by Sonication. Cell. Mol. Bioeng., 9, pp. 315-324.

[0243] O'Brien, K., Breyne, K., Ughetto, S., Laurent, L.C., 2020. RNA delivery by extracellular vesicles in mammalian cells and its applications. Nat. Rev., 21, pp. 585-606.

[0244] Rand, R. P., Fuller, N. L., Gruner S. M. and Parsegian, V. A., Membrane Curvature, Lipid Segregation, and Structural Transitions for Phospholipids under Dual-Solvent Stress, Biochemistry, 29, 76, 1990.

[0245] Sodt, J., Pastor, R. W., 2013, Bending Free Energy from Simulation: Correspondence of Planar and Inverse Hexagonal Lipid Phases, Biophys. J., 104(10), 2202-11.

[0246] Théry et al., 2018, Minimal information for studies of extracellular vesicles, J Extracellular Vesicles, 7(1): 1535750.

[0247] Wu et al., 2023. “Advances in Extracellular Vesicle Nanotechnology for Precision Theranostics” Adv. Sci. 2023, 10, 2204814.

Claims

1. A pH-induced structural transition non-layered lipid nanocarrier (LNV), comprising: (a) At least one ionizable cationic lipid; (b) At least one phospholipid with a critical packing parameter (CPP) value greater than 1; and (c) At least one nonionic surfactant with a CPP of less than 1, having a molar concentration of 20% to 50%.

2. The LNV as described in claim 1, wherein: (a) The apparent acid dissociation constant (pKa) of the at least one ionizable cationic lipid before incorporation into LNV is 5 to 7.5; (b) The at least one phospholipid is a phosphatidylethanolamine (PE) lipid; (c) The at least one surfactant is a fatty acid polyethylene glycol ester or a polysorbate; or (d) A combination of at least two of (a) to (c).

3. The LNV as described in claim 1 or 2, wherein: (a') The at least one ionizable cationic lipid is D-Lin-MC3-DMA (MC3); (b') The at least one phospholipid is dioleoylphosphatidylethanolamine (DOPE); (c') The at least one nonionic surfactant is a fatty acid polyethylene glycol ester, such as polyethylene glycol 12-hydroxystearate, preferably Kolliphor. ® HS15 (KLP); or Combinations of at least two of (d') (a') to (c').

4. The LNV according to any one of claims 1-3, wherein it is loaded with a drug or diagnostic agent, preferably wherein the drug or diagnostic agent is a nucleic acid molecule.

5. A composition comprising an LNV as defined in any one of claims 1 to 4 and at least one pharmaceutically acceptable excipient.

6. A method for preparing an LNV as defined in any one of claims 1 to 3, comprising mixing: (a) an organic phase comprising at least one ionizable cationic lipid, at least one phospholipid and at least one surfactant, and dissolved in an organic solvent; and (b) an aqueous medium.

7. The method of claim 6, wherein, At least one of the following conditions must be met: (i) The organic phase further comprises a lipophilic drug or diagnostic agent; and (ii) The aqueous phase further comprises a hydrophilic drug or diagnostic agent, preferably wherein the hydrophilic drug or diagnostic agent is a nucleic acid molecule.

8. The method of claim 7, wherein (ii) the aqueous phase further comprises a hydrophilic drug or diagnostic agent, and the pH of the aqueous medium is from about 4 to about 6.

9. The method according to any one of claims 6-8, wherein, The hybrid operation utilizes microfluidic devices.

10. A method for preparing semi-synthetic extracellular vesicles (ssEVs), comprising mixing an LNV as defined in any one of claims 1-4 or a composition as defined in claim 5 with an extracellular vesicle (EV) at a pH value higher than 6 and up to about 10, thereby generating the ssEVs.

11. Semi-synthetic extracellular vesicles (ssEVs), prepared by the method defined in claim 10.

12. Semi-synthetic extracellular vesicles (ssEVs) are essentially composed of all or part of the following components: (a) A lipid nanocarrier as defined in any one of claims 1-4; and (b) Extracellular vesicles (EVs).

13. A composition comprising ssEV as defined in claim 11 or 12 and at least one pharmaceutically acceptable excipient.

14. Reagent kit, containing: (A) (a) The LNP as defined in any one of claims 1-4 or the composition as defined in claim 5; and (b) (i) The solution used to hydrate (a); (ii) Extracellular vesicles (EVs) or a composition comprising said EVs and at least one pharmaceutically acceptable excipient; (iii) Use the instructions in (a) and (i) and / or (ii); or, (iv) Combinations of at least two of (i) to (iii); or, (B) (a') the ssEV as defined in claim 11 or 12, or the composition as defined in claim 13; and (b') (i') The solution used to hydrate (a'); (ii') Use the instructions for (a') and (i'); or, The combination of (iii'), (i'), and (ii').

15. The ssEV as defined in claim 11 or 12 or the composition as defined in claim 13, used as a pharmaceutical or diagnostic agent.

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