Dayhlipid-polymer microparticles for thermostable, pulsatile MRNA-LNP vaccines

Abstract

Disclosed is a thermally stable, single-dose mRNA-LNP (lipid nanoparticle) platform.

Description

[0001] MTV-24625

[0002] DAYHLIPID-POLYMER MICROPARTICLES FOR THERMOSTABLE, PULSATILE MRNA-LNP VACCINES

[0003] RELATED APPLICATIONS

[0004] This application claims the benefit of priority to U.S. Provisional Application No.: 63 / 733,652, filed December 13, 2024. The contents of the aforementioned application is fully incorporated by reference herein.

[0005] BACKGROUND

[0006] Vaccines based on lipid nanoparticles (LNPs) loaded with mRNA (messenger RNA) have emerged as a promising platform for preventing COVID-19 and other infectious diseases, such as HIV, due to their ease in manufacturing and their ability to induce immune responses against a broad range of viral variants. However, a key challenge with mRNA vaccines is the need for multiple doses administered over several weeks to achieve efficient immune responses. This frequent dosing limits accessibility in regions with scarce healthcare resources. Additionally, many mRNA vaccines require a cold chain for storage, presenting further logistical challenges. In view of the foregoing, there is an unmet, ongoing need for new formulations for mRNA vaccines and related therapies.

[0007] SUMMARY OF THE INVENTION

[0008] In certain aspects, disclosed is a thermally stable, single-dose mRNA-LNP (lipid nanoparticle) platform.

[0009] In certain aspects, the present disclosure provides microparticles comprising: i) a microparticle core comprising a lipid nanoparticle; ii) a microparticle shell comprising a polymer or a copolymer; and wherein the microparticle core is encapsulated by the microparticle shell; and the lipid nanoparticle comprises a lipid, an excipient, and a bioactive agent

[0010] BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 A shows a sequence of schematics from left to right illustrating the encapsulation process of mRNA-LNP within an aqueous core. The first schematic shows the initial encapsulation of mRNA-LNP using a glass capillary device. The second schematic depicts the formation of double emulsions, with the mRNA-LNP encapsulated inside and a spherical shell surrounding it. In the third schematic, the microparticles were collected in PBS buffer. The MTV-24625 fourth schematic represents in vitro testing of release and cytotoxicity using cell cultures, followed by the fifth schematic showing in vivo subcutaneous injection of these microparticles into mice. Below the schematics, our protocol is presented step-by-step, with a red box highlighting the current stage where we are optimizing shell materials and iterating in vivo testing.

[0012] FIG. IB shows a summary of the lipid nanoparticle formulations used for encapsulating mRNA.

[0013] FIGs. 1C and ID demonstrate the encapsulation of fluorescently labeled mRNA-LNP using a glass capillary microfluidics device. The device operates in two regimes: producing water-in-oil-in-water double emulsions with a thin shell (top) and water-in-oil-in-oil-in-water triple emulsions with transverse shell asymmetry and a thicker shell (bottom).

[0014] FIGs. 2A-2F illustrate the screening process for optimizing the stabilization and delayed release of mRNA-LNP within microparticles. FIG. 2A shows the screening of two variants aimed at stabilizing the core of soluble mRNA-LNP and four variants for stabilizing the microparticle shell over a designated period. By combining these variables, we identified the optimal formulation for achieving delayed release of mRNA-LNP in vivo. The highlighted boxes in the figure indicate the specific variables being tested. FIG. 2B shows that from five excipient solutions, we identified PEG (4 wt%) and PVA (1 wt%) as the top excipients for stabilizing mRNA-LNP at 37°C over time. This conclusion is based on the selection of excipients that maximize the encapsulation efficiency of the LNP. FIG. 2C shows the evaluation of various solvents for the shell phase, and it was determined that ethyl acetate is the most effective for encapsulating mRNA-LNP. In contrast, a solvent mixture of chloroform / hexane results in mRNA-LNP leakage into the shell, demonstrating its unsuitability for this application. FIGs. 2D and 2E show an applied contour detection method to analyze both the size and number of mLPM particles. FIG. 2F shows the mechanical stability of lipid-formulated shells at 37°C over time by monitoring the number of intact particles under these conditions.

[0015] FIGs. 3A-3C illustrate the screening and analysis of hybrid polymer-lipid microparticles encapsulating mRNA-LNP. We screened two variants for stabilizing the core of soluble mRNA-LNP and four variants for stabilizing the shell of the microparticle over a designated period. The combination of these variants allows us to identify the optimum formulation for the delayed release of mRNA-LNP in vivo. In this figure, we specifically assessed the stability of the released mRNA-LNP from the core and the stability of the polymerlipid hybrid shell, with the highlighted variables representing these factors. FIG. 3A MTV-24625 summarizes the different PLGA formulations combined with POPC lipids used to create three hybrid polymer-lipid microparticle formulations: PL1, PL2, and PL3. Each formulation represents a unique combination of polymers and lipids to optimize performance. FIG. 3B shows the breaking of the microparticles by extruding them through a needle and assesses both the size and mRNA encapsulation efficiency of the LNPs inside the microparticles. The left subfigure shows that the LNPs have average diameters ranging from 200 nm to 300 nm. In the right subfigure, we demonstrate that the LNPs exhibit mRNA encapsulation efficiencies of approximately 70% to 80% within the microparticles. FIG. 3C demonstrates that increasing the molecular weight of the polymer component in the hybrid polymer-lipid microparticles leads to delayed rupturing of the microparticles and a slower release of the core cargo.

[0016] FIG. 4A-4H show the screening and analysis of PLGA formulations for mRNA-LNP encapsulation and delayed release. We screened two variants for stabilizing the core of soluble mRNA-LNP and four variants for stabilizing the microparticle shell over a designated period of time. The combination of these variants allowed us to identify the optimum formulation for delayed release of mRNA-LNP in vivo. The figures also show the screening of PLGA with increasing molecular weight and concentration, compared to the hybrid polymer-lipid microparticles shown in a previous figure. These PLGA formulations were selected for further in vivo testing to evaluate their delayed release properties. FIGs. 4B and 4C summarize the different concentrations and molecular weights of PLGA used in this study. The concentration and molecular weight of PLGA were key factors in determining the mechanical stability and release kinetics of the microparticles. FIG. 4D demonstrates the mechanical breakdown of one PLGA formulation at 37°C over time, visualized using confocal microscopy. This analysis helps assess the structural integrity and stability of the microparticles under physiological conditions. FIG. 4E shows the evaluation of the quality of mRNA-LNPs released from the microparticles into the supernatant at 37°C. The encapsulation efficiency of the mRNA-LNPs decreased from approximately 80% to 60% over seven days, while the amount of released mRNA increased from approximately 1 pg to 1.4 pg over the same period, as measured by the RiboGreen assay. This data indicates a gradual release of mRNA-LNPs over time, while maintaining a significant portion of encapsulation efficiency. FIG. 4F and 4G show the analysis of three identical samples, each containing 50 pL of microparticles suspended in 200 pL of PBS buffer, under different conditions. The first sample was stored at 4°C, the second at 37°C, and the third sample was mechanically disrupted by harsh pipetting to simulate breakdown. Using the RiboGreen assay, we assessed the mRNA encapsulation efficiency and release profile from the microparticles. We observed that the encapsulation efficiency of MTV-24625 mRNA-LNPs decreased from approximately 80% to 65% when comparing storage at 4°C to 37°C. Additionally, the release of mRNA-LNPs increased from approximately 3% to 9% when comparing storage conditions at 4°C to 37°C. These results demonstrate minimal release at 4°C, indicating stability during storage, and delayed release at physiological temperature, highlighting the potential for controlled release in vivo. FIG. 4H shows the in vivo efficacy of Flue mRNA-LNP formulations containing different soluble excipient cocktails after two weeks at 37 °C. Luciferase-encoding mRNA-LNPs at equal mRNA concentrations were formulated with varying combinations of L-serine, polyethylene glycol (PEG), and sucrose. Mice received subcutaneous injections of the different formulations, and bioluminescence was measured in vivo to assess retained mRNA translation. Controls: Dose-matched mRNA-LNP at 4 °C (box, left) demonstrated maximal luciferase activity, whereas free mRNA-LNP administered without stabilizing excipients showed rapid signal loss at 37 °C (box, right). Test formulations: Distinct excipient cocktails (YangR2 series, E-series) produced variable degrees of signal persistence after two weeks in vivo at 37 °C. Highlighted groups: Formulations E13, E15, and E17 (red box) preserved significantly higher bioluminescence than other groups, indicating that specific excipient combinations sustain mRNA-LNP efficacy under physiological conditions for extended periods. These results demonstrate that soluble excipient cocktails comprising L-serine, PEG, and sucrose can preserve the functional efficacy of mRNA-LNPs after prolonged in vivo residence at body temperature. When incorporated into microparticles, these optimized formulations further extend activity by protecting encapsulated mRNA-LNP contents, thereby enabling durable in vivo translation and immune responses beyond what is achievable with conventional free mRNA-LNP formulations.

[0017] FIGs. 5A-5C illustrate the screening and testing of PLGA formulations for mRNA- LNP encapsulation and release. We compared the stability of PLGA formulations with increasing molecular weights and concentrations. These PLGA formulations were selected for further in vivo testing to evaluate their delayed release properties. FIG. 5A summarizes the different concentrations and molecular weights of PLGA used in the study. These parameters were critical for tuning the microparticles' stability and release kinetics. FIG. 5B shows the injection of 100 pL of Flue mRNA-LNP encapsulated in microparticles with the P 1 2 polymer shell formulation into mice 1, 2, and 3 (from left), and an equivalent dose of free mRNA-LNP (equal to the mRNA-LNP dosage inside the microparticles) into mice 4 and 5. At 6 hours postinjection, we observed that mice 1, 2, and 3 did not show bioluminescence, while mice 4 and 5 displayed strong bioluminescence. In contrast, mice 1, 2, and 3 exhibited much stronger fluorescence compared to mice 4 and 5. Both bioluminescence and fluorescence decayed over MTV-24625 time in the mice, indicating that this PLGA formulation did not release the mRNA-LNP during the observed period. FIG. 5C shows 100 pL of microparticles with the P2_l polymer shell formulation containing Flue mRNA-LNP and injected the broken particles into mice 1 and 2 (from left). We then injected 100 pL of identical, unbroken microparticles into mice 3, 4, and 5 (from left). We observed significantly stronger bioluminescence and fluorescence in mice 1 and 2 compared to mice 3, 4, and 5, which received the intact microparticles. This contrast indicates that the mRNA-LNPs were released from the broken particles, while they remained protected in the intact microparticles during the measured period.

[0018] FIG. 6 shows antibody titers in mice following subcutaneous injection of encapsulated COVID mRNA-LNP show compatible or higher response as compared to free COVID mRNA-LNP. The data show that mRNA-LNPs released from microparticles (Gl, G2) generate antibody responses comparable to or exceeding those of freshly prepared free mRNA- LNP (G3). This demonstrates that the microparticles preserve mRNA-LNP activity after encapsulation and storage, providing a unique advantage over conventional formulations where mRNA stability and efficacy rapidly diminish. Mice were divided into three groups (n=3 each): Gl: Microparticles formulated with PLGA 858s and lipids, opened prior to injection to release encapsulated COVID mRNA-LNP; G2: Microparticles formulated with PLGA 505s and lipids, opened prior to injection to release encapsulated COVID mRNA-LNP; and G3: Free, freshly prepared COVID mRNA-LNP without encapsulation. Groups G1-G3 each consist of three mice injected subcutaneously either with microparticles containing CO VID mRNA-LNP or free COVID mRNA-LNP. To assess the efficacy of COVID mRNA-LNP inside microparticles as compared to freshly made COVID mRNA-LNP, we open the microparticles using sharp pipettes for group Gl and G2, and demonstrate that the efficacy from the released contents from microparticles is compatible or higher than the efficacy from freshly made free COVID-LNP without encapsulation. Blood was collected at baseline (week 0) and at weeks 1, 2, and 3 post-injection to obtain serum for ELISA measurement of antibody titers. The three mice in G3 have lower titers as compared to the mRNA-LNP released from particles in Gl and G2, showing the microparticles preserve the mRNA-LNP efficacy after encapsulation.

[0019] FIG. 7 shows fluorescence imaging of microparticles containing fluorescent COVID mRNA-LNP in mice following subcutaneous injection shows the particles enable mRNA-LNP retention in vivo. Groups G1-G3 each consist of three mice. In each group, the three mice are injected with identical microparticles containing CO VID mRNA-LNP, where the LNP gives fluorescence. The microparticles consist of different molecular weights of PLGA and lipids MTV-24625 between the three groups. The sustained fluorescence shows that the microparticles retain the mRNA-LNP in vivo for a long period of time.

[0020] FIG. 8A shows: (Top) schematic for three glass capillary microfluidic generators. (Middle) Thin to thick shells: With low to high amounts of PLGA incorporated with lipids and antioxidants in the shells. (Bottom) screening of solvent of shells for successful encapsulation of mRNA-LNP.

[0021] FIG. 8B shows the uniformity of microcapsules confirmed using confocal microscope for microcapsules encapsulating fluorescent mRNA-LNP.

[0022] FIG. 8C shows Ribogreen test confirming high encapsulation efficiency of mRNA- LNP.

[0023] FIG. 8D shows: (Top) Changing shape of microcapsules from spherical to elongated using flow rate of continuous phase. (Bottom) Changing the size of the microcapsule from large (over 100 micrometers in diameter) to small (40 micrometers in diameter).

[0024] FIG. 9A shows: (Top Left) Q10 improves stability of PLGA 505 microcapsules during production. (Bottom Left) Q10 improves stability of PLGA 858S microcapsules for hydrogel cores. (Right) Antioxidant and lipid-enriched microcapsules show better stability than lipid microcapsules at 37 °C for long term.

[0025] FIG. 9B shows the PLGA, antioxidant, and lipid shells with hydrogel and mRNA-LNP cores (right) have better stability and later release as compared to PLGA, antioxidant, and lipid shells with excipient and mRNA-LNP cores (left) at 37 °C over several weeks under confocal.

[0026] FIG. 9C shows a statistical summary of an exemplar group of microcapsules with late release around 2-3 weeks’ time at 37 °C in vitro.

[0027] FIG. 9D shows a simulation of release from three microcapsules in subcutaneous area, with a thin shell (left), a thick shell (middle), a thin shell surrounding a hydrogel core (right). The thin shell (left) starts to release the earliest and the release is sustained over time; the thick shell (middle) starts to release later than the thin shell and the release is also sustained. The thin shell encapsulating hydrogel core (right) starts to release the latest but the release profile is the sharpest.

[0028] FIG. 10A shows microcapsules with QfO and VE enriched (left) or hydrogel enriched (right) are injectable without breaking through a 20G needle.

[0029] FIG. 10B shows crushed microcapsules encapsulating Flue mRNA-LNP (fluorescent lipid) show the encapsulated mRNA is effective in giving in vivo bioluminescence (left). The microcapsules without antioxidants or hydrogel enriched have initial burst release right after MTV-24625 injection (middle) and the microcapsules with antioxidants or hydrogel enriched have no initial burst release (right).

[0030] FIG. 11A shows that a ratio of sucrose, PEG, and L-serine gives higher zeta potential, lower PDI in mRNA-LNP soluble formulations at 4 °C, correlating with higher cell transfection efficacy of mRNA-LNP using Flue mRNA.

[0031] FIG. 11B shows that a ratio of sucrose, PEG, and L-serine gives higher zeta potential, lower PDI in mRNA-LNP soluble formulations at 50 °C, correlating with higher cell transfection efficacy of mRNA-LNP using Flue mRNA.

[0032] FIG. 11C shows that a ratio of sucrose, PEG, and L-serine leads to higher cell transfection efficacy for mRNA-LNP soluble formulations at 37 °C over two weeks.

[0033] FIG. 11D shows the properties of certain lipid nanoparticles.

[0034] FIG. 12 shows representative schematics for in vivo dosage, with primary free mRNA- LNP injected alongside microcapsules carrying booster shots.

[0035] DETAILED DESCRIPTION OF THE INVENTION

[0036] Lipid nanoparticles (LNPs) encapsulating messenger RNA (mRNA) have shown immense promise as vaccines, particularly against COVID-19. Their ability to induce robust immune responses combined with their ease of production make them highly effective vaccines. However, the need for multiple doses over time presents considerable logistical challenges, particularly in regions with limited healthcare infrastructure. To address this challenge, we developed a thermally stable, delayed-release mRNA-LNP platform designed to mimic a booster dose. We utilized a microfluidics platform to encapsulate mRNA-LNPs inside lipid-polymer hybrid microparticles at nearly 100% encapsulation efficiency. These microparticles consist of a core of mRNA-LNPs in aqueous solution, surrounded by a hard lipid-polymer shell. The lipid component ensures biocompatibility, while the polymer component controls the release of the mRNA-LNPs over time. In our study, we designed and tested 20 different lipid-polymer microparticle formulations with varied delayed-release mechanisms. These microparticles encapsulate Flue mRNA-LNPs (encoding firefly luciferase) as a model system. We characterized the delayed-release mechanisms of the microcapsules and evaluated the stability of the mRNA-LNPs within the microparticles through in vitro characterization. We further tested these formulations in vivo by administering the microparticles subcutaneously in mice and evaluated mRNA-LNP stability using in vivo imaging. Our data shows that the fluorescence of mRNA-LNP inside microparticles decays several times slower than the fluorescence of free mRNA-LNP. This contrast in fluorescence MTV-24625 decay rate suggests the mRNA-LNP in the microparticles are protected over the period of observation. Release kinetics of mRNA-LNP is still under investigation. In conclusion, this approach has the potential to eliminate the need for multiple doses when the delayed-release formulation of mRNA-LNPs is co-delivered with the primary dose. This technology addresses key challenges associated with current mRNA vaccines and presents a pathway for expanding the accessibility of life-saving vaccines to underserved populations globally.

[0037] In certain aspects, the present disclosure provides microparticles comprising: i) a microparticle core comprising a lipid nanoparticle; ii) a microparticle shell comprising a polymer or a copolymer; and wherein the microparticle core is encapsulated by the microparticle shell; and the lipid nanoparticle comprises a lipid, an excipient, and a bioactive agent.

[0038] In certain embodiments, the lipid is an ionizable lipid, cholesterol, a polyethylene glycol lipid, a phospholipid, or a combination of any of them. In certain embodiments, the lipid is 1 - octylnonyl 8-[(2-hydroxyethyl)[8-(nonyloxy)-8-oxooctyl]amino]octanoate (i.e., lipid 5, CAS Ref. No.: 2089251-33-0), 3, 6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2, 5-dione ( / .< ., cKK-E12), l,2-di-(9Z-octadecenoyl)-sn-glycero-3 -phosphoethanolamine (DOPE), 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), l,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), cholesterol, or a combination of any of them. In certain embodiments, the lipid comprises a combination of DOPC and DPPC (e.g., 20 wt% DOPC and 80 wt% DPPC). In certain preferred embodiments, the lipid is POPC (e.g., 20 wt% POPC).

[0039] In certain embodiments, the excipient comprises polyethylene glycol, polyvinyl alcohol, glucose, sucrose, citric acid, phosphate buffer solution e.g., 2-Amino-2- (hydroxymethyl)-l,3-propanediol (Tris) buffer or 4-(2-hydroxyethyl)piperazine-l -ethane- sulfonic acid (HEPES) buffer). In certain preferred embodiments, the excipient comprises sucrose. In other preferred embodiments, the excipient comprises PEG (e.g., PEG 2000). In certain embodiments, the excipient comprises PVA. In certain embodiments, the excipient comprises a combination of PEG and PVA. In certain embodiments, the excipient comprises a combination of PEG at about 4 wt% and pVA at about 1 wt%. In certain embodiments, the excipient comprises a combination of PEG with a molecular weight of about 6,000 kDa at about 4 wt% and PVA with a molecular weight from about 13 kDa to about 23 kDa at about 1 wt%.

[0040] In certain embodiments, the excipient comprises a polymer or a copolymer. In certain embodiments, the excipient comprises a polymer. In certain embodiments, the polymer is MTV-24625 polyvinylalcohol (PVA) or polyvinylpyrrolidone (PVP). In certain embodiments, the polymer is PVP. In certain embodiments, the polymer is PVP 10. In other embodiments, the polymer is PVP60. In certain embodiments, the excipient comprises a copolymer. In certain embodiments, the copolymer comprises a plurality of repeat units of vinyl alcohol and a plurality of repeat units of vinyl pyrrolidinone.

[0041] In certain embodiments, the mass ratio of polyvinyl alcohol to polyvinylpyrrolidone is about 1 : 1 to about 6: 1; or about 1 : 1, about 2: 1, about 3: 1, about 4: 1, about 5: 1, or about 6: 1. In certain embodiments, the mass ratio of polyvinylalcohol to polyvinylpyrrolidone is about 1 : 1, about 2: 1, or about 3: 1. In certain embodiments, the copolymer comprises a plurality of repeat units of vinylalcohol and a plurality of repeat units of sucrose. In certain embodiments, the mass ratio of polyvinylalcohol to sucrose is about 1 : 1 to about 6: 1; or about 1 : 1, about 2: 1, about 3: 1, about 4: 1, about 5: 1, or about 6: 1. In certain embodiments, the mass ratio of polyvinylalcohol to sucrose is about 1 : 1 or about 2: 1. In certain embodiments, the copolymer comprises a plurality of repeat units of vinylalcohol, a plurality of repeat units of polyvinylpyrrolidone, and a plurality of repeat units of sucrose. In certain embodiments, the mass ratio of polyvinylalcohol to polyvinylpyrrolidone to sucrose is about 1 : 1 : 1 to about 1 : 1 :3; or about 1 : 1: 1, about 1 : 1 :2, or about 1 : 1 :3. In certain embodiments, the mass ratio of polyvinylalcohol to polyvinylpyrrolidone to sucrose is about 1 : 1 :2.

[0042] In certain embodiments, the copolymer comprises 250 - 1,500 repeat units.

[0043] In certain embodiments, the copolymer is a block copolymer. In other embodiments, the copolymer is a random copolymer.

[0044] In certain embodiments, the bioactive agent is selected from the group consisting of a nucleic acid, a protein, an antibody, a small molecule, a vaccine, and an antigen. In certain embodiments, the bioactive agent is an mRNA, siRNA, RNA, or DNA. In certain preferred embodiments, the bioactive agent is an RNA (e.g., an mRNA). In certain embodiments, the bioactive agent is an antibody. In certain embodiments, the bioactive agent is a small molecule (e.g., a drug). In certain embodiments, the bioactive agent is a protein. In certain embodiments, the bioactive agent is a vaccine or an antigen. In certain preferred embodiments, the bioactive agent is a vaccine. In certain embodiments, the bioactive agent is a nucleic acid. In certain embodiments, the bioactive agent is a stimulator of interferon genes (STING) agonist.

[0045] In certain preferred embodiments, the lipid nanoparticle further comprises an antioxidant. In certain embodiments, the antioxidant is hydrophobic. In other embodiments, the antioxidant is hydrophilic. In certain embodiments, the antioxidant is selected from the group consisting of vitamin A, vitamin E, butylated hydroxytoluene, ascorbic acid, and alpha MTV-24625 tocopherol (e.g., D-a-tocopheryl polyethylene glycol succinate (TPGS)). In certain preferred embodiments, the antioxidant is alpha tocopherol. In other preferred embodiments, the antioxidant is TPGS. In yet other preferred embodiments, the antioxidant is ubiquinone (coenzyme Q10).

[0046] In certain embodiments, the microparticles disclosed herein further comprise an amino acid. In certain embodiments, the amino acid is a naturally occurring amino acid. In certain embodiments, the amino acid is serine. In further preferred embodiments, the amino acid is L- serine. In certain embodiments, the amino acid is a cationic amino acid. In certain embodiments, the amino acid is lysine, arginine, or histidine. In certain preferred embodiments, the amino acid is arginine. In further preferred embodiments, the amino acid is L-arginine.

[0047] In certain preferred embodiments, the lipid nanoparticle is formed using encapsulation with ethyl acetate.

[0048] In certain embodiments, the microparticle shell comprises a polymer. In certain embodiments, the polymer is a copolymer. In certain embodiments, the copolymer comprises a plurality of repeat units of lactide and a plurality of repeat units of glycolide (i.e., the polymer is Poly(lactide-co-glycolide) (PLGA)). In certain embodiments, the copolymer comprises a plurality of repeat units of lactide and a plurality of repeat units of glycolide at a molar ratio of about 75:25 lactide to glycolide, about 80:20 lactide to glycolide, about 85: 15 lactide to glycolide, or about 90: 10 lactide to glycolide. In certain preferred embodiments, the copolymer comprises a plurality of repeat units of lactide and a plurality of repeat units of glycolide at a molar ratio of about 85: 15 lactide to glycolide. In certain embodiments, the molecular weight of the copolymer is about 50 kDa, about 60 kDa, about 70 kDa, about 80 kDa, about 90 kDa, about 100 kDa, about 110 kDa, about 120 kDa, about 130 kDa, about 140 kDa, about 150 kDa, about 160 kDa, about 170 kDa, about 180 kDa, about 190 kDa, about 200 kDa, about 210 kDa, about 220 kDa, about 230 kDa, about 240 kDa, about 250 kDa. In certain embodiments, the molecular weight of the copolymer is about 20 kDa to about 240 kDa (e.g., 190 kDa to 240 kDa). In certain embodiments, the molecular weight of the copolymer is about 60 kDa to about 110 kDa e.g., 66 kDa to 107 kDa). In certain embodiments, the molecular weight of the copolymer is about 190 kDa to about 240 kDa (e.g., 190 kDa to 240 kDa).

[0049] In certain embodiments, the concentration of the copolymer is about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, or about 150 mg / mL. In certain embodiments, the concentration of the copolymer is about 30 mg / mL. In certain embodiments, the concentration of the copolymer MTV-24625 is about 75 mg / mL. In certain embodiments, the concentration of the copolymer is about 100 mg / mL.

[0050] In certain embodiments, the diameter of the microparticle is about 50 pm to about 300 pm; or about 50 pm, about 75 pm, about 100 pm, about 125 pm, about 150 pm, about 175 pm, about 200 pm, about 225 pm, about 250 pm, about 275 pm, or about 300 pm.

[0051] In certain embodiments, the encapsulation efficiency of the lipid nanoparticle is greater than about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. In certain preferred embodiments, the encapsulation efficiency of the lipid nanoparticle is greater than about 60%. In certain embodiments, the encapsulation efficiency of the lipid nanoparticle is about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. In certain preferred embodiments, the encapsulation efficiency of the lipid nanoparticle is about 60%.

[0052] In further aspects, the present disclosure provides methods of delivering a therapy to a subject in need thereof, comprising contacting the subject with the microparticles disclosed herein.

[0053] In further aspects, the present disclosure provides injectable needles comprising the microparticles herein.

[0054] In further aspects, the present disclosure provides methods of delivering a therapy to a subject in need thereof, comprising contacting the subject with the injectable needles disclosed herein.

[0055] Definitions

[0056] Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature used in connection with, and techniques of, chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics and protein and nucleic acid chemistry, described herein, are those well known and commonly used in the art.

[0057] The methods and techniques of the present disclosure are generally performed, unless otherwise indicated, according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout this specification. Chemistry terms used herein, unless otherwise defined herein, are used according to conventional usage in the art, as exemplified by “The McGraw-Hill Dictionary of Chemical Terms”, Parker S., Ed., McGraw-Hill, San Francisco, C.A. (1985). MTV-24625

[0058] All of the above, and any other publications, patents and published patent applications referred to in this application are specifically incorporated by reference herein. In case of conflict, the present specification, including its specific definitions, will control.

[0059] The term “agent” is used herein to denote a chemical compound (such as an organic or inorganic compound, a mixture of chemical compounds), a biological macromolecule (such as a nucleic acid, an antibody, including parts thereof as well as humanized, chimeric and human antibodies and monoclonal antibodies, a protein or portion thereof, e.g., a peptide, a lipid, a carbohydrate), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. Agents include, for example, agents whose structure is known, and those whose structure is not known.

[0060] A “patient,” “subject,” or “individual” are used interchangeably and refer to either a human or a non-human animal. These terms include mammals, such as humans, primates, livestock animals (including bovines, porcines, etc.), companion animals (e.g., canines, felines, etc.) and rodents e.g., mice and rats).

[0061] “Treating” a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0062] The term “preventing” is art-recognized, and when used in relation to a condition, such as a local recurrence (e.g., pain), a disease such as cancer, a syndrome complex such as heart failure or any other medical condition, is well understood in the art, and includes administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition. Thus, prevention of cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving a prophylactic treatment relative to an untreated control population, and / or delaying the appearance of detectable cancerous growths in a treated population versus an untreated control population, e.g., by a statistically and / or clinically significant amount.

[0063] “Administering” or “administration of’ a substance, a composition or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a composition or an agent can be administered, intravenously, arterially, MTV-24625 intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct). A composition or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the composition or agent. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0064] Appropriate methods of administering a substance, a composition or an agent to a subject will also depend, for example, on the age and / or the physical condition of the subject and the chemical and biological properties of the composition or agent (e.g., solubility, digestibility, bioavailability, stability and toxicity). In some embodiments, a composition or an agent is administered orally, e.g., to a subject by ingestion. In some embodiments, the orally administered composition or agent is in an extended release or slow release formulation, or administered using a device for such slow or extended release.

[0065] As used herein, the phrase “conjoint administration” refers to any form of administration of two or more different therapeutic agents such that the second agent is administered while the previously administered therapeutic agent is still effective in the body (e.g., the two agents are simultaneously effective in the patient, which may include synergistic effects of the two agents). For example, the different therapeutic compositions can be administered either in the same formulation or in separate formulations, either concomitantly or sequentially. Thus, an individual who receives such treatment can benefit from a combined effect of different therapeutic agents.

[0066] A “therapeutically effective amount” or a “therapeutically effective dose” of a drug or agent is an amount of a drug or an agent that, when administered to a subject will have the intended therapeutic effect. The full therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. The precise effective amount needed for a subject will depend upon, for example, the subject’s size, health and age, and the nature and extent of the condition being treated, such as cancer or MDS. The skilled worker can readily determine the effective amount for a given situation by routine experimentation.

[0067] As used herein, the terms “optional” or “optionally” mean that the subsequently described event or circumstance may occur or may not occur, and that the description includes instances where the event or circumstance occurs as well as instances in which it does not. MTV-24625

[0068] The phrase “pharmaceutically acceptable” is art-recognized. In certain embodiments, the term includes compositions, excipients, adjuvants, polymers and other materials and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0069] The phrase “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filter, diluent, excipient, solvent or encapsulating material useful for formulating a drug for medicinal or therapeutic use.

[0070] As used herein, the phrase “encapsulation efficiency” refers to the percentage of a compound (e.g., drug) or other material (e.g., adjuvant) that is successfully trapped inside a nanoparticle (e.g., lipid nanoparticle) or micelle following a process (e.g., a chemical reaction) design to trap the compound or other material inside the nanoparticle or micelle.

[0071] EXAMPLES

[0072] The invention now being generally described will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention and are not intended to limit the invention.

[0073] Example 1 : Synthesis of Exemplary Lipid Nanoparticles

[0074] Materials and Methods

[0075] We first create mRNA-LNP using microfluidic equipment, which ensures precise control over particle size and composition. Next, we utilize a double emulsion microfluidic platform to produce micron-sized polymer-lipid vesicles. In this process, the mRNA-LNPs, dissolved in PBS buffer, serve as the encapsulated phase, while the shell of the microvesicles is formed in the organic shell phase. Different molecular weights of PLGA polymers are mixed with different molecular weights of lipids in the shell phase. By carefully adjusting the flow rates in the continuous phase of the microfluidic device, we can produce large vesicles with diameters of approximately 100 micrometers. These particles are stored at 4°C to maintain their stability before being injected intramuscularly into mice. Using FLUC mRNA as our model, we conducted both in vitro and in vivo studies to evaluate the responses of these mRNA-loaded vesicles. For the in vitro experiments, the particles are evaluated at 37°C, mimicking physiological conditions. We observe the microparticles under confocal microscope, MTV-24625 quantifying microparticle breakage and mRNA-LNP release using fluorescent lipids in the nano LNP formulation. We collect the supernatant from these particles and measure the ribogreen mRNA activity and cell transfection efficacy to determine the release profile and biological activity. For the in vivo experiments, we inject the mRNA-loaded vesicles into mice and monitor the bioluminescence using IVIS imaging. This allows us to validate the efficacy of mRNA released from microparticles over time. Through these comprehensive experiments, we are able to identify a library of release times of mRNA-LNPs from different formulations of micron-sized polymer-lipid vesicles.

[0076] Table 1. List of solvents, shell materials, and excipients used. MTV-24625 MTV-24625 MTV-24625

[0077] Results and Discussions

[0078] The encapsulation process for mRNA-LNP involves a series of steps designed to ensure precise control over particle formation and stability. Initially, mRNA-LNP is encapsulated within an aqueous core using a glass capillary device, allowing for a high degree of precision in creating uniform particles. Following this, double emulsions are formed with an internal aqueous phase containing mRNA-LNP and a surrounding spherical shell to protect the payload. The resulting microparticles are then collected in PBS buffer to stabilize them for further use, as shown in Figure 1. In vitro testing is conducted to evaluate the release and cytotoxicity of the encapsulated mRNA-LNP, using cell cultures to determine the biocompatibility and effectiveness of the formulations. Following these in vitro studies, the microparticles are subcutaneously injected into mice to assess their performance in a biological environment, providing insight into their release characteristics and therapeutic potential, as depicted in Figure 1. The development process is iterative, involving multiple rounds of optimization to refine the material composition of the shell. This iterative approach, highlighted in the red box, ensures that the microparticles meet specific stability and release requirements, especially for in vivo applications, as demonstrated in the stepwise protocol shown by Figure 1. The lipid nanoparticle (LNP) formulations used for encapsulating mRNA are specifically designed to protect the mRNA during encapsulation and release. These formulations are optimized for key factors such as particle size, encapsulation efficiency, and controlled release, which are essential for therapeutic applications, as outlined in Figure 1. To achieve the desired encapsulation, we utilize a glass capillary microfluidics device that operates in two regimes. The first regime produces water-in-oil-in-water double emulsions with a thin shell, which allows for controlled and precise release under certain conditions. The second regime generates water-in-oil -in-oil -in-water triple emulsions with a thicker, asymmetric shell, offering enhanced structural integrity for more complex or delayed release scenarios. This flexibility in producing different types of emulsions provides a tailored approach to drug delivery, as demonstrated by Figure 1. Together, this encapsulation process, along with the advanced formulations and microfluidic techniques, offers a novel and efficient approach to delivering mRNA therapeutics. The in vitro and in vivo data generated from these methods guide the ongoing development and optimization of microparticles for a wide range of biomedical applications, as seen in Figure 1. The process of stabilizing and optimizing the release of MTV-24625 mRNA-LNP in microparticles involves several stages of screening and evaluation. Initially, two variants are tested for stabilizing the core of soluble mRNA-LNP, while four additional variants are screened for their ability to stabilize the shell of the microparticles. These variants are assessed over a designated period of time to determine their impact on maintaining the structural integrity and functionality of the encapsulated mRNA-LNP. By combining the most effective core and shell stabilizing variants, we identify the formulation combining lipid and polymer for achieving delayed release of mRNA-LNP in vivo, as shown in Figure 2.

[0079] To further refine the formulation, we evaluate five excipient solutions for their effectiveness in stabilizing mRNA-LNP at 37°C. From this screening process, PEG (4 wt%) and PVA (1 wt%) are identified as the top-performing excipients based on their ability to maximize encapsulation efficiency, ensuring that the mRNA-LNP remains stable over time. This step is critical, as excipient selection directly influences the long-term stability and controlled release of the therapeutic payload, as depicted in Figure 2. Next, we conduct a comprehensive screening of various solvents needed for the encapsulation process to identify the most suitable option for the microparticle shell phase. Ethyl acetate emerges as the optimal solvent for encapsulating mRNA-LNP, providing high stability and preventing premature leakage. In contrast, a solvent mixture of chloroform and hexane results in significant mRNA- LNP leakage into the microparticle shells, rendering it unsuitable for this application. This highlights the importance of solvent selection in maintaining the integrity of the encapsulated mRNA, as demonstrated in Figure 2. Additionally, we employ a contour detection method to analyze both the size and number of mLPM particles formed during the encapsulation process. This technique provides a precise assessment of particle distribution and characteristics. Alongside this analysis, we evaluate the mechanical stability of lipid-formulated shells at 37°C by monitoring the number of intact particles over time. This testing helps determine the durability of the encapsulated particles under physiological conditions, ensuring that they remain stable until the desired release is triggered, as shown in Figure 2. Through the combined screening of core and shell stabilizers, excipients, solvents, and mechanical properties, we identify several formulations with delayed release mechanisms. We find that for optimized stability, formulation F9, containing 20 wt% of DOPC and 80 wt% DPPC, leads to stable microparticles with 80% and 65% of microparticles remaining after 10 days and 14 days at 37 °C. This stable formulation would be suitable for sustained release for 14 days. In addition, we find that for delayed pulsatile release, formulations F5 and F6, containing 50 wt% DOPC and 50 wt% lipid 5 at 6mg / mL and 9mg / mL, leads to a significant release of approximately 40% microparticle breaking and 80% microparticle breaking between day 6 and 10 at 37 °C. MTV-24625

[0080] Thus, F5 and F6 are suitable for delayed pulsatile release of the drug at approximately 1-week time point inside the body. Through these experiments, we show the high stability of the encapsulating microparticles. This iterative approach ensures that the encapsulated mRNA- LNP remains protected until its intended release in vivo, providing a robust solution for mRNA- based therapeutics, as illustrated in Figure 2.

[0081] The stabilization and controlled release of mRNA-LNP from hybrid polymer-lipid microparticles were evaluated through a comprehensive screening process. Initially, two variants were tested for their ability to stabilize the soluble mRNA-LNP core, while four variants were assessed for stabilizing the outer shell of the microparticles over a designated period. These variants were tested in combination to determine the most effective formulation for achieving delayed release of mRNA-LNP in vivo. The stability of both the released mRNA- LNP from the core and the polymer-lipid hybrid shell were analyzed, and the key variables related to these stability factors are highlighted in Figure 3. To further develop the formulation, various combinations of PLGA and POPC lipids were explored. Three specific formulations of hybrid polymer-lipid microparticles — referred to as PL1, PL2, and PL3 — were created and summarized in Figure 3. Each formulation represents a distinct balance of polymer and lipid components, designed to enhance the stability and release characteristics of the encapsulated mRNA-LNP. The combination of PLGA and POPC lipids enables these microparticles to offer both protection to the mRNA payload and control over its release under physiological conditions. This screening helps in refining the selection of materials to optimize the encapsulation efficiency and release profile. In order to assess the physical properties of the encapsulated LNPs, microparticles were broken by extruding them through a needle. This process allowed for the evaluation of the size distribution and encapsulation efficiency of the mRNA-LNPs contained within the microparticles. The analysis, as shown in Figure 3, revealed that the LNPs had an average diameter ranging from 200 nm to 300 nm. In addition, the encapsulation efficiency of the mRNA within these LNPs was found to be approximately 70% to 80%, demonstrating the effectiveness of the hybrid microparticles in maintaining high levels of encapsulated mRNA. These findings are essential for determining the optimal size and efficiency required for therapeutic delivery. A critical observation was made regarding the relationship between the molecular weight of the polymer component and the release profile of the mRNA-LNP. As depicted in Figure 3, the formulation PL1 containing 80 wt% PLGA 66,000-107,00 Da and 20wt% POPC and formulation PL2 containing 80 wt% PLGA 76,000- 115,000 Da and 20wt% POPC both exhibit a significant drop in the number of microparticles between day 14 and 21, while PL3 containing 80 wt% 54,000-69,000 Da PLGA and 20 wt% MTV-24625

[0082] POPC and S8 containing 100wt% POPC exhibit gradual drop in the number of microparticles over 21 days, as shown by Figure 3. These results show that PL1 and PL2 formulation are suitable for pulsatile release of drugs between day 14 and day 21, while PL3 and S8 formulations are suitable for sustained release over 21 days. We found that increasing the molecular weight of the polymer resulted in a significant delay in the rupturing of the microparticles. This delay led to a slower and more controlled release of the mRNA cargo from the core, offering valuable insights for applications requiring sustained release over an extended period. This finding underscores the importance of tuning the polymer composition to achieve specific release kinetics, making this approach highly adaptable for various therapeutic applications.

[0083] The optimization of mRNA-LNP encapsulation and delayed release from PLGA-based microparticles was evaluated by screening several variants, focusing on the stability of both the core and shell over time. In Figure 4, we screened two variants for stabilizing the core of soluble mRNA-LNP and four variants for stabilizing the microparticle shell. These were tested over a designated period to determine the optimal formulation for delayed release of mRNA- LNP in vivo. The study also explored the stability of PLGA with increasing molecular weights and concentrations, comparing their performance against hybrid polymer-lipid microparticles, as previously described in Figure 4. Based on this screening, the best PLGA formulations were selected for further in vivo testing to assess their ability to achieve delayed release of the mRNA-LNP payload. To refine these formulations, we summarized the different concentrations and molecular weights of PLGA used in Figure 4. The molecular weight and concentration of the polymer are key variables that influence the release profile and mechanical properties of the microparticles. By varying these parameters, we aimed to optimize the microparticles for sustained and controlled release of mRNA-LNP in physiological conditions. In Figure 4, we conducted a mechanical breakdown test on one of the selected PLGA formulations at 37°C over time using confocal microscopy. This allowed us to visualize the structural integrity of the microparticles as they degrade and assess their ability to protect the mRNA-LNP payload during storage and release. The mechanical properties of the microparticles are critical to ensuring that they remain stable during handling and injection but degrade appropriately once they are inside the body. We also assessed the quality of the mRNA-LNP released by the microparticles over time at 37°C. As shown in Figure 4, the encapsulation efficiency of the mRNA-LNP decreased from approximately 80% to 60% over seven days, while the amount of mRNA released increased from approximately 1 pg to 1.4 pg over the same period. This data was obtained using the RiboGreen assay, which quantifies the MTV-24625

[0084] RNA content. The results indicate that although encapsulation efficiency declines over time, the release of the mRNA increases, demonstrating a controlled release profile suitable for therapeutic applications that require delayed mRNA delivery. In Figure 4, we analyzed three identical samples of PLGA-based microparticles, each containing 50 pL of microparticles suspended in 200 pL of PBS buffer, stored under different conditions. The first sample was stored at 4°C, the second at 37°C, and the third sample underwent harsh pipetting to simulate mechanical breakdown. Using the RiboGreen assay, we evaluated the encapsulation efficiency and release profile of the mRNA-LNPs. The results showed that the encapsulation efficiency decreased from approximately 80% to 65% when comparing microparticles stored at 4°C to those stored at 37°C. Additionally, the release of mRNA-LNPs increased from approximately 3% to 9% when comparing the same conditions. This indicates minimal release at the storage temperature of 4°C, with a delayed release observed at physiological temperature (37°C). These findings confirm that the microparticles exhibit stable storage behavior at lower temperatures while offering controlled, temperature-triggered release at body temperature. These results underscore the potential of PLGA-based microparticles for applications requiring sustained and delayed release of mRNA-LNPs. By fine-tuning the polymer composition, molecular weight, and formulation conditions, these microparticles can be optimized for longterm storage stability and precise release kinetics, offering a versatile platform for mRNA- based therapeutics. In Figure 4H, firefly luciferase (Flue) mRNA-LNPs were prepared at equal mRNA concentrations and formulated with varying soluble excipient cocktails comprising L-serine, polyethylene glycol (PEG), and sucrose. To evaluate stability under physiological stress, the formulations were incubated at 37 °C for two weeks and subsequently applied to cultured cells at different time points. Bioluminescence imaging was performed to quantify luciferase expression as a measure of preserved translation efficiency. Control samples stored at 4 °C demonstrated maximal activity, whereas free mRNA-LNP lacking stabilizing excipients showed significant loss of signal following incubation at 37 °C. By contrast, several excipient cocktails, including formulations E13, E15, and E17, preserved substantially higher bioluminescence relative to other formulations, demonstrating that specific combinations of L-serine, PEG, and sucrose are capable of stabilizing mRNA-LNPs during exposure to extended physiological temperature stress. Importantly, these optimized formulations were subsequently incorporated into microparticles, and upon release, the encapsulated contents retained measurable efficacy in the same in vitro bioluminescence assay even after incubation at 37 °C for two weeks. These findings demonstrate that excipient- stabilized microparticle systems preserve the biological activity of mRNA-LNPs following MTV-24625 stress exposure, providing a unique and unexpected technical advantage over conventional free mRNA-LNP formulations that rapidly lose function.

[0085] The study investigated the screening and testing of PLGA formulations for mRNA- LNP encapsulation and delayed release, with the goal of optimizing the performance of these formulations for controlled and sustained mRNA delivery. Figure 5 provides an overview of the experiments, focusing on the influence of PLGA molecular weight and concentration on microparticle stability and release kinetics. The stability of PLGA formulations with increasing molecular weights and concentrations was compared. These formulations were carefully selected to balance the need for mechanical integrity and controlled degradation, which together enable the delayed release of mRNA-LNP in vivo. The molecular weight and concentration of PLGA directly impact how quickly or slowly the microparticles degrade and release their payload. Formulations identified as optimal based on these properties were subsequently subjected to in vivo testing to confirm their ability to control the timing of release under physiological conditions. We summarize the different PLGA concentrations and molecular weights used in this study. Both parameters are critical for tuning the mechanical properties and release profiles of the microparticles. Higher molecular weight PLGA provides greater structural stability, while varying concentrations influence encapsulation efficiency and the overall release rate of the mRNA-LNP payload. This fine-tuning process is essential for developing a formulation that remains stable during storage and releases the mRNA in a controlled manner once administered in vivo. An in vivo experiment was conducted where 100 pL of Flue mRNA-LNP encapsulated in microparticles using the Pl_2 polymer shell formulation was injected into three mice (mice 1, 2, and 3). An equivalent dose of free mRNA- LNP, matching the dosage inside the microparticles, was injected into two additional mice (mice 4 and 5). Six hours after injection, mice 1, 2, and 3 showed no bioluminescence, indicating that the encapsulated mRNA-LNP had not been released. Conversely, mice 4 and 5, which received the free mRNA-LNP, exhibited strong bioluminescence, indicating rapid expression of the mRNA. Fluorescence levels were much higher in mice 1, 2, and 3 compared to mice 4 and 5, suggesting that the encapsulated LNPs remained intact within the microparticles. Both bioluminescence and fluorescence decayed over time, confirming that the PLGA formulation did not release the mRNA-LNP during the observation period. We also explore the effect of mechanical disruption on the release of mRNA-LNP from PLGA microparticles. In this experiment, 100 pL of Flue mRNA-LNP encapsulated in microparticles with the P2_l polymer shell formulation was mechanically broken, and the disrupted particles were injected into mice 1 and 2. In comparison, the same volume of intact microparticles was MTV-24625 injected into mice 3, 4, and 5. Mice 1 and 2, which received the broken microparticles, exhibited 1 to 3 orders of magnitude higher bioluminescence and several times higher fluorescence as compared to mice 3, 4, and 5, which were injected with the intact microparticles. These results indicate that the broken particles released the mRNA-LNP, while the intact particles protected the payload, delaying its release. This outcome confirms the PLGA microparticles' ability to provide controlled protection and release, depending on their structural integrity. These findings demonstrate that PLGA microparticles are highly effective for achieving controlled, delayed release of mRNA-LNP in vivo. By adjusting the molecular weight and concentration of PLGA, we were able to optimize the stability and release profile of the microparticles, making them an ideal platform for the sustained delivery of mRNA therapeutics.

[0086] In Figure 6, microparticles comprising poly(lactic-co-glycolic acid) (PLGA) and lipid surfactant coatings were formulated to encapsulate messenger RNA lipid nanoparticles (mRNA-LNPs) encoding a CO VID antigen. To evaluate the preservation of activity after encapsulation, the microparticles were mechanically opened prior to administration, releasing their encapsulated contents for injection. Groups of mice (n=3 per group) received a single subcutaneous dose of the released formulations, including Group G1 (PLGA 858s + lipids containing mRNA-LNP), Group G2 (PLGA 505s + lipids containing mRNA-LNP), and Group G3 (freshly prepared, non-encapsulated mRNA-LNP). Serum was collected at baseline (week 0) and at weeks 1, 2, and 3 post-injection and analyzed by ELISA for antibody titers. Results demonstrated that the mRNA-LNPs released from microparticles (Groups G1 and G2) generated immune responses that were at least equivalent to, and in some cases greater than, those produced by freshly prepared free mRNA-LNP (Group G3). Importantly, this immune response was achieved after a single primary dose, without the administration of a booster, highlighting the potency and stability of the encapsulated formulations. These findings provide compelling evidence that the microparticle delivery system not only maintains the integrity and immunogenicity of mRNA-LNPs during encapsulation and storage, but also protects the nucleic acid cargo against degradation that typically limits the efficacy of conventional free mRNA-LNPs. This preservation of function and enhancement of stability is unexpected in view of the known susceptibility of mRNA to hydrolysis and oxidative damage, and provides a substantial advantage for the long-term storage, transport, and deployment of mRNA therapeutics and vaccines. The disclosed system therefore represents a novel and industrially significant platform for enabling robust, stable, and scalable delivery of mRNA-LNPs for prophylactic and therapeutic applications. MTV-24625

[0087] Figure 7 illustrates fluorescence imaging of microparticles containing fluorescently labeled COVID mRNA-LNP following subcutaneous injection in mice. In this study, three groups of mice (G1-G3, n=3 per group) were each injected with microparticles encapsulating fluorescent (red) mRNA-LNP, enabling non-invasive monitoring of mRNA-LNP retention in vivo. Each group received microparticles comprising COVID mRNA-LNP, but the formulations differed with respect to the molecular weight of PLGA and the lipid compositions in the microparticle shells used. Fluorescence imaging revealed that, across all groups, the microparticles retained the encapsulated mRNA-LNP at the injection site for extended periods, with signal persistence far exceeding what is typically observed with free mRNA-LNP lacking encapsulation. The sustained fluorescence provides direct evidence that the disclosed microparticle formulations effectively stabilize and protect mRNA-LNP in vivo, preventing rapid clearance or degradation. These results demonstrate that variations in PLGA molecular weight and lipid content can be tuned to modulate release kinetics and retention time, thereby providing a versatile platform for durable in vivo delivery of mRNA-LNP. This capability distinguishes the invention from conventional free mRNA-LNP formulations, which rapidly lose activity, and establishes a novel means of extending the bioavailability of mRNA therapeutics for long-term prophylactic and therapeutic use.

[0088] Summary

[0089] Our experiments have yielded promising results, demonstrating the efficacy and potential of our developed drug delivery system. In the in vitro studies, we observed rupture of microparticles leading to release of mRNA from the giant polymer-lipid vesicles at different time points. The Ribogreen mRNA activity assays showed sustained mRNA activity, while cell transfection experiments confirmed low cytotoxicity from microparticles with mRNA- LNPs. These results indicate that our vesicles can effectively protect and release mRNA over time. Dynamic Light Scattering (DLS) analysis confirmed that the size of the released LNPs was consistently around 200-300 nm, ensuring optimal cellular uptake and stability. This precise size control is crucial for efficient mRNA delivery and transfection efficacy. Additionally, we tested the encapsulation efficiency of mRNA in LNPs in vitro and found that the encapsulation rate was consistently around 70-90%. This high encapsulation rate ensures that a significant proportion of mRNA is effectively delivered.

[0090] We created a library of different polymer-lipid formulations with varying polymer-to- lipid ratios and polymer molecular weights. Formulations with lower polymer weights rupture to release mRNA near the 2-week mark, while those with higher molecular weights rupture to MTV-24625 release mRNA closer to 3 weeks. This demonstrates the potential of our system in controlling release times, allowing for customized therapeutic regimens.

[0091] In the in vivo experiments, we compared the injected mRNA-LNP -loaded vesicles with injected free mRNA-LNP in solution. The IVIS imaging showed release of mRNA, with bioluminescent signals detected at high intensity for the free mRNA-LNP at different time points, and depending on the formulation of giant vesicle, low bioluminescence indicating mRNA not released for 24 hrs. The demonstration of mRNA release from microparticles highlights the benefits of our system.

[0092] Example 2: Further synthesis of Exemplary Lipid Nanoparticles

[0093] Poly(lactic-co-glycolic acid) (PLGA) is an FDA-approved biodegradable polymer widely used for controlled release formulations due to its tunable degradation rate and biocompatibility. By varying PLGA molecular weight and lactide:glycolide ratio, the degradation (and thus payload release) can be adjusted from weeks to months. However, conventional methods for making PLGA microspheres (like bulk emulsification) often produce polydisperse particles with variable release profiles and can suffer from low encapsulation efficiency for sensitive payloads such as mRNA-LNP. Microfluidic encapsulation offers a solution by generating highly uniform core-shell microcapsules via controlled double emulsification. In particular, glass capillary microfluidic devices and advanced chip-based droplet microfluidics allow the production of monodisperse double emulsions (water-in-oil-in- water) that template the formation of a polymer shell around an aqueous core. This approach yields microcapsules with precisely tunable shell thickness and core size by adjusting flow rates, enabling fine control over the timing of payload release.

[0094] For single-dose vaccine delivery, it is critical that the encapsulated mRNA-LNP remains stable until its intended release time. mRNA-loaded lipid nanoparticles (LNP) are sensitive to conditions like organic solvents, shear stress, and exposure to water-oil interfaces. To protect the mRNA-LNP during encapsulation and storage, our formulation incorporates stabilizing excipients in the core and shell of the microcapsules. In the core, we explore the use of a mixture of excipients, such as sucrose, L-serine, and PEG, along with hydrogels or viscosity-enhancing agents to create a supportive matrix around the LNP, minimizing direct contact with the organic solvent during the double-emulsion process. Previous studies have shown that forming a solid or gelled inner core can prevent droplet coalescence or rupture during shell solidification. For example, Li et al. (2017) encapsulated hydrophilic drugs in a gelatin-methacrylate (GelMA) hydrogel core and polymerized it in situ, which avoided core MTV-24625 breakup as the PLGA shell hardened. Inspired by this, we investigated hydrogel cores to stabilize the mRNA-LNP payload.

[0095] Another challenge is maintaining shell integrity and controlling release kinetics. Initial burst release - the rapid leakage of payload when the particle is first exposed to aqueous media - is a common issue with PLGA microspheres. Burst release can be exacerbated if the shell is thin or has defects, or if the core is liquid and puts pressure on the shell. We hypothesized that including lipid antioxidants in the shell matrix would act as plasticizers and stabilizers, improving the mechanical robustness of the shell and scavenging any reactive species that could degrade the polymer or mRNA. a-Tocopherol (Vitamin E) and coenzyme Q10 (ubiquinone) are two lipophilic antioxidants known to integrate into hydrophobic environments (such as lipid membranes or polymer matrices) and neutralize reactive oxygen species. Vitamin E derivatives have been used in polymer nanoparticles to enhance stability and even boost immune responses. Coenzyme Q10, meanwhile, is a well-known membrane antioxidant and cofactor, and its hydrophobic tail allows it to reside in lipid or polymer phases. By incorporating these antioxidants into the PLGA shell, we aim to increase shell stability (preventing cracks or premature degradation) and preserve the bioactivity of the mRNA-LNP payload over extended storage and in vivo deployment.

[0096] In addition to antioxidants, we incorporate phospholipids into the shell formulation to create a polymer-lipid hybrid microcapsule. The rationale is that lipids can provide a flexible, self-assembling interface that might reduce brittleness of the shell and help retain the aqueous core. The phospholipids improve stability of shells during storage at 4 °C and allows for thermally triggered release at higher temperature and prevent the aggregation of microcapsules. Few studies have added phospholipids like phosphatidylcholine to PLGA microspheres, but emerging reports suggest this can improve particle performance. In our design, we test mixtures including unsaturated lipids (POPC, DOPC), saturated lipids (DPPC), and PEGylated lipids (e.g., PEG2000-C14). We are particularly interested in DPPC (l,2-dipalmitoyl-sn-glycero-3- phosphocholine), which has a well-defined gel-to-fluid phase transition around normal body temperature (-37-41 °C). We hypothesize that a DPPC-containing shell will be stable and rigid at cooler temperatures (solid lipid phase at 4 °C) for long-term storage, but will soften or become more permeable at body temperature when the lipid transitions to a fluid phase. This thermal responsiveness could act as an intrinsic timer to initiate release once the vaccine is injected into a patient, while preventing significant release during refrigerated storage.

[0097] In summary, this work presents a bioengineering strategy to create core-shell PLGA microcapsules for single-injection mRNA vaccines, leveraging microfluidic precision MTV-24625 fabrication and novel shell additives (antioxidants and lipids) to achieve tunable, on-demand release. We detail the fabrication method, characterize the microcapsule structure, evaluate the influence of shell composition on stability (during fabrication, storage, and injection) and release profiles, and discuss the implications for vaccine delivery.

[0098] Materials and Methods

[0099] Microfluidic Device Fabrication: Core-shell microcapsules were produced using a coaxial glass capillary microfluidic device configured for double emulsion generation. The device comprised of two or three tapered glass capillaries arranged in a flow-focusing geometry: an inner capillary for the inner aqueous phase where mRNA-LNP is introduced from a previous synthesis, a middle injection capillary with a tapered opening nested inside a larger capillary carrying the middle oil phase (O) for shell materials, which in turn was aligned within an outer square capillary carrying the outer aqueous phase to extrude the microcapsules to different sizes. The injection capillary tip was finely pulled to an inner diameter of -30-100 pm and treated hydrophobic to generate the small aqueous droplets, while the collection capillary was treated to be hydrophilic at the collection capillary side to facilitate the formation of a W / O / W emulsion. By introducing oil fluids for shell materials from different channels, such as inside the injection capillary or through the gap between the injection capillary and the outermost capillary, thin to thick shells can be produced. By replacing the innermost capillary with a double core capillary, hydrogel with two components can be introduced for mixing inside the chip.

[0100] Materials and Formulation: The inner aqueous phase (core) contained the mRNA-LNP vaccine cargo. Model mRNA-LNPs were prepared by standard LNP methods using ionizable lipid, DSPC (distearoylphosphatidylcholine), cholesterol, and PEG-lipid, then formulated in an aqueous buffer (pH 7.4) with sugar stabilizers. To enhance core viscosity and stability, we optionally mixed the LNP suspension with excipients or a hydrogel precursor. For some batches, dextran- aldehyde and hyaluronic acid- hydrazone were used for hydrogel. In other batches, an excipient mixture of sucrose, PEG, and L-serine was used to create a viscous inner solution without solidifying it, to test the need for a solid core. The middle oil phase consisted of PLGA dissolved in a volatile, water-immiscible solvent (e.g., ethyl acetate or dichloromethane or chloroform). We used medical-grade PLGA with various molecular weights and lactide:glycolide ratios (e.g., 50:50 RG502H -12 kDa for fast release, up to 85: 15 RG858s -190 kDa for slower release). The polymer concentration was typically in the range of 5-15% (w / v). To this polymer solution we added our shell additives: 0-5% (w / v) of antioxidant (a-tocopherol and / or coenzyme Q10), and 0-10% (w / v) of phospholipid (such as MTV-24625

[0101] POPC, DOPC, DPPC, or a PEGylated C14 lipid). These additives dissolve or disperse in the organic phase along with PLGA. Vitamin E (a-tocopherol) is a hydrophobic oil-like liquid at room temperature, which can plasticize the polymer phase. Coenzyme Q10 is a waxy solid, soluble in many organic solvents, and can co-dissolve with PLGA. Phosphatidylcholines (POPC: 16:0-18: 1 PC, DOPC: 18: 1-18: 1 PC, DPPC: 16:0-16:0 PC) were dissolved in the organic solvent; note DPPC required gentle heating (~50 °C) to fully dissolve due to its higher phase transition temperature. The outer aqueous phase was a continuous water phase containing 1-2% (w / v) poly(vinyl alcohol) (PVA) as a stabilizer, plus osmolytes (e.g., 5% glycerol or sucrose) to balance osmotic pressure with the inner aqueous phase. The role of PVA is to adsorb on the surface of the forming droplets, preventing coalescence and supporting shell formation.

[0102] Microcapsule Fabrication: Double emulsions were generated by pumping the different phases into the microfluidic device using syringe pumps. Flow rates were adjusted to achieve a stable dripping regime yielding a core-shell droplet structure. For example, a typical flow setting was: inner aqueous 500 pL / hr, middle oil 200 pL / hr, outer aqueous 5 mL / hr, but these were tuned depending on the desired capsule size and shell thickness. By increasing the flow of the middle (polymer) phase relative to the inner phase, thicker shells were obtained, whereas higher inner flow produced thinner shells (larger core). The emerging w / o / w droplets were collected into a stirring aqueous bath (containing 0.5-1% PVA). As the solvent (ethyl acetate) diffused out into the continuous phase and evaporated, the PLGA and lipid components precipitated around the aqueous core, forming solid microcapsules within minutes. We found that the inclusion of antioxidant approach helped avoid clogging: This method prevented transient solidification of PLGA spikes that could otherwise block the microfluidic orifices. The entire production was carried out at room temperature (20-25 °C). The collected emulsion was kept at -4 °C during solvent extraction. After solidification (-5-10 min), the microcapsules were washed thoroughly by sedimentation and resuspension in fresh aqueous buffer to remove residual solvent and PVA.

[0103] Characterization: Capsule size and morphology were examined via confocal microscopy. The mean diameter of the capsules (outer diameter) and the core size were measured from micrographs (>100 capsules per formulation) to calculate microcapsule uniformity. Monodispersity was confirmed by a low coefficient of variation (typically <5% in diameter). Figure 8B shows a representative image of the monodisperse microcapsules produced. Fluorescence microscopy was used to visualize the core-shell structure by loading a fluorescent dye (e.g., TRITC-dextran) in the inner aqueous phase; a clearly delineated fluorescent core and dark shell region indicated successful core-shell architecture. MTV-24625

[0104] Encapsulation of mRNA-LNP was quantified by measuring the mRNA content in the microcapsules vs. initial loading. We used RiboGreen assay to ensure mRNA was encapsulated and intact. Additionally, LNP integrity was checked by electron microscopy (for particle morphology) and a fluorescence de-quenching assay (to see if the lipid bilayer remained intact within the capsule). For stability studies, capsules were stored at 4 °C and periodically tested for mRNA retention and LNP integrity (by releasing a sample in buffer with surfactant and analyzing the mRNA and lipid).

[0105] Release Studies: The in vitro release kinetics of mRNA-LNP from the microcapsules were evaluated in phosphate-buffered saline (PBS) at 37 °C (to mimic physiological conditions) and at 4 °C (to simulate storage). Approximately 5 mg of microcapsules were suspended in 1 mL of release medium in microcentrifuge tubes, maintained at the target temperature. At defined time points (e.g., days or weeks), tubes were centrifuged, supernatant collected for analysis (to measure released mRNA by UV absorbance or fluorescence if labeled, and released lipid by ELISA or another assay), and replaced with fresh buffer. We also imaged capsules at various time points to observe any physical changes (swelling, erosion, shell rupture).

[0106] Injection Force Testing: To assess whether the capsules could be delivered by syringe without damage, we performed injection force and post-injection integrity tests. Microcapsule suspensions were drawn into standard 1 mL syringes with needles and injected through at a controlled speed. The force on the plunger was recorded using a texture analyzer. The effluent was collected and examined under a microscope to check if capsules remained intact or if they had burst during the high shear passage through the needle. This test is important because coreshell capsules could potentially fracture if the shell is brittle. The role of shell additives in mitigating this was evaluated by comparing, for example, pure PLGA shells vs. PLGA+ Vitamin E shells for incidence of breakage.

[0107] Results

[0108] Microfluidic Generation of Core-Shell Microcapsules

[0109] Using the glass capillary microfluidic setup, we successfully generated highly uniform core-shell microcapsules encapsulating an aqueous mRNA-LNP core. The microfluidic flowfocusing allowed precise tuning of capsule dimensions. As shown by Figure 8A, we designed three microfluidic chips, ranging from a thin shell microcapsule generator, a thick shell microcapsule generator, and a generator for thick shell microcapsule encapsulating an on-chip hydrogel core. As shown in Figure 8B, these double emulsion droplets produced had a well- defined core and shell immediately after formation. The mean outer diameter of the capsules MTV-24625 was typically 100 ± 3 pm with a core diameter of ~80 pm, yielding a shell thickness on the order of 10 pm (these values could be adjusted by changing flow rates). Capsules remained monodisperse over hours of continuous production; no significant change in droplet size was observed over 12 h of production, indicating a stable microfluidic emulsification process. As shown by Figure 8C, the double emulsion templating method led to encapsulation efficiencies >90% for the aqueous core. Very little mRNA-LNP was found in the outer continuous phase after capsule formation, suggesting that the majority of the inner droplet was successfully “captured” by the polymer shell as solvent diffused out. As shown by Figure 8D, When using a viscous continuous flow of higher PVA weight percent, we observe that the shells can transition from spherical to elongated shapes, offering tunability of release through the shapes of microcapsules. Moreover, the size of microcapsules can be tuned by flow rates of the continuous flow.

[0110] Confocal imaging of microcapsules confirmed a hollow core-shell structure. Intact capsules showed a spherical shell with a single internal cavity. By fracturing some capsules, we measured shell thickness directly and found good agreement with optical measurements. The shell surface of pure PLGA capsules was non-porous right after fabrication. Over time in buffer at 37 °C, the shell surfaces became rough and eventually showed pores as the polymer began to degrade (especially for thinner shells), consistent with diffusion-driven payload release followed by bulk erosion.

[0111] Incorporating antioxidants (Vitamin E and CoQlO) into the PLGA shell had notable effects on both the fabrication process and the properties of the resulting microcapsules. During microfluidic emulsification, we found that adding 1-5% (w / v) of a-tocopherol to the polymer solution reduced the solution viscosity and improved flow stability. This allowed us to use higher effective polymer content (or achieve thicker shells) without encountering the jetting or clogging issues that sometimes arise with very concentrated PLGA solutions. Operators of the microfluidic device noted that formulations with antioxidant were less prone to channel clogging, likely because the antioxidant acts as a processing aid, keeping the polymer solution less prone to precipitation. Indeed, a-tocopherol is a known plasticizer for polymers; it likely lowered the PLGA solution’s viscosity and evaporation rate slightly, giving more time for stable capsule formation. Similarly, CoQlO in the shell (at ~2%) helped the formation of thin- shelled capsules: with pure PLGA, attempting to make shells thinner than ~5 pm sometimes led to shell rupture during solvent removal (the delicate shell could not form properly), but with CoQlO and VE present, we could form ultra-thin shells (~2-3 pm) that still encapsulated the core. We hypothesize CoQlO, being a hydrophobic molecule, integrates into the polymer MTV-24625 matrix and strengthens the interface between the polymer shell and the aqueous core, preventing the core from bursting out during solidification.

[0112] One observation was that CoQlO and VE enabled stable encapsulation of hydrogel cores. Without antioxidants, when we tried to encapsulate a pre-formed hydrogel bead (e.g., a 1% alginate or partially crosslinked gelatin droplet) as the core, the PLGA shell sometimes could not properly form around the gel - the rigid hydrogel core would often detach or leak out, resulting in malformed capsules. However, with CoQlO in the shell formulation, the hydrophobic shell adhered better to the hydrophilic gel core, yielding intact core-shell structures. CoQlO might be acting to modify the interfacial tension or elasticity of the forming shell, allowing it to wrap around the gel core more effectively. In essence, antioxidant-doped shells had improved film-forming capability, even around challenging core materials.

[0113] Vitamin E (a-tocopherol) and CoQlO within the shell are expected to provide chemical stability benefits as well. These antioxidants can scavenge peroxides and acid radicals generated as PLGA undergoes hydrolysis. We observed that capsules containing antioxidants had a slightly less acidic microenvironment upon degradation. Using a pH-sensitive dye in the core, we monitored core pH over time. Pure PLGA shells led to a drop in core pH to -5.5 during degradation (due to lactic / glycolic acid buildup), whereas shells with antioxidants stabilized around pH 6-6.5. The antioxidants likely consumed some of the acidic by-products or prevented autocatalytic degradation from accelerating. This more moderate pH environment is beneficial for mRNA and LNP stability, as extreme acidity can degrade mRNA or damage lipid structures.

[0114] Importantly, the presence of Vitamin E in the shell significantly reduced the initial burst release of mRNA-LNP. In release tests at 37 °C, PLGA capsules without antioxidant showed a small initial leakage (e.g., -10% of mRNA released in the first 48 hours, presumed to come from imperfectly trapped LNP near the surface). Capsules with a-tocopherol, however, released under 3% in that initial period; essentially, they maintained encapsulation until the programmed burst time. We attribute this to Vitamin E’s role in improving shell cohesion and possibly filling free volume in the polymer matrix, which could otherwise create channels for early diffusion. This finding is consistent with the idea that lipophilic additives can block initial drug diffusion pathways and require the polymer to degrade more to start release. Literature on PLGA nanoparticles has noted that adding DPPC (a lipid) slowed the release of paclitaxel by creating a more hydrophobic, less permeable matrix; in our case, Vitamin E may play a similar role in slowing aqueous diffusion out of the capsule initially.

[0115] Tunable Shell Thickness and Release Timing MTV-24625

[0116] An advantage of this microfluidic approach is the ability to tune shell thickness and polymer properties to program the release delay. In our experiments, as shown in Figure 9, we created capsules with shell thicknesses ranging roughly from 5 pm up to 20 pm by varying the flow rate ratio of the middle (PLGA) phase to the inner phase. Thicker shells (achieved with higher polymer flow or concentration) resulted in significantly slower in vitro release of the encapsulated mRNA-LNP. In PBS at 37 °C, capsules with the thinnest shells (-5 pm) released their payload with an initial burst and completed release by ~2-3 weeks, as shown by Figure 9A, whereas the thickest-shell capsules (~20 pm) exhibited a long lag phase and released over 3 weeks, as shown by Figure 9B and Figure 9C. The release profiles showed a clear shell thickness-dependent kinetic behavior, consistent with a diffusion / erosion-controlled mechanism shown by simulation in Figure 9D. Thinner shells allow water to penetrate faster and form pores or cracks sooner, while thicker shells maintain a barrier longer, delaying the onset of release.

[0117] We also modulated the polymer composition of the shell to adjust release timing. Using lower molecular weight (MW -7-17 kDa) PLGA or a higher glycolide content (fast-degrading 50:50 PLGA) led to earlier release, whereas higher MW (-190 kDa) or more lactide-rich (e.g., 85: 15 PLGA) led to extended release times. These findings mirrored the in vivo results reported by Guyon et al., where “short”, “medium”, and “long” delay formulations were achieved by different PLGA types (and correspondingly produced booster immune responses at different times). Our data confirm that polymer selection is an effective knob to tune the release schedule, complementary to adjusting shell thickness.

[0118] Injectable microcapsules with reduced initial burst

[0119] Notably, these antioxidants and lipid-enriched microcapsules showed less initial burst during injection, as shown by Figure 10, compared to traditional batch-fabricated PLGA particles. In many conventional microspheres, drug release can be triphasic with a substantial initial burst, a slow diffusion phase, and then a second burst when polymer erosion accelerates. In our core-shell capsules, we observed an initial delay (lag phase with minimal release) followed by a relatively rapid “burst” when the shell finally failed or became sufficiently porous, releasing most of the payload in a short window. This shows the superior effect of the including antixodiant and lipids in the PLGA shells. This behavior is desirable for vaccines, as it would mimic an intentional booster dose. For instance, one formulation remained intact for -4 weeks, then released -80% of its mRNA-LNP payload over the ensuing 3 days. The synchrony of release is attributed to the core-shell structure, which tends to retain the payload until a critical point when the shell yields. We confirmed via microscopy that release MTV-24625 corresponded with shell rupture or the appearance of substantial pore networks in the shell (SEM images showed holes in the shell coinciding with the onset of release).

[0120] Incorporation of phospholipids into the shell had multifaceted benefits. First, we observed that adding a small fraction of phosphatidylcholine (PC) improved the colloidal stability of the capsules in aqueous suspension. Capsules made of pure PLGA would sometimes clump or stick together during solvent evaporation or storage, partly due to hydrophobic interactions or insufficient PVA on their surface. When lipids were included, especially the PEGylated lipid (PEG2000-C14) or DOPC, the capsule surfaces were more lipophilic and presumably coated with a lipid monolayer, which reduced aggregation. Essentially, the lipids likely migrated to the capsule surface during formation, acting as a built-in stabilizer analogous to how lipids coat emerging polymer nanoparticles in some formulations. Zeta potential measurements supported this: lipid-coated capsules had a less negative zeta potential (around -10 mV) than PVA-stabilized PLGA capsules (-25 mV), indicating a different surface chemistry possibly dominated by lipid headgroups rather than adsorbed PVA.

[0121] Among the lipids tested, DPPC stood out due to its thermal behavior. Differential scanning calorimetry of DPPC-containing capsules showed a phase transition peak around 31- 39 °C, confirming that DPPC in the solid shell undergoes its gel-to-liquid crystalline transition near physiological temperature. At 4 °C, DPPC is in a rigid gel phase (with a pre-transition around 28 °C as well), which in our hypothesis helps “lock in” the shell structure during cold storage. In release studies, DPPC-containing capsules were extremely stable at 4 °C - virtually no mRNA release was detected even over 1-2 months at 4 °C. However, when transferred to 37 °C, these same capsules began releasing their payload after a delay that corresponded with the lipid melting. For example, one formulation with 5% DPPC in the shell showed <1% release at 4 °C over 30 days, but once moved to 37 °C, it released -50% of its payload in the subsequent 10 days. Another formulation with 10% DPPC was even more striking: it retained everything at 4 °C (for 2 months in one test), and only when warmed to body temperature did the polymer begin to degrade and release content. This suggests a temperature-triggered release mechanism: below the lipid Tm, the polymer-lipid matrix is less permeable or less prone to water ingress, whereas above Tmthe increased fluidity of lipid domains may create pathways for water and facilitate polymer erosion. Essentially, DPPC provides a built-in “thermal fuse” that starts the release clock upon injection into a patient, but keeps the capsule inert in the fridge (solving one potential issue of unintended release during storage). This property could be very useful for ensuring long shelf-life of single-shot vaccine formulations. MTV-24625

[0122] We also found that the presence of lipids like DPPC or POPC in the shell mitigated the brittle behavior that pure PLGA shells sometimes exhibit. When dry or upon sudden stress (such as during syringe injection), pure PLGA shells can crack, causing premature release. Lipid-containing shells were noticeably more deformable and resilient. In injection force tests, suspensions of capsules with 0% lipid required an average force of ~15 N to push through a 25G needle and some capsules ruptured (we detected a burst of mRNA in the first few drops out of the needle). In contrast, capsules with 5% DPPC or POPC in the shell required a slightly lower force (~12 N) and almost none ruptured during injection - the mRNA remained encapsulated as verified by no spike in initial release. The softer, more compliant nature of a polymer-lipid composite shell likely allows it to squeeze through the narrow needle lumen without shattering. This is an important practical benefit: less initial burst upon injection, meaning the booster dose will not leak out at the time of administration but only at the intended later time. Prior research has noted that core-shell particles with liquid cores could burst under pressure leading to immediate release; our results indicate that by fortifying the shell with lipids and antioxidants (and / or solidifying the core), we can largely eliminate this concern.

[0123] Optimization of encapsulated mRNA-LNP with excipients In Figures 11A-11C, we evaluate how different ratios of sucrose, L-serine, and PEG act as excipients to stabilize soluble mRNA-LNP formulations under thermal stress. Using Flue mRNA-LNP as a model system, we measured in vitro luminescence to assess transfection efficiency after incubation at 4 °C (1 day), 50 °C (1 day), and 37 °C (up to 2 weeks). Each condition was characterized by particle size, PDI, and zeta potential, showing how excipient composition influenced colloidal stability and surface charge. The luminescence profiles at elevated temperatures revealed that specific combinations of sucrose, L-serine, and PEG provided superior protection against aggregation and loss of activity, preserving both mRNA integrity and LNP morphology. After 2 weeks at 37 °C, several optimized formulations maintained high luminescence and stable physicochemical parameters, identifying the best excipient ratios for long-term stabilization of soluble mRNA-LNPs. These optimized formulations were subsequently used as the core matrices for encapsulation inside the microcapsule systems, ensuring both storage stability and functional release performance.

[0124] Discussion

[0125] We have developed a microfluidic encapsulation platform for single-injection vaccine delivery that addresses several key challenges in the field of controlled release vaccines. Our approach combines the precision of microfluidics (to achieve uniform, programmable release microcapsules) with innovative formulation additives (antioxidants and phospholipids) that MTV-24625 enhance the stability and performance of the capsules. The result is a versatile system where booster dose timing can be dialed in by design, and the integrity of both the capsule and the sensitive mRNA-LNP payload is maintained throughout fabrication, storage, and administration.

[0126] Stability Considerations'. A contribution of this work is addressing mRNA-LNP stability in a controlled release format. LNPs are typically delivered fresh, and their encapsulation in a polymer device raises questions about mRNA integrity over time. We mitigated this through multiple means: (1) microfluidic encapsulation is gentle and high-yield, avoiding shear or extensive exposure to damaging solvents; (2) inclusion of antioxidants like vitamin E and Q10 helps prevent oxidative damage to the unsaturated lipids in LNP and the mRNA (which can be prone to hydrolysis by reactive species); (3) keeping the microcapsules solid and at refrigerated conditions until use effectively “pauses the clock” on mRNA degradation, especially with DPPC providing a stable barrier at 4 °C. Our observation that core pH remained closer to neutral in antioxidant-loaded capsules is encouraging, as acidification in degrading PLGA often inactivates biomolecules. Future studies could directly measure mRNA integrity (via RT-PCR or in vitro translation assays) after various storage times in the capsules to quantify this benefit.

[0127] Reduced Burst and Improved Safety. From a safety and efficacy standpoint, minimizing the initial burst release is crucial. An uncontrolled early release of the booster dose could not only reduce the booster effect but also potentially cause local reactogenicity by delivering too much payload at once. By solidifying the core (with hydrogels) and fortifying the shell (with antioxidants and lipids), we achieved microcapsules that largely retain their cargo until the intended time. The injection test is particularly important - it simulates the shear and pressure of administration. Our lipid-containing shells preventing capsule rupture on injection is a practical improvement that any translational effort in this area would need to incorporate. It’s also noteworthy that even with a solid or gel core, a brittle shell could crack; thus, the approach of chemically modifying the shell’s mechanical properties (essentially making it less glassy and more tough by adding plasticizing oils) is validated by our results.

[0128] Release Kinetics and Immune Response '. The release profiles we obtained tend towards an “all-or-none” burst after a programmable delay, which is ideal for triggering a booster immune response. There may be cases where a more continuous release is desired (for instance, to simulate multiple smaller boosters or a prolonged antigen presence). Our system could be tuned for that by blending polymers or shell components that erode more gradually rather than bursting. For example, using some fraction of poly(caprolactone) or other polymers could yield MTV-24625 a multi-phasic release. Additionally, multiple populations of capsules with different shell thicknesses could be mixed to provide sequential boosts (one could imagine a single injection containing, say, 50 pm capsules releasing at 1 month and 70 pm capsules releasing at 3 months). This modularity is one of the strengths of microfluidic production - one can dial in multiple distinct capsule types and combine them in a formulation.

[0129] We conducted preliminary immunogenicity tests in mice to verify that the delayed release function translates to a booster immune response. Mice were injected with a single dose containing a mixture of free mRNA-LNP (prime dose) and encapsulated mRNA-LNP (booster dose in microcapsules). For a formulation designed to release at ~4 weeks, we observed a strong secondary rise in antigen-specific antibody titers around weeks 5-6, achieving about 80% of the titer level of a traditional two-shot regimen (prime at week 0, boost at week 4). Importantly, in our mRNA vaccine context, the antibody response indicates that the mRNA released from the capsules at the delayed time point was still functional - it successfully transfected cells and expressed the antigen, despite having been encapsulated for weeks. This speaks to the protective role of our formulation: the mRNA-LNP remained potent, likely aided by the antioxidant and low-temperature storage prior to release. Control mice that received only the prime (and no booster) had a much lower antibody titer, confirming the need for the booster dose. Additionally, no adverse inflammation was noted at the injection site attributable to the microcapsules; they appear to be well-tolerated, as expected for PLGA-based materials.

[0130] Summary

[0131] We have expanded the capabilities of core-shell PLGA microcapsule technology for single-shot vaccines by incorporating antioxidant and lipid excipients into the shell. Our microfluidic fabrication method yields uniform microcapsules with tunable release intervals, capable of protecting and then releasing fragile mRNA-LNP vaccine payloads in a controlled manner. Microfluidic precision: The use of a glass capillary microfluidic device enables the production of monodisperse core-shell microcapsules with adjustable shell thickness, which in turn allows programmed release timings from a few weeks up to several months after injection. Antioxidant-stabilized shells: Incorporating a-tocopherol and coenzyme Q10 into the PLGA shell formulation improves shell formation and integrity. These antioxidants act as plasticizers and stabilizers, preventing microcapsule rupture during fabrication and reducing initial burst release of the payload. They also help maintain a less acidic microenvironment within degrading capsules, which is beneficial for mRNA and LNP stability. Hydrogel or viscous cores: Encapsulating the mRNA-LNP in a hydrogel or viscous matrix (e.g., crosslinked GelMA or dextran solution) in the core dramatically enhances encapsulation efficiency and MTV-24625 prevents core leakage. Solidified cores remain intact during shell solidification, avoiding the “rupture or fusion” of inner droplets that can occur with liquid cores. Phospholipid-enhanced stability: Adding phospholipids (POPC, DOPC, DPPC, PEGylated lipids) to the shell creates a polymer-lipid hybrid capsule that is more stable against aggregation and mechanical stress. In particular, DPPC provides thermally gated release - capsules are stable at refrigeration temperatures and then become permeable at body temperature due to the lipid phase transition around ~37 °C. This feature ensures long-term stability during storage and a triggered release upon injection. Reduced burst on injection: The combined effect of a tougher, lipid-plasticized shell and a possibly gelled core means the microcapsules can withstand the shear forces of injection through a fine needle without breaking. This minimizes any unintended early release (burst) at the time of administration, preserving the booster dose for its intended release schedule. Immunogenic efficacy: In a proof-of-concept in vivo test (mouse model), a single injection of prime + encapsulated booster elicited a strong immune response comparable to a two-injection regimen, validating that the delayed release of mRNA-LNP can act as an effective booster immunization. The mRNA-LNP payload remained potent after weeks of encapsulation, likely due to the protective formulation.

[0132] In summary, this bioengineering approach addresses many challenges of single-dose vaccine delivery. The microcapsules are injectable, scalable, and programmable, aligning with the criteria needed for real-world deployment. By fine-tuning the composition of the capsule shells, we achieved a balance of stability and triggerability - keeping the vaccine secure until the right moment, then releasing it in a timely fashion to provide the necessary booster stimulus. These findings pave the way for further development of single-injection vaccines for various diseases, which could have a profound impact on global vaccination strategies by simplifying logistics and increasing compliance. The principles demonstrated here - combining microfluidic engineering with smart material design - may also be extended to other controlled release applications where maintaining the integrity of a therapeutic payload over extended periods is crucial.

[0133] Overall, our work contributes a significant step toward the realization of “dose-on- demand” vaccines and underscores the importance of interdisciplinary innovation at the interface of engineering, materials science, and immunology in addressing public health challenges. MTV-24625

[0134] INCORPORATION BY REFERENCE

[0135] All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0136] EQUIVALENTS

[0137] While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

MTV-24625We claim:

1. A microparticle, comprising: i) a microparticle core comprising a lipid nanoparticle; and ii) a microparticle shell comprising a polymer or a copolymer; wherein the microparticle core is encapsulated by the microparticle shell; and the lipid nanoparticle comprises a lipid, an excipient, and a bioactive agent.

2. The microparticle of claim 1, wherein the lipid is an ionizable lipid, cholesterol, a polyethylene glycol lipid, a phospholipid, or a combination of any of them.

3. The microparticle of claim 1 or 2, wherein the lipid is 1 -octylnonyl 8-[(2- hydroxyethyl)[8-(nonyloxy)-8-oxooctyl]amino]octanoate (i.e., lipid 5, CAS Ref. No.: 2089251-33-0), 3, 6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2, 5-dione (i.e., cKK-E12), l,2-di-(9Z-octadecenoyl)-sn-glycero-3 -phosphoethanolamine (DOPE), 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), l,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), cholesterol, or a combination of any of them.

4. The microparticle of any one of claims 1-3, wherein the lipid comprises a combination of DOPC and DPPC (e.g., 20 wt% DOPC and 80 wt% DPPC).

5. The microparticle of any one of claims 1-3, wherein the lipid is POPC (e.g, 20 wt% POPC).

6. The microparticle of any one of claims 1-5, wherein the excipient comprises polyethylene glycol, polyvinyl alcohol, glucose, sucrose, citric acid, phosphate buffer solution (e.g, 2-Amino-2-(hydroxymethyl)-l,3-propanediol (Tris) buffer or 4-(2- hydroxyethyl)piperazine-l -ethane-sulfonic acid (HEPES) buffer).

7. The microparticle of any one of claims 1-6, wherein the excipient comprises sucrose.

8. The microparticle of any one of claims 1-7, wherein the excipient comprises PEG (e.g., PEG 2000).MTV-246259. The microparticle of any one of claims 1-8, wherein the excipient comprises PVA.

10. The microparticle of any one of claims 1-9, wherein the excipient comprises a combination of PEG and PVA.

11. The microparticle of any one of claims 1-10, wherein the excipient comprises a combination of PEG at about 4 wt% and pVA at about 1 wt%.

12. The microparticle of any one of claims 1-10, wherein the excipient comprises a combination of PEG with a molecular weight of about 6,000 kDa at about 4 wt% and pVA with a molecular weight from about 13 kDa to about 23 kDa at about 1 wt%.

13. The microparticle of any one of claims 1-12, wherein the excipient comprises a polymer or a copolymer.

14. The microparticle any one of claims 1-13, wherein the excipient comprises a polymer.

15. The microparticle of claim 14, wherein the polymer is polyvinyl alcohol (PVA) or polyvinyl pyrrolidone (PVP).

16. The microparticle of claim 14, wherein the polymer is PVP.

17. The microparticle of claim 14, wherein the polymer is PVP10.

18. The microparticle of claim 14, wherein the polymer is PVP60.

19. The microparticle of any one of claims 1-12, wherein the excipient comprises a copolymer.

20. The microparticle of claim 19, wherein the copolymer comprises a plurality of repeat units of vinyl alcohol and a plurality of repeat units of vinyl pyrrolidinone.

21. The microparticle of claim 20, wherein the mass ratio of polyvinyl alcohol to polyvinylpyrrolidone is about 1 : 1 to about 6: 1; or about 1 : 1, about 2: 1, about 3: 1, about 4: 1, about 5: 1, or about 6: 1.MTV-2462522. The microparticle of claim 20, wherein the mass ratio of polyvinyl alcohol to polyvinylpyrrolidone is about 1 : 1, about 2: 1, or about 3: 1.

23. The microparticle of claim 19, wherein the copolymer comprises a plurality of repeat units of vinyl alcohol and a plurality of repeat units of sucrose.

24. The microparticle of claim 23, wherein the mass ratio of polyvinyl alcohol to sucrose is about 1 : 1 to about 6: 1; or about 1 : 1, about 2: 1, about 3: 1, about 4: 1, about 5: 1, or about 6: 1.

25. The microparticle of claim 23, wherein the mass ratio of polyvinyl alcohol to sucrose is about 1 : 1 or about 2: 1.

26. The microparticle of claim 19, wherein the copolymer comprises a plurality of repeat units of vinyl alcohol, a plurality of repeat units of polyvinylpyrrolidone, and a plurality of repeat units of sucrose.

27. The microparticle of claim 26, wherein the mass ratio of polyvinyl alcohol to polyvinylpyrrolidone to sucrose is about 1: 1 : 1 to about 1 : 1 :3; or about 1 : 1 : 1, about 1: 1 :2, or about 1 : 1 :3.

28. The microparticle of claim 26, wherein the mass ratio of polyvinyl alcohol to polyvinylpyrrolidone to sucrose is about 1: 1 :2.

29. The microparticle of any one of claims 19-28, wherein the copolymer comprises 250 - 1,500 repeat units.

30. The microparticle of any one of claims 19-29, wherein the copolymer is a block copolymer.

31. The microparticle of any one of claims 19-29, wherein the copolymer is a random copolymer.MTV-2462532. The microparticle of any one of claims 1-31, wherein the bioactive agent is selected from the group consisting of a nucleic acid, a protein, an antibody, a small molecule, a vaccine, and an antigen.

33. The microparticle of any one of claims 1-31, wherein the bioactive agent is an mRNA, siRNA, RNA, or DNA.

34. The microparticle of any one of claims 1-31, wherein the bioactive agent is an RNA (e.g., an mRNA).

35. The microparticle of any one of claims 1-31, wherein the bioactive agent is an antibody.

36. The microparticle of any one of claims 1-31, wherein the bioactive agent is a small molecule (e.g., a drug).

37. The microparticle of any one of claims 1-31, wherein the bioactive agent is a protein.

38. The microparticle of any one of claims 1-31, wherein the bioactive agent is a vaccine or an antigen.

39. The microparticle of any one of claims 1-31, wherein the bioactive agent is a nucleic acid.

40. The microparticle of any one of claims 1-31, wherein the bioactive agent is a stimulator of interferon genes (STING) agonist.

41. The microparticle of any one of claims 1-40, wherein the lipid nanoparticle further comprises an antioxidant.

42. The microparticle of claim 41, wherein the antioxidant is hydrophobic.

43. The microparticle of claim 41, wherein the antioxidant is hydrophilic.MTV-2462544. The microparticle of claim 41, wherein the antioxidant is selected from the group consisting of vitamin A, vitamin E, butylated hydroxytoluene, ascorbic acid, and alpha tocopherol (e.g., D-u-tocopheryl polyethylene glycol succinate (TPGS)).

45. The microparticle of claim 41, wherein the antioxidant is alpha tocopherol.

46. The microparticle of claim 41, wherein the antioxidant is TPGS.

47. The microparticle of claim 41, wherein the antioxidant is ubiquinone (coenzymeQ10).

48. The microparticle of any one of claims 1-47, further comprising an amino acid.

49. The microparticle of claim 48, wherein the amino acid is a naturally occurring amino acid.

50. The microparticle of claim 48, wherein the amino acid is serine.

51. The microparticle of claim 48, wherein the amino acid is L-serine.

52. The microparticle of claim 48, wherein the amino acid is a cationic amino acid.

53. The lipid nanoparticle of any one of claims 1-49, wherein the amino acid is lysine, arginine, or histidine.

54. The microparticle of claim 53, wherein the amino acid is arginine.

55. The microparticle of any one of claims 1-54, wherein the lipid nanoparticle is formed using encapsulation with ethyl acetate.

56. The microparticle of any one of claims 1-55, wherein the microparticle shell comprises a polymer.

57. The microparticle of claim 56, wherein the polymer is a copolymer.MTV-2462558. The microparticle of claim 57, wherein the copolymer comprises a plurality of repeat units of lactide and a plurality of repeat units of glycolide (i.e., the polymer is Poly(lactide- co-glycolide (PLGA)).

59. The microparticle of claim 58, wherein the copolymer comprises a plurality of repeat units of lactide and a plurality of repeat units of glycolide at a molar ratio of about 75:25 lactide to glycolide, about 80:20 lactide to glycolide, about 85: 15 lactide to glycolide, or about 90: 10 lactide to glycolide.

60. The microparticle of claim 58, wherein the copolymer comprises a plurality of repeat units of lactide and a plurality of repeat units of glycolide at a molar ratio of about 85: 15 lactide to glycolide.

61. The microparticle of any one of claims 57-60, wherein the molecular weight of the copolymer is about 50 kDa, about 60 kDa, about 70 kDa, about 80 kDa, about 90 kDa, about 100 kDa, about 110 kDa, about 120 kDa, about 130 kDa, about 140 kDa, about 150 kDa, about 160 kDa, about 170 kDa, about 180 kDa, about 190 kDa, about 200 kDa, about 210 kDa, about 220 kDa, about 230 kDa, about 240 kDa, about 250 kDa.

62. The microparticle of any one of claims 57-60, wherein the molecular weight of the copolymer is about 20 kDa to about 240 kDa (e.g., 190 kDa to 240 kDa).

63. The microparticle of any one of claims 57-60, wherein the molecular weight of the copolymer is about 60 kDa to about 110 kDa (e.g., 66 kDa to 107 kDa).

64. The microparticle of any one of claims 57-60, wherein the molecular weight of the copolymer is about 190 kDa to about 240 kDa (e.g., 190 kDa to 240 kDa).

65. The microparticle of any one of claims 57-64, wherein the concentration of the copolymer is about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, or about 150 mg / mL.

66. The microparticle of any one of claims 57-64, wherein the concentration of the copolymer is about 30 mg / mL.MTV-2462567. The microparticle of any one of claims 57-64, wherein the concentration of the copolymer is about 75 mg / mL.

68. The microparticle of any one of claims 57-64, wherein the concentration of the copolymer is about 100 mg / mL.

69. The microparticle of any one of claims 1-68, wherein the diameter of the microparticle is about 50 pm to about 300 pm; or about 50 pm, about 75 pm, about 100 pm, about 125 pm, about 150 pm, about 175 pm, about 200 pm, about 225 pm, about 250 pm, about 275 pm, or about 300 pm.

70. The microparticle of any one of claims 1-69, wherein the encapsulation efficiency of the lipid nanoparticle is greater than about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%.

71. The microparticle of any one of claims 1-69, wherein the encapsulation efficiency of the lipid nanoparticle is greater than about 60%.

72. The microparticle of any one of claims 1-69, wherein the encapsulation efficiency of the lipid nanoparticle is about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%.

73. The microparticle of any one of claims 1-69, wherein the encapsulation efficiency of the lipid nanoparticle is about 60%.

74. A method of delivering a therapy, comprising contacting a subject in need thereof with the microparticle of any one of claims 1-73.

75. The method of claim 74, wherein the therapy is a vaccine.

76. The method of claim 74, wherein the therapy is an mRNA vaccine.

77. An injectable needle, comprising the microparticle of any one of claims 1-76.MTV-2462578. A method of delivering a therapy, comprising contacting a subject in need thereof with the injectable needle of claim 77.

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