Microfluidic platform for lipid nanoparticle unipatch aerosolization for messenger RNA inhalation

Abstract

Lungs offer significant advantages for gene therapy delivery via inhalation, leveraging their vast surface area. Clinical nebulizers employing vibrating mesh technology are the standard choice for converting liquid drugs into aerosols. However, they have limitations when delivering mRNA via inhalation, including severe damage to nanoparticles due to shear forces. Microfluidic aerosolization platforms (MAPs) can maintain the structural and physicochemical integrity of lipid nanoparticles, enabling safe and efficient delivery of mRNA to the respiratory system. Results show that this novel microfluidic aerosolizer outperforms traditional vibrating mesh nebulizers by avoiding issues such as particle aggregation, mRNA encapsulation loss, and nanoparticle morphology deformation. Notably, aerosolized nanoparticles generated by the microfluidic aerosolization platform result in enhanced transfection efficiency across multiple cell lines. In vivo experiments in mice inhaling these aerosolized nanoparticles demonstrated successful lung-specific mRNA transfection without observable signs of toxicity. This groundbreaking MAP represents a significant advance in lung gene therapy, enabling precise and efficient delivery of aerosolized nanoparticles.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 614,049, filed December 22, 2023, the entire contents of which are expressly incorporated herein by reference. Technical Field

[0003] This disclosure pertains to the field of medical aerosolization. Background Technology

[0004] The successful development of mRNA vaccines against SARS-CoV-2 has transformed the perspectives of researchers and clinicians on the use of mRNA as a therapeutic tool. What was previously considered a theoretical approach has now become a promising and feasible clinical application option. In particular, mRNA has gained significant attention in the field of pulmonology due to its potential in treating genetic diseases, including cystic fibrosis (CF) and α-1 antitrypsin deficiency, as well as in the field of vaccinology for intranasal vaccination. Summary of the Invention

[0005] In some respects, microfluidic aerosolization systems can provide delivery of therapeutic media to the pulmonary airways. A microfluidic aerosolization system may include: a microfluidic chip having multiple microfluidic chambers and corresponding orifices; a heat source thermally connected in the form of a resistor to each microfluidic chamber, each microfluidic chamber being individually addressable for droplet ejection; a cartridge housing the microfluidic chip and multiple storage units holding therapeutic media configured to deliver the therapeutic media into the microfluidic chambers; and a processor configured to generate an activation pulse heating mode; wherein the microfluidic chambers eject multiple droplets in response to the activation pulse mode.

[0006] In some examples, the chip is fabricated monolithically on a silicon substrate.

[0007] In some examples, the activated pulse heating mode has a pulse range of approximately 5 volts to approximately 40 volts and a timing range of approximately 0.1 microseconds to approximately 10 microseconds. In some examples, the heating mode has a frequency of at least approximately 10 kHz.

[0008] In some examples, the chambers discharge droplets at a rate of at least approximately 5 million droplets per second. In some examples, the microfluidic aerosolization system comprises at least approximately 500 microfluidic chambers and corresponding pores.

[0009] In some examples, at least some pores have a diameter of no more than about 15 micrometers. In some examples, the cassette contains a volume of therapeutic medium in one or more reservoirs. In some examples, the therapeutic medium comprises mRNA encapsulated within lipid nanoparticles. In some examples, the processor is configured to deliver said volume of medium broken down into multiple individual patient doses.

[0010] In some examples, the box includes at least two reservoirs. In other examples, the first reservoir contains a first treatment medium, while the second reservoir contains a different second medium. In still other examples, each reservoir contains the same treatment medium.

[0011] In some examples, at least one memory includes a buffer. In some examples, all orifices can be individually programmed using activation pulse modes to control at least one of drop size, drop size composition, drop ejection frequency, and drop volume. In some examples, the processor can execute instructions to individually control the pulses of each microfluidic chamber. In some examples, the processor can control at least one of drop size, drop size composition, drop ejection frequency, and drop volume. In some examples, the processor is configured to activate multiple microfluidic chambers and orifices simultaneously or sequentially. In some examples, the processor can control the applied voltage and pulse width such that the heating mode has a frequency ranging from 1 kHz to 15 kHz. In some examples, the processor can control the applied voltage and pulse width such that the heating mode produces droplets with diameters ranging from 7.3 μm to 24.4 μm. In some examples, the processor can control the applied voltage and pulse width such that the heating mode produces droplets with diameters ranging from 7.3 μm to 19.4 μm. In some examples, the processor can control the applied voltage and pulse width, so that the heating mode produces droplets with volumes ranging from 0.2 pL to 3.8 pL.

[0012] In some examples, the system includes an orifice having a profile that matches the chip size to influence the shape of the plume as it exits the orifice.

[0013] In some examples, the therapeutic medium includes a drug. In other examples, the therapeutic medium includes a small molecule drug. In some examples, the therapeutic medium includes a bioactive agent. In other examples, the bioactive agent includes mRNA. In still other examples, the bioactive agent includes encapsulated mRNA.

[0014] In some examples, the processor can control the applied voltage and pulse width. In some examples, the applied voltage and pulse width cause the formation of microbubbles, which nucleate, expand, and collapse to eject fluid droplets into the microcavity. In some other examples, the applied voltage is less than about 5 V and the pulse width is less than about 10 μs. In some other examples, the applied voltage includes a waveform that is at least one of square, sawtooth, or sinusoidal. In still other examples, the applied voltage and pulse width cause the deflection of a microelectromechanical membrane, which in turn ejects fluid droplets into the microcavity. In some examples, individual chamber addressing modes and ejection frequencies are adjustable (e.g., by the processor) to regulate the formation of multiple droplets into a plume, the depth of droplet penetration into the lung pathway, and the aggregation of droplets within the plume.

[0015] In some aspects, the pulmonary approach can deliver therapeutic mediators. This method may include the steps of: introducing a therapeutic mediator into at least one microfluidic chamber having a pore; and heating the microfluidic chamber to cause at least a portion of the mediator to evaporate and expel at least one droplet through the pore and into the pulmonary airway, wherein the pore has a diameter of no more than about 20 micrometers.

[0016] In some examples, the pores have a diameter of no more than about 15 micrometers. In some examples, the droplets have a diameter of no more than about 20 micrometers. In other examples, the droplets have a diameter of at least about 50 micrometers. In some examples, the method includes introducing a therapeutic medium into at least about 100 microfluidic chambers. In other examples, the method includes introducing a bioactive medium into at least about 500 microfluidic chambers. In still other examples, the method includes discharging at least about 1 million droplets per second. In still other examples, the method includes discharging at least about 5 million droplets per second. In some examples, the method includes applying a voltage and pulse width to the microfluidic chambers using a processor, such that the heating mode has a frequency ranging from 1 kHz to 15 kHz. In some examples, the method includes applying a voltage and pulse width to the microfluidic chambers using a processor, such that the heating mode produces droplets with a diameter ranging from 7.3 μm to 24.4 μm. In some examples, the processor can control the applied voltage and pulse width, such that the heating mode produces droplets with a diameter ranging from 7.3 μm to 19.4 μm. In some examples, the method involves using a processor to apply voltage and pulse width to a microfluidic chamber, such that a heating mode produces droplets with volumes ranging from 0.2 pL to 3.8 pL.

[0017] In some examples, the therapeutic medium comprises an mRNA vaccine. In other examples, the therapeutic medium comprises an mRNA vaccine encapsulated within lipid nanoparticles. In some examples, the therapeutic medium comprises a drug. In other examples, the therapeutic medium comprises a small molecule drug. In some examples, the therapeutic medium comprises a bioactive agent. In some examples, the bioactive agent comprises mRNA. In some examples, the bioactive agent comprises encapsulated mRNA.

[0018] In some examples, the method includes controlling the heating of the microfluidic chambers using a processor that executes instructions. In some examples, the processor can adjust the activation pulse pattern. In some examples, the processor can cause simultaneous or sequential activation of multiple microfluidic chambers and orifices. In some examples, the activation pulse pattern can be used to individually program all orifices to control at least one of drop size, drop size composition, drop ejection frequency, and drop volume. Attached Figure Description

[0019] The following figures and related descriptions are provided for illustrative purposes and not to limit the scope of the claims.

[0020] Figure 1 A shows a view of the chips included in the example system.

[0021] Figure 1 B is a cross-sectional view of the microfluidic chamber of the chip.

[0022] Figure 1 C illustrates a layered structure with a microfluidic chamber exhibiting bubble formation.

[0023] Figure 1 D is a schematic diagram illustrating the formation of bubbles on a chip, comprising the following steps: (1) bubble nucleation, (2) bubble growth, (3) droplet ejection, and (4) droplet detachment and refilling.

[0024] Figure 2 A to Figure 2 C shows a comparison of droplet formation from a microfluidic cartridge with a 4-pL nozzle at three different frequencies: 1 kHz, 10 kHz, and 15 kHz.

[0025] Figure 2 D to Figure 2 F plots the droplet diameter, volume, and droplet volume at three different generation frequencies.

[0026] Figure 2 G and Figure 2 H shows a simulation-based analysis used to estimate the temperature change of the fluid in the thermal boundary layer during an electric pulse, taking into account the effects of time and geometry.

[0027] Figure 3A is a cartoon depicting lipid nanoparticles and their components.

[0028] Figure 3 B to Figure 3 F compares the hydrodynamic size distribution, zeta potential, and RNA encapsulation of nanoparticles generated using a vibrating mesh nebulizer or a microfluidic aerosolization platform.

[0029] Figure 3 G compares RNA encapsulation of nanoparticles produced by microfluidic aerosolization systems or vibrating mesh nebulizers with protein blotting of untreated nanoparticles and free mRNA.

[0030] Figure 3 H is a cartoon image of a liposome containing two DOPE-conjugated FRET probes, 7-nitrobenzo-2-oxa-1,3-diazole (NBD-PE), and rhodamine B (Rho-PE), which is prepared as a "FRET liposome".

[0031] Figure 3 I plotted the fusion percentage of liposomes to assess the frequency of membrane fusion events in NPLs in droplets generated by the microfluidic aerosolization system compared to acoustically treated, vibrating mesh, Triton X-100, and untreated liposomes.

[0032] Figure 4 A to Figure 4 C is an image of the untreated LNP / mRNA sample, the vibrating mesh nebulizer, and the droplets of the microfluidic aerosolization system.

[0033] Figure 4 D is a fluorescence image of cells exposed to untreated droplets, LNP solution, droplets generated by a vibrating mesh nebulizer, and droplets generated by a microfluidic aerosolization system.

[0034] Figure 4 E describes Figure 4 mRNA transfection in experiment D.

[0035] Figure 4 F depicts the mRNA transfection efficiency across five different cell lines.

[0036] Figure 5 A is a cartoon illustration of the inhalation system of a whole-body rodent, which includes a 3 L container connected to a microfluidic aerosolization system.

[0037] Figure 5 B is a confocal image of mouse lung tissue, showing the presence of Nluc transcripts throughout the lung exposed to LNP / Nluc aerosols.

[0038] Figure 5C is a confocal image showing no detected signal in the lungs exposed to PBS aerosols.

[0039] Figure 5 D and Figure 5 E showed that Nluc expression in mouse lungs was directly proportional to the dose of LNP / Nluc aerosolization.

[0040] Figure 5 F and Figure 5 G showed that luciferase expression was higher 24 hours after treatment compared to 48 hours after treatment.

[0041] Figure 5 I and Figure 5 H shows exposure to LNP / Nluc aerosol containing 1 mg mRNA ( Figure 5 H) or an equal volume of PBS ( Figure 5 Histopathological images of mouse lungs (I).

[0042] Figures 6 to 9 A view of a portable aerosolizer is shown.

[0043] Figure 10 It shows Figures 6 to 9 The portable aerosol aerator's display and user controls.

[0044] Figure 11 Showing from Figures 6 to 9 The airflow at the mouth of the portable aerosol aerator.

[0045] Figure 12A It shows Figures 6 to 9 An exploded side view of a portable aerosolizer.

[0046] Figure 12B It shows Figures 6 to 9 Two views of the droplet ejector of a portable aerosol aerator.

[0047] Figure 13 It shows Figures 6 to 9 A cross-sectional side view of the droplet ejector of a portable aerosol agglomerator.

[0048] Figure 14 This illustrates the process during droplet nucleation. Figures 6 to 9 A cross-sectional view of the droplet ejector of a portable aerosol agglomerator.

[0049] Figure 15 This is an image sequence of aerosols generated from a microfluidic device.

[0050] Figure 16 The representative size distribution of liposomes was depicted.

[0051] Figure 17 A to Figure 17 C is a graph showing the size distribution of aerosol droplets at different distances from the device.

[0052] Figure 18 A to Figure 18 D is a graph showing the size distribution of aerosol droplets by changing the aerosolization frequency or the number of nozzles.

[0053] Figure 19 A to Figure 19 F is a size distribution diagram of aerosol droplets over multiple time periods during aerosolization.

[0054] Figure 20 These are images of agarose gel electrophoresis analysis of LNP-encapsulated mRNA after aerosolization using a mesh nebulizer or microfluidic device.

[0055] Figure 21 It is an image showing the visual change in the opacity of the LNP solution after atomization.

[0056] Figure 22 Cell viability was described when LNP was transfected using atomized LNP.

[0057] Figure 23 A to Figure 23 Figure B shows the effect of nebulized LNP delivery to the relevant lung cell lines.

[0058] Figure 24 The cell viability of various cell lines after treatment with atomized LNP was described.

[0059] Figure 25 A to Figure 25 B is an image showing lung delivery of aerosolized LNP / Nluc via spontaneous inhalation into the mouse lungs.

[0060] Figure 25 C describes the quantized luminescence signal from the captured image.

[0061] Figure 26A and Figure 26B It involves collecting histopathological images of mouse lungs over 24 hours. Figure 26A The inhalation of LNP / Nluc was shown when 1 mg of mRNA was aerosolized. Figure 26B This demonstrates the use of a microfluidic platform to aspirate an equal volume of sterile PBS. Detailed Implementation

[0062] The microfluidic aerosolizer disclosed herein avoids problems such as particle aggregation, loss of mRNA encapsulation, and deformation of nanoparticle morphology. Furthermore, the aerosolized nanoparticles generated by the microfluidic aerosolizer can improve transfection efficiency. The aerosolized nanoparticles generated by the microfluidic aerosolizer can allow for successful lung-specific mRNA transfection without observable signs of toxicity. The microfluidic aerosolizer can enable lung gene therapy, thereby allowing for the precise and efficient delivery of aerosolized nanoparticles.

[0063] A major challenge in the clinical translation of mRNA-based therapeutic treatments lies in identifying safe and effective delivery strategies. Recent advances in the development of novel nanoparticles capable of targeting the lungs after systemic administration demonstrate the great potential for lung-specific mRNA delivery. However, concerns remain regarding the potential risk of off-target delivery of these RNA vectors.

[0064] Given that many lung diseases are closely related to the condition of lung epithelial cells, inhalation may be the most direct, safe, and effective method for administering mRNA therapy. Inhalation allows for high concentrations of treatment in the lung epithelial tissue, enabling targeted and localized treatment of respiratory conditions while minimizing systemic exposure. This localized administration holds significant promise for treating the lung system with mRNA therapy. Clinical trials based on inhalation-mediated mRNA delivery (NCT05712538 and NCT05737485) demonstrate growing interest in this approach. However, inhaled mRNA therapy still faces challenges related to shear damage caused by inhalers, particularly nebulizers (which are typically used to generate aerosols from aqueous drugs). Both jet nebulizers and vibrating mesh nebulizers involve atomization processes that apply strong shear forces to nanoparticles, resulting in a significant loss of their mRNA delivery efficiency. Therefore, addressing and mitigating these challenges could help maximize the efficacy and safety of inhaled mRNA therapy for lung diseases. Research focusing on optimizing nanoparticles has shown promising results in improving their tolerance to shear forces applied by nebulizers. Increasing the molar concentration of polyethylene glycol (PEG) in lipid nanoparticle (LNP) formulations can enhance mRNA transfection after aerosolization. High PEG content in LNPs can improve their resilience to damage during aerosolization and mitigate the negative impact of shear forces on mRNA delivery efficiency.

[0065] While fine-tuning nanoparticles can salvage their delivery capabilities during atomization, an alternative solution to this challenge lies in developing novel aerosolization platforms. Atomization via a vibrating mesh essentially involves guiding fluid flow through a fine sieve to atomize aerosols at the microscale. In this context, exploring other possible fluid dynamics for generating plumes offers promising opportunities for developing novel aerosolization devices. Microfluidic devices have been widely used to produce a variety of nanoparticles, providing a simple method for the continuous, controllable, and reproducible fabrication of small-sized nanoparticles with narrow size distributions. Without being bound by specific theories, systems may be able to utilize microfluidics to flow LNPs through microchannels, thereby providing the potential for mRNA aerosolization without subjecting nanoparticles to damaging shear forces.

[0066] The microfluidic-based aerosolization (“MAP”) platform generates a homogeneous aerosol containing mRNA encapsulated within LNPs (LNP / mRNA), effectively avoiding destructive shear forces. Connecting a microfluidic chip to a cartridge containing an LNP / mRNA solution provides on-demand droplet generation with precise dose control for potential use in individual chronic treatments or large-scale vaccination. Unlike the sonication methods in vibrating mesh nebulizers or the jet explosion methods in jet nebulizers, the droplet generation process in this microfluidic aerosolization platform expels droplets with minimal shear-affected volume, making it highly suitable for delivering macromolecule-based therapies such as nucleic acids, proteins, and nanoparticles. An example microfluidic aerosolization platform is compared to a clinical-grade vibrating mesh nebulizer based on sonication. For research, ionizable lipids with previously optimized formulations for siRNA and mRNA delivery are used to highlight the clinical translation potential of the microfluidic platform provided in this disclosure. This novel platform promises to address the clinical need for inhaled nanoparticles for a broad range of RNA therapeutics and vaccines by providing controlled and efficient aerosolization without compromising the integrity of the nanoparticles.

[0067] Overview

[0068] Inhalable gene therapy holds immense potential for a range of drug development endeavors, including inhalable vaccines and treatments for inherited lung diseases such as cystic fibrosis (CF). Even with recent findings that selective organ-targeting of LNPs leads to effective mRNA transfection of the lungs, inhalation remains a prominent non-invasive and efficient method, specifically reaching the bronchioles and lung parenchyma while avoiding unintended systemic transfection. Given the expanding applications of mRNA in gene editing, the focus on achieving highly precise delivery via inhalation has grown significantly, particularly due to its potential to provide durable solutions for inherited lung diseases. However, ensuring adequate therapeutic doses remains a significant hurdle. One possible explanation for limited lung delivery could be the sensitivity of nanoparticles to instability during aerosolization. Researchers have actively sought solutions to this impasse, developing more robust nanoparticles through fine formulation tuning and the exploration of various biomaterials. While these efforts are promising, ultimately, resolving this challenge may stem from innovations in advanced medical devices capable of generating aerosols while maintaining the integrity of the nanoparticles.

[0069] A microfluidic aerosolization platform (“MAP”) can generate nanoparticle aerosols suitable for inhaled mRNA therapy. The findings confirm the MAP’s ability to generate uniform nanoparticle aerosols without causing deformation of the nanostructures or loss of encapsulated mRNA. Unlike conventional nebulizers, such as vibrating mesh nebulizers and jet nebulizers, MAP employs individually addressable nozzles for precise droplet delivery, allowing for the generation of LNP / mRNA-containing aerosols at lower operating frequencies. Through this low-shear aerosolization method, MAP prevents nanoparticle destruction, lipid aggregation, and mRNA leakage that occur in other nebulizers. That is, even after the aerosolization process, the MAP maintains the LNP size distribution and mRNA encapsulation. This was validated by cryoTEM imaging, which showed minimal impact on LNP morphology compared to the deformation and dissociation observed with mesh nebulizers. This maintenance of nanoparticle integrity is crucial not only for efficient mRNA delivery to cells but also for preventing undesirable side effects in the respiratory system. Furthermore, MAP provides precise control over droplet and plume sizes, ensuring accurate drug delivery. Importantly, it demonstrates significantly higher mRNA delivery efficiency to cells compared to conventional nebulizers. While mesh nebulizers resulted in a drastic 100-fold reduction in mRNA delivery, MAP consistently maintained intracellular mRNA delivery efficiency. These results highlight how the integrity of nanoparticles directly impacts mRNA delivery efficiency. Finally, despite the liver-specific tropism of LNPs, the LNP aerosol generated by MAP successfully delivered mRNA to the mouse lungs via inhalation. This was achieved in a dose-dependent manner, exhibiting selective lung transfection without inducing significant inflammation. These findings underscore the potential utility of platforms for administering lung-based mRNA therapy.

[0070] The system offers several advantages. First, to mitigate potential thermal denaturation, it may be necessary to minimize the temperature rise during bubble formation to prevent any adverse effects on LNP structure and mRNA stability. MAP-generated aerosolized LNP / mRNA shows minimal changes in its physicochemical properties. Furthermore, the short aerosolization time applied to LNP / mRNA limits its thermal impact on efficacy. Second, to address various clinical needs in pulmonology, the nozzle geometry needs to be improved to accommodate a wide range of droplet diameters. Recent studies have demonstrated that intratracheal vaccination induces peripheral and mucosal immunity against the SARS-CoV-2 antigen, suggesting that MAP can avoid the need for endoscopy during administration. Additionally, to improve the precision of therapeutic delivery to deeper lung tissues, the MAP nozzle geometry needs to be designed for smaller droplet diameters. This could potentially be more effective in treating the lower respiratory tract, such as the bronchioles and alveoli.

[0071] Recent advances in LNP chemistry have identified the role of PEG molar concentration in RNA delivery. PEG incorporation into LNPs can help maintain nanoparticle stability during self-assembly. Furthermore, PEG molecules inhibit interactions between LNPs and serum proteins, prolonging nanoparticle circulation time. However, they also inhibit the formation of a biomolecular corona on the nanoparticle surface, which delays LNP endocytosis and subsequent mRNA delivery. In the case of LNP nebulization, PEG molecules are thought to contribute to the recovery or stabilization of nebulized nanoparticles through steric effects. This complex interaction of PEG in LNP chemistry increases the complexity of formulation design for inhalation. Even with meticulous optimization, the nebulization process can compromise the integrity of LNPs, obscuring key properties necessary for effective lung transfection. MAP can help eliminate uncertainties in formulation discovery by preventing LNP deformation during nebulization. This platform shows promise in enabling optimized LNP formulations developed for systemic administration to function effectively in inhalation. Alternatively, formulation screening based on intratracheal instillation can provide greater accuracy in predicting efficacy via aerosol delivery. Notably, preserving the integrity of the mRNA payload throughout aerosolization can facilitate precise drug delivery and reduce the risk of immunogenicity associated with activated RNA sensors in the patient's lungs. Overall, MAP plays a crucial role in accelerating the development of inhalable nanoparticles. Furthermore, utilizing this platform has the potential to achieve significant breakthroughs in overcoming translational challenges related to lung gene therapy.

[0072] Furthermore, MAP can help enable optimized LNP formulations developed for systemic administration to function effectively under inhalation conditions. Intratracheal instillation-based formulation screening offers higher accuracy in predicting delivery via aerosols, partly due to the continuous generation of aerosols prepared by MAP. Preservation of mRNA payload integrity throughout the aerosolization process facilitates precise dosing and reduces the risk of immunogenicity associated with activating RNA sensors in the patient's lungs.

[0073] Example aerosolization system

[0074] To achieve efficient delivery of LNP-assisted gene therapy in the form of micrometer-sized droplets, a microfluidic device was used, consisting of a CMOS-based chip with integrated microfluidic structures. The device includes an array of 960 droplet ejectors, each individually addressable, enabling the generation of droplet plumes containing LNP / mRNA. Figure 1 A). In short, it involves monolithically fabricating a microfluidic chip on a silicon substrate using standard photolithography techniques. This chip includes a polymer manifold and nozzle structure. It is a specialized device that utilizes microfluidics and thermodynamic principles to allow liquid to flow through a fluid manifold and generate and eject small droplets. Each of the 960 droplet ejectors consists of a microfluidic cavity holding the liquid and a nozzle or orifice (approximately 10 μm in diameter), through which droplets are ejected. Figure 1 B. Figure 1 C). Below the chamber, multiple thin-film layers of heating elements and electrical materials are integrated. The device is digitally actuated by applying short electrical pulses to individual heating elements, generating microbubbles that propel droplets out of the nozzle. Specifically, when current flows through the resistance heating element, the temperature of the heater surface rises rapidly, causing the liquid in substantial contact with the heater surface to evaporate. As the liquid evaporates, it forms bubbles within the microfluidic channels, and the sudden expansion of these bubbles propels droplets out through the nozzle. Figure 1 D). Proper control of the duration and amplitude of the electrical pulses continuously produces droplets of specific size and velocity, while the actuation of individual injectors allows for precise and controlled distribution. For example, if each of 960 injectors is individually and repeatedly actuated at a frequency of 10 kHz per nozzle, droplets are produced at a rate of 9.6 million droplets per second, thus creating a plume. (See also...) Figure 15 By specifying the exact number of droplets to be ejected from each nozzle (e.g., by adjusting the pulse width or pulse frequency applied to the nozzles using a processor), the total dispensing volume can be adjusted and easily controlled. Throughout the process, the liquid is precisely controlled and delivered to the nozzles using microfluidics, ensuring consistent and uniform droplet size and shape. Furthermore, microfluidics enables precise control of plume size, droplet velocity and size, and the applied dosage.

[0075] In inhalation-mediated drug delivery, droplet size plays a crucial role in determining its accumulation location within lung tissue. Larger droplets tend to deposit in the upper airways and are easily removed by mucociliary clearance before reaching deeper lung tissue. In contrast, smaller droplets have a higher chance of penetrating deeper into the lungs and reaching the alveolar regions. Therefore, the ideal droplet size for inhalation-mediated drug delivery depends on the specific target of action. For conditions such as asthma, chronic obstructive pulmonary disease (COPD), or CF, smaller droplets capable of reaching the lower airways and alveoli are preferred. On the other hand, larger droplets may be more suitable for local treatment of upper respiratory tract infections or inhaled inoculation. Therefore, precise control of droplet size in aerosols is essential for the successful development of an effective delivery platform for inhalation-mediated drug therapy. To evaluate the droplet sizes generated by MAP, their size distribution was analyzed using two different techniques: the JetXpert droplet observer and the Spraytec. ® Droplet size measurement system. For droplet size characterization, liposomes (DSPC:cholesterol:DSPE-PEG2K = 52:45:3) were used to simulate the physical properties of the bulk LNP suspension. The average size of the liposomes was approximately 80 nm, and their approximate polydispersity index (PDI) was 0.1. Figure 16 The droplet observer system allows us to capture precise images of individual droplets in flight, measure their shapes, and estimate their volumes, even at the picoliter scale. Droplet formation was performed and compared from nozzles ejected from a microfluidic chamber at two different operating frequencies, with each nozzle actuated at 1 kHz and 15 kHz respectively, for visualization of single-droplet ejection events. Figure 2 A to Figure 2 B). During flight, it was observed that the droplets generated at 1 kHz split into 4 droplets (B). Figure 2 A), while at 15 kHz, those droplets broke into 3 droplets ( Figure 2 B). Analysis of images of the jet fluid captured upon exiting the nozzle opening revealed that the four droplets formed at a jet frequency of 1 kHz comprised 59%, 30%, 8%, and 3% of the total jet volume, respectively. Figure 2 C). Similarly, when operating at a frequency of 15 kHz, the three droplets formed in each jet represent 72%, 17%, and 11% of the total jet volume, respectively. Figure 2D). It is worth noting that because droplets have different velocities, they collide with each other, causing them to break up or merge in the air. As a result, droplet observer systems are limited to analyzing droplet breakup when ejected from the nozzle opening. To accurately determine the droplet size of the aerosol, the Spraytec® aerosol-based laser diffraction size determination method was used. Droplet size was measured using all nozzles at two ejection frequencies (7.5 kHz and 15 kHz). Figure 2 E, Figure 2 F). At 7.5 kHz, 50% of the total number of droplets is less than 13.0 μm (Dn(50)) and 90% of the droplets is less than 20.9 μm (Dn(90)) Figure 2 E). Similarly, at 15 kHz, 50% of the total number of droplets is less than 14.6 μm ((Dn(50)) and 90% of the droplets is less than 24.4 μm (Dn(90)) ( Figure 2 F). In some examples, 19.4 μm and 24.4 μm are alternative upper limits for droplet diameter, depending on the required clinical application. The droplet size distribution is characterized by variations in measurement parameters, including measurement distance, number of nozzles, and time elapsed for aerosolization. When measurements are taken near the nozzle plate, the distribution becomes slightly narrower (F). Figure 17 It was observed that reducing the aerosolization frequency or the number of nozzles tended to slightly reduce the droplet size. Figure 18 Furthermore, the size distribution during aerosolization was measured over time. The size distribution at the beginning of aerosolization was smaller compared to the middle and end, suggesting that the aerosolization process becomes more stable as distribution continues. Figure 19 Notably, in all cases, the effect of the studied variables on droplet size remained less than 2 μm, demonstrating the robust aerosolization performance of the microfluidic platform. Figures 17 to 19 ).

[0076] The temperature changes of the fluid in the thermal boundary layer during the electric pulse were also analyzed by simulation, taking into account the effects of time and geometry. The temperature distribution of the fluid in the ejector chamber is non-uniform, with higher temperatures closer to the heater surface. Figure 2 G, Figure 2 H). The temperature rise is minimal when the distance from the heater surface exceeds 1 μm. Furthermore, the temperature rise is temporally correlated with the height of the electrical signal actuated by the jetting droplet applied to the heater, and exhibits a peak temperature near the end of the jetting sequence. Figure 2 G, Figure 2 H). Although the temperature only experiences a localized increase to 300°C for a few microseconds, this increase is limited to the region near the heater, which is unlikely to impair the overall stability of the LNP / mRNA to be aerosolized.

[0077] To investigate any potential effects of thermal changes and shear forces generated by the microfluidic platform, LNP was aerosolized and the encapsulated mRNA was delivered to cells. The LNP formulation consisted of DLin-MC3-DMA, cholesterol, DSPC, and DMG-PEG in a molar ratio of 50:38.5:10:1.5. 2000 Composition (i.e., lipid composition of patisiran), in which mRNA is present at an N / P ratio of 5.30 ( Figure 3 A). After aerosolizing LNP / mRNA using a vibrating mesh nebulizer or a microfluidic platform according to this disclosure, the hydrodynamic size distribution, zeta potential, and RNA encapsulation of nanoparticles were compared with those of untreated nanoparticles. Figure 3 B to Figure 3 G). Untreated LNP / mRNA had a hydrodynamic diameter < 100 nm and a narrow distribution (PDI < 0.2). Figure 3 B to Figure 3 D). When atomized via a vibrating mesh atomizer, the size distribution of nanoparticles changes significantly. As previously shown, the ultrasonic treatment of the mesh atomizer results in large, unstable, and aggregated LNPs. The atomized LNPs / mRNA exhibit a hydrodynamic diameter of approximately 700 nm with a broad distribution (PDI > 0.7). Figure 2 B to Figure 2 D). In contrast, the size distribution of nanoparticles is almost unaffected by microfluidic atomization. Figure 3 B to Figure 3 D). The hydrodynamic size of nanoparticles aerosolized by microfluidics is < 100 nm and their distribution is narrow (PDI < 0.2). Figure 3 C to Figure 3 D). Although slightly reduced after aerosolization, the zeta potential of all LNP samples remained in the neutral range (i.e., between +10 mV and -10 mV). Figure 3 E). These significant differences in dynamic light scattering (DLS) analysis indicate that the microfluidic platform offers a clear benefit in preserving the physicochemical properties of LNP / mRNA during aerosolization compared to a conventional mesh nebulizer. The encapsulation state of mRNA after LNP nebulization was characterized. Although untreated LNP / mRNA exhibited high encapsulation (>95%), sonication of the mesh nebulizer resulted in a significant loss of mRNA encapsulation (approximately 43%). Figure 3 F). Furthermore, microfluidic aerosolization rescued the encapsulated mRNA from leakage, demonstrating its protective effect on the mRNA load during aerosolization. To confirm the preservation of load encapsulation, agarose gel electrophoresis analysis was performed on the LNP / mRNA samples ( Figure 3 G and Figure 20The mRNA solution (“mRNA only” group) and LNP / mRNA treated with Triton X-100 detergent showed mRNA migration in the gel, producing two bands: one for its linear form and the other for its secondary structure. Untreated LNP / mRNA did not migrate but remained in the pores, indicating stable encapsulation of mRNA within the nanoparticles. When testing LNP / mRNA exposed to aerosolization via a vibrating mesh, mRNA bands appeared, indicating leakage of mRNA during aerosolization of the nanoparticles. Figure 3 G). In contrast, LNP / mRNA atomized by the microfluidic platform showed very weak bands in the gel, supporting negligible mRNA loss during atomization (G). Figure 3 These results indicate that the microfluidic platform can atomize LNPs without mRNA loss (G). Figure 3 G), combined with the results of mRNA encapsulation assay ( Figure 3 F). Without being bound by a specific theory, the loss of mRNA during nebulization may be caused by the aggregation and rearrangement of lipids incorporated into the LNP. The LNP / mRNA was tested to examine whether it could fuse with the membrane during nebulization via fluorescence resonance energy transfer (FRET). Liposomes containing two DOPE-conjugated FRET probes, 7-nitrobenzo-2-oxa-1,3-diazole (NBD-PE) and rhodamine B (Rho-PE), were prepared to obtain "FRET liposomes". Figure 3 H). Due to FRET occurring between NBD and Rhodamine B, the proximity of the two probes in the liposomes leads to a decrease in NBD fluorescence. After membrane fusion, the distance between the two probes increases, resulting in a subsequent increase in NBD fluorescence signal. To assess membrane fusion events, LNP / mRNA was mixed with FRET liposomes and subjected to various conditions: Triton X-100, bath sonication, vibrating mesh atomization, and microfluidic aerosolization. Figure 3 I). Signals obtained from untreated samples represent 0% fusion, while Triton X-100 treatment represents 100% fusion. Of the methods tested, vibrating mesh atomization induced the highest degree of membrane fusion ( Figure 3 (I) Notably, it significantly outperformed bath sonication, likely due to the additional shear stress generated by the mesh. On the other hand, microfluidic aerosolization resulted in a significantly lower fusion level (approximately 16% lower), indicating its minimal impact on the lipid membrane structure during LNP aerosolization. In summary, the microfluidic platform according to this disclosure offers significant advantages in the aerosolization of LNP / mRNA, achieved by maintaining the physicochemical properties and structural integrity of the nanoparticles and providing better protection for the encapsulated mRNA load, in which respects it surpasses conventional vibrating mesh nebulizers.

[0078] Next, the morphology of aerosolized LNP / mRNA was examined using cryotransmission electron microscopy (CryoTEM). Untreated LNP / mRNA samples showed a spherical shape with a single bilayer. Figure 4 A), and it was found that the particle diameter was less than 100 nm, consistent with the results of DLS analysis. Figure 2 C). However, ultrasonic disruption using a mesh atomizer causes dissociation and aggregation of nanoparticles (C). Figure 4 B). The density of nanoparticles in each field of view was also significantly reduced (data not shown). Some captured particles showed diameters in the hundreds of nanometers range, and the images had a generally low signal-to-noise ratio, indicating a difference in electron density of the sample on the grid. In contrast, the morphology of LNP / mRNA after microfluidic aerosolization was very similar to that of the untreated sample, despite the presence of several large nanoparticles ( Figure 4 C). Macroscopic observation of LNP / mRNA samples further supports the conclusion that sonication induced LNP dissociation and aggregation. In contrast, untreated samples and samples subjected to microfluidic aerosolization appeared optically translucent. However, samples aerosolized by a mesh nebulizer became opaque. Figure 21 This indicates the presence of large particles capable of scattering light.

[0079] Without being bound by specific theories, intact nanostructures provided by microfluidic platforms can generate larger mRNA transfections than nanostructures damaged by mesh nebulizers. To test this hypothesis, 293T / 17 cells were treated with aerosolized or unaerosolized LNPs containing ZsGreen1 mRNA (LNP / ZsGreen1). ZsGreen1 mRNA transfection was assessed using fluorescence microscopy 24 h after treatment. The negative control group showed no green fluorescence, while cells treated with LNP / ZsGreen1 solution showed bright green fluorescence in the field of view. Figure 4 D). When comparing vibrating mesh nebulizers and microfluidic platforms, the latter clearly leads to increased expression of the ZsGreen1 protein in the treated cells. Figure 4D). Furthermore, the ZsGreen1 expression levels observed in cells treated with aerosolized LNP / ZsGreen1 using a microfluidic platform appeared comparable to those observed in cells treated with LNP / ZsGreen1 solution. This suggests that the aerosolization of LNP / mRNA by the microfluidic platform has little effect on the efficiency of nanoparticle-mediated mRNA delivery to cells. On the other hand, the vibrating mesh nebulizer significantly hindered nanoparticle transfection of mRNA in vitro during aerosolization. Further measurements were performed to examine the advantages of the microfluidic platform over the vibrating mesh nebulizer in mRNA delivery. A consistent pattern was again observed when delivering firefly luciferase (Fluc) mRNA. In 293T / 17 cells, the group treated with the vibrating mesh nebulizer showed an almost 100-fold reduction in mRNA transfection compared to the LNP solution treatment group. Figure 4 E). In contrast, the group treated with the microfluidic platform showed similar mRNA transfection levels to the LNP solution-treated group, although a reduction of approximately 7-fold was observed only at the lowest mRNA dose (E). Figure 4 E). All treatments had almost no effect on the cell viability of 293T / 17 cells. Figure 22 To confirm the benefits of the microfluidic platform in aerosolizing LNP / mRNA, experiments were repeated in more biologically relevant cells. In human bronchial epithelial cells (16HBE14o-), the microfluidic platform showed comparable efficiency to LNP solution treatment in delivering mRNA. Figure 23 In contrast, vibrating mesh nebulizers significantly impaired the ability of nanoparticles to deliver mRNA at all tested mRNA doses. Subsequent screening in multiple cell lines showed similar results, indicating that the microfluidic platform achieved approximately 5 to 187 times higher mRNA delivery efficiency than vibrating mesh nebulizers. Figure 4 F and Figure 24 In summary, the microfluidic platform according to this disclosure clearly demonstrates significant advantages over vibrating mesh nebulizers because it exhibits superior efficiency in delivering mRNA to cells.

[0080] The overall relative advantage of the microfluidic platform according to this disclosure in generating nanoparticle-containing aerosols for mRNA delivery to mouse lungs has been established. For this purpose, a whole-body rodent inhalation system was used, the system comprising a 3 L container connected to the microfluidic platform. Figure 5A). This system facilitates the controlled administration of aerosolized LNP / mRNA to mice, enabling the evaluation of its potential applicability for inhalable mRNA therapy. A microfluidic platform is programmed via a controller to generate a plume and load LNPs (LNP / Nluc) containing Nluc mRNA into a cartridge. Mice are placed in a chamber and exposed to an aerosol containing LNP / Nluc, after which they spontaneously inhale the nanoparticles. To confirm successful mRNA delivery to the lungs using a rodent inhalation system facilitated by the microfluidic platform according to this disclosure, RNAScope in situ hybridization (ISH) analysis was performed.

[0081] LNP / Nluc aerosol containing 1 mg mRNA was aerosolized into a chamber and mice were allowed to inhale the aerosol. Twenty-four hours later, mouse lungs were collected, fixed, and prepared for staining. The presence of Nluc mRNA transcripts in the lungs was observed using confocal imaging. Additionally, rod-shaped cells (a specific type of bronchial epithelial cell) were labeled to assess the distribution of Nluc mRNA transcripts within the lung epithelial tissue. Confocal images clearly showed the presence of Nluc transcripts throughout the entire lung exposed to the LNP / Nluc aerosol. Figure 5 B). In contrast, no signal was detected in the lungs exposed to PBS aerosol, confirming the specificity of the assay. Figure 5 C). Furthermore, the delivered Nluc mRNA transcripts were primarily located in rod-shaped cells, indicating that the inhaled mRNA was effectively deposited in the lung epithelial tissue. Figure 5 C). These findings strongly support the effectiveness of the microfluidic platform according to this disclosure in generating sufficiently fine aerosols to reach mouse lung epithelial cells via spontaneous respiration, thus demonstrating its effectiveness in lung mRNA delivery. To evaluate the effectiveness of the delivered mRNA in cell transfection, bioluminescent imaging was performed on collected lungs. Similarly, LNP / Nluc was aerosolized into a chamber to deliver a dose of 1 mg Nluc mRNA. In vitro imaging of mouse lungs was performed 24 hours after inhalation. Bioluminescent imaging showed consistent luciferase expression in lungs collected from all mice. This consistent expression in the lungs supports the robust performance of the microfluidic platform according to this disclosure in delivering mRNA to the lungs and achieving successful protein expression in mouse respiratory tissues. Figure 25 Further investigation was conducted to assess whether mRNA transfection was dose-dependent. Results showed that Nluc expression in the lungs was directly proportional to the dose of LNP / Nluc aerosolization. Figure 5 D and Figure 5 E). This indicates that increasing the dose of LNP / mRNA leads to a corresponding increase in protein expression in the lungs. Furthermore, the site of mRNA transfection achieved via inhaled LNP / Nluc is limited to the lungs ( Figure 5D). Conventional LNP formulations containing DLin-MC3-DMA exhibited intrinsic tropism for liver transfection upon systemic administration. However, results confirmed that inhaled LNPs could deliver mRNA to the respiratory system, consistent with previous studies. Luminescence measurements were performed at 24 and 48 hours post-treatment. The results showed that luciferase expression was significantly higher at 24 hours post-treatment compared to 48 hours post-treatment. Figure 5 F and Figure 5 G). This observation indicates the transient nature of mRNA transfection, where the expression of the delivered mRNA peaks at an early time point and then gradually declines over time. Subsequently, the possibility of acute lung injury following LNP / Nluc inhalation was examined by histopathology. Mice were exposed to LNP / Nluc aerosol containing 1 mg of mRNA (G). Figure 5 H) or an equal volume of PBS ( Figure 5 I). After 24 hours of treatment, lung tissue was collected, lung sections were prepared, and stained with hematoxylin and eosin (I). Figure 5 H and Figure 5 E) Used to assess any potential histological changes or tissue damage. In H&E-stained lung sections, the lungs in both groups appeared histologically normal. However, minimal mononuclear cell infiltration was observed in both groups around the terminal bronchioles, interstitium, or peribronchial regions (Fig. 26). Furthermore, the lungs showed a slight increase in lymphocytes in the bronchial-associated lymphoid tissue, but this was not significantly different between the two groups, suggesting that these abnormalities may be artifacts and unrelated to treatment. These minor changes could be attributed to excessive fluid accumulation in the tissue, leading to mild congestion in the lungs. Overall, these findings collectively enhance the lung efficacy of the microfluidic aerosolization platform according to this disclosure, providing a safe, effective, and direct way to deliver mRNA therapy for respiratory conditions while avoiding undesirable effects in other organs.

[0082] Materials and methods

[0083] This document discusses numerous working examples. These working examples are not intended to limit the scope of this disclosure, but rather to illustrate various aspects. Any feature discussed below with reference to the working examples can be combined with implementations discussed anywhere in this disclosure, and vice versa.

[0084] Material

[0085] Firefly luciferase (Fluc) mRNA was purchased from Trilink Biotechnologies. Cholesterol was obtained from Sigma-Aldrich (MO, USA), and DSPE-PEG was also used. 2K and DMG-PEG 2KPurchased from NOF America. DLin-MC3-DMA was purchased from Biofine International Inc. (BC, Canada). 1,2-Distearayoyl-sn-glycero-3-phosphocholine (DSPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(rhodamine B sulfonyl) (Rhod-PE), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE) were obtained from Avanti Polar Lipids, Inc. (AL, USA).

[0086] Characterization of droplets generated by a microfluidic aerosolization platform

[0087] Liposomes were prepared as an alternative to LNPs when characterizing droplets formed via a microfluidic aerosolization platform. In short, the lipid phase consisted of the following components: DSPC, cholesterol, and DSPE-PEG. 2K The lipid solutions were diluted to 25 mM in an EtOH / DMF mixture (97.5:2.5) at a molar ratio of 52:45:3. Sterile PBS and the lipid solutions were heated at 65 °C and mixed at a 2:1 volume ratio using a NanoAssemblr Benchtop system (Precision Nanosystems, BC, Canada), then dialyzed overnight at 4 °C in a 10 kDa Slide-a-Lyzer G2 box (Thermo Fisher, MA, USA) with sterile PBS. The total batch size of liposomes was 1.2 L. The hydrodynamic size and polydispersity of the liposomes were determined by DLS using a Zetasizer Nano ZSP device (Malvern Panalytical, UK).

[0088] Droplet formation and ejection were evaluated using a drop watcher system (JetXpert Dropwatcher, ImageXpert, NH). Droplets dispensed by a microfluidic head were examined, and the behavior of individual droplets was captured using the drop watcher camera. The system was calibrated to a ratio of 1 pixel to 0.001034 mm using built-in software and a manufacturer-supplied calibration target. Slits of known width were imaged at determined magnification and working distance. This allowed for the determination of droplet size and estimation of droplet volume using the manufacturer-supplied software.

[0089] Using Spraytec ®The droplet size measurement system (Malvern Panalytical, UK) measures droplet size. The system measures the size distribution of aerosolized droplets via laser diffraction. This requires measuring the angular intensity of light scattered from the spray as it passes through the laser beam. The recorded scattering pattern is then analyzed and plotted using the manufacturer's software.

[0090] Thermal simulations were performed using proprietary FMS ejector modeling code (Funai Lexington, KY) with the following settings: bubble detachment time of 1236 ns, voltage of 11 V, chip temperature of 45 °C, pre-pulse duration of 200 ns, pulse delay of 800 ns, and main thermal pulse duration of 600 ns. The hydrodynamics of the microfluidic aerosolization platform were characterized using a proprietary aqueous dye formulation with well-defined thermophysical properties. Data were plotted using Origin 2022 (version 9.9).

[0091] In vitro transcription of mRNA

[0092] In vitro transcription of mRNA was performed as described. In short, a nano-luciferase (containing a T7 promoter) was used. Nluc Linearized plasmids were used as templates for in vitro transcription. RNA was synthesized using the HiScribe T7 High-Yield RNA Synthesis Kit (New England Biolabs Inc., MA, USA) and CleanCap Reagent AG (Trilink Biotechnologies) according to the manufacturer's instructions. Nluc mRNA was purified using the Monarch RNA Cleaning Kit (New England Biolabs) and stored at -80°C. Measurements were taken using a multi-well microplate reader (Tecan Trading AG, Switzerland). Nluc mRNA concentration. Agarose gel electrophoresis was performed to visualize the mRNA and assess degradation. 1 μg of IVT mRNA or RiboRuler high-range RNA ladder (Thermo Fisher, MA, USA) was denatured and loaded onto a 1.5% agarose-formaldehyde gel pre-stained with GelRed (Biotium, CA, USA). The gel was electrophoresed at 85 V for 2 h, followed by UV development.

[0093] LNP formulations

[0094] LNPs were prepared using microfluidic mixing as described above. The lipid phase consisted of the following components: DLin-MC3-DMA, cholesterol, and DMG-PEG. 2KThe mRNA was diluted to 5.5 mM with DSPC in 100% ethanol at a molar ratio of 50:38.5:1.5:10. The mRNA was then diluted in sterile 50 mM citrate buffer. The mRNA and lipid solutions were mixed at a volume ratio of 3:1 using a NanoAssemblr Benchtop system (Precision Nanosystems, BC, Canada), and then dialyzed overnight against sterile PBS in a 10 kDa Slide-a-Lyzer G2 box (Thermo Fisher, MA, USA) at 4°C.

[0095] LNP characterization

[0096] Hydrodynamic size and polydispersity of LNPs were determined by DLS in a Zetasizer Nano ZSP device (Malvern Panalytical, UK). mRNA encapsulation was quantified using a modified protocol with the Quant-iT RiboGreen RNA Assay Kit (Thermo Fisher, MA, USA) and a multi-functional microplate reader. mRNA encapsulation was qualitatively measured using agarose gel migration before and after aerosolization.

[0097] LNP aerosolization

[0098] To aerosolize LNP via ultrasound, LNP was added dropwise to a vibrating mesh nebulizer (Aerogen, Ireland) as described above. 4 For microfluidic-based aerosolization using a microfluidic device, an LNP is added to a single channel of the previously described microfluidic cartridge, which is connected to a microfluidic aerosolization platform (FMS, Lexington, KY). Droplet ejection is controlled using software provided by FMS with the following settings: a frequency of 1.2 kHz, a target temperature of 25 °C, a voltage of 11 V, a preheating pulse settling time of 100 ns, a preheating pulse duration of 200 ns, a main thermal pulse settling time of 800 ns, and a main thermal pulse duration of 600 ns.

[0099] FRET-based lipid membrane fusion assay

[0100] FRET assays were used to evaluate shear stress-induced lipid mixing and membrane fusion during aerosolization. In short, DOPE-conjugated FRET probes NBD-PE and Rho-PE were incorporated into FRET liposomes. This formulation caused a decrease in NBD fluorescence due to FRET transfer to rhodamine. An increase in the NBD signal was detected when lipid fusion occurred due to the increased distance between the two probes. To prepare the FRET liposomes, a DOPC:NBD-PE:Rho-PE mixture was mixed in chloroform at a molar ratio of 99:0.5:0.5 in a round-bottom flask. Chloroform was removed by a constant gas flow and flask rotation, resulting in the formation of a homogeneous lipid agglomerate at the bottom of the flask. Vacuum was applied for 2 h to ensure complete chloroform evaporation. After drying, the lipid membrane was sonicated for 20 min, hydrated with HEPES-buffered saline, and extruded using a small extruder (Avanti Polar Lipids). The liposome solution was passed through a 100 nm membrane 11 times. Subsequently, FRET liposomes were mixed with LNP / mRNA at a 1:1 (v / v) ratio and exposed to different conditions: no treatment, 2% Triton X-100, 20 min sonication in a bath, vibrating mesh nebulizer, or microfluidic aerosolization platform. The resulting samples were added to black 96-well plates (80 μl per well) for fluorescence measurement. Fluorescence measurements were performed at Ex / Em = 465 / 535 nm using a multi-functional microplate reader. The results of untreated and Triton X-100 treated samples were set as negative controls. ) and positive control ( The fusion percentage is calculated as follows: .

[0101] Cryo-transmission electron microscopy (CryoTEM)

[0102] After plasma cleaning of the grid, 2 μl of sample was deposited onto the grid in the FEIVitrobot chamber at 100% relative humidity and allowed to stand for 10 seconds. The grid was then blotted with filter paper for 1 second, followed by immersion in liquid ethane cooled with liquid nitrogen. Visible defects on the frozen grid were examined, and the complete grid was then assembled into a cassette. CryoTEM was performed at 200 kV using a Glacios cryo-electron microscope equipped with a Gatan K3 camera.

[0103] Cell culture

[0104] HeLa cells were donated by Professor Robert Langer of the Massachusetts Institute of Technology (MIT). A549 cells were provided by Professor Adam Alani of Oregon State University. 16HBE14o- and CFBE41o- cells were obtained from Professor Kelvin MacDonald of OHSU. HEK293T / 17 cells were purchased from the American Type Culture Collection (ATCC). HeLa and HEK293T / 17 cells were cultured in DMEM supplemented with 10% heat-inactivated FBS, 1% penicillin / streptomycin, and 10 mM HEPES buffer. 16HBE14o- and CFBE41o- cells were maintained in MEM / EBSS supplemented with 10% heat-inactivated FBS, 1% penicillin / streptomycin / glutamine, 1% sodium pyruvate, and 10 mM HEPES buffer. A549 cells were cultured in RPMI-1640 supplemented with 10% heat-inactivated FBS, 1% penicillin / streptomycin and 10 mM HEPES buffer.

[0105] Characterization of in vitro mRNA delivery

[0106] Cells were seeded at 4,000 cells / well in white 96-well plates, 50,000 cells / well in clear 12-well plates, or 40,000 cells / well in 8-well microplates (Ibidi), followed by overnight incubation for adhesion. mRNA aerosolization was performed using one of two methods: a vibrating mesh nebulizer (Aerogen, Ireland) or a microfluidic aerosolization platform (Funai Lexington, KY). Cells, particularly those in the plates, were treated with the appropriate dose of mRNA. For cells in 96-well plates, aerosolized particles were collected from the output of the nebulizer unit and added to the seeded cells. For cells in 12-well plates, the nebulizer unit was maintained, and LNPs encapsulating mRNA were directly dispensed onto the cells, followed by incubation for 24 h. Cell viability and in vitro luciferase expression were evaluated using the ONE-Glo + Tox luciferase reporter gene and cell viability assay kit (Promega) and a multi-functional microplate reader.

[0107] animal

[0108] All animals used in the studies were conducted at Oregon Health and Sciences University and approved by the Institutional Animal Care and Use Committee (IACUC, IP0001707).

[0109] In vivo LNP / mRNA delivery via microfluidic aerosolization

[0110] To characterize the delivery of LNP to mice via aerosolization, a whole-chamber aerosolization system was selected as described. 12 In short, all mice in the group were placed in a 3 L chamber at a time. LNPs encapsulating mRNA were prepared at 0.25 mg / mL and diluted in PBS. LNPs were loaded into a single channel of a microfluidic cassette in 200 μl volumes each time. LNPs were aerosolized into the chamber at a rate of 25 μl / 2 min until the total dose of mRNA delivered was 1 mg. All mice in the group were unrestrained and not sedated. Twenty-four h post-treatment, animals were sacrificed and lungs were collected for further characterization. To detect Nluc expression in the lungs, the collected lungs were gently and briefly washed in sterile PBS, and each lung was incubated for 5 min at room temperature in 200 μl of a 40-fold diluted solution of Nano-Glo substrate (Promega) in PBS. In vitro bioluminescence imaging was then performed using an IVIS Lumina XRMS (PerkinElmer). The collected lungs were then homogenized by adding 150 μl of PBS to each lung and homogenizing thoroughly on ice with a hand-held homogenizer until slurry was observed. Samples transferred to microcentrifuge tubes were centrifuged at 17,000 xg for 30 min at 4 °C. On white 96-well plates, 30 μl of supernatant and 60 μl of 80-fold diluted Nano-Glo substrate in PBS were incubated at room temperature for 5 min. The luminescence was read using a multi-well microplate reader. The luminescence was normalized to total protein concentration and measured using a BCA protein assay kit (Thermo Fisher).

[0111] Histopathology

[0112] BALB / c mice were exposed to LNP / mRNA aerosols generated by a microfluidic device until the total dose of mRNA delivered was 1 mg, or until an equal volume of sterile PBS was delivered. After 24 h, the mice were humanely euthanized, and their lungs were perfused with sterile PBS through the right ventricle. A 20G catheter was inserted into the trachea for lung inflation using 10% neutral buffered formalin at a pressure of 25 cm above the surgical plane. The inflated lungs were carefully removed, placed in a tissue embedding cassette, and immersed in 70% ethanol for dehydration. Subsequently, the tissues were embedded in paraffin, sectioned, placed on slides, stained with H&E, and covered with a slide for histopathological evaluation (IDEXX BioAnalytics, MO, USA). Whole-slide imaging of the samples was performed using a slide scanner (Leica Biosystems), and the images were analyzed using Aperio ImageScope v12.4.6.5003 (Leica Biosystems) and Fiji.

[0113] RNAscope in situ hybridization

[0114] Following the manufacturer's protocol, delivered Nluc mRNA and endogenous scgb1a1 mRNA transcripts were visualized in formalin-fixed paraffin-embedded (FFPE) lung tissue sections using the RNAscope Multiplex Fluorescent Reagent Kit v2 (ACD). Nluc probes (catalog number 885981) and scgb1a1 probes (catalog number 420351-C3) were prepared, and Nluc and scgb1a1 transcripts were visualized using tyramine signal amplification (TSA) based Opal fluorophores Opal 570 (Akoya Biosciences, #FP1488001KT, 1:800 dilution) and Opal 690 (#FP1487001KT, 1:1,500 dilution). Confocal images were obtained using a ZEISS LSM 880 (Carl Zeiss AG).

[0115] Figure 1 A to Figure 1 D. A microfluidic system for aerosolizing LNP / mRNA. Figure 1 A) Microscopic image of a microfluidic chip containing 960 nozzles. The nozzles are arranged in three fluid channels, each consisting of two rows of nozzles, and each channel connects to a separate fluid chamber. Each nozzle has a diameter of approximately 10 μm. Figure 1B) An illustration of a cross-sectional view of a single nozzle fabricated using conventional semiconductor CMOS processes, proprietary FMS thin-film processes, and proprietary FMS MEMS lithography processes. The thin film consists of multiple layers, including a protective layer, a cavitation layer, a dielectric film, electrodes, and a heating film. When the heater is activated, the blue arrows indicate the primary direction of bubble growth. Figure 1 C) Illustration of the layered structure of a microfluidic capsule head with bubbles. Scale bar indicates 10 μm. Figure 1 D) shows a schematic diagram of the sequential mechanism of LNP / mRNA droplet ejection (aerosolization) at the nozzle cross section: (1) bubble nucleation, (2) bubble growth, (3) droplet ejection, and (4) droplet detachment and refilling.

[0116] Figure 2 A to Figure 2 H. Characterization of droplets generated from a microfluidic platform. In ( Figure 2 A) 1 kHz and ( Figure 2 B) Representative images of a single plume ejected at 15 kHz. Black arrows indicate the direction of the plume. Estimate the volume percentage of each droplet from the captured images and record the volume percentage at each ejection. Figure 2 C) 1 kHz and ( Figure 2 D) Volume percentage of the injection at 15 kHz. Figure 2 E and Figure 2 F) in ( Figure 2 E) 7.5 kHz and ( Figure 2 F) Laser diffraction-based determination of the size distribution of a jet of liquid droplets ejected at a frequency of 15 kHz. Figure 2 G and Figure 2 H) Electrothermal simulation of fluid temperature changes within the thermal boundary layer during the pulse, based on geometry and time. (G) Surface plot of fluid temperature response during the pulse and (H) contour plot. Time is expressed as the duration from the start of the pulse, in nanoseconds (ns).

[0117] Figure 3 A to Figure 3 I. Physicochemical characterization of LNP / mRNA aerosols generated by conventional nebulizers and microfluidic aerosolization platforms. Figure 3 A) Schematic diagram of a single LNP / mRNA. PEG lipids (blue); ionizable lipids (green); structural lipids (pink); cholesterol (yellow). Figure 3 B) Representative size distribution of LNP / mRNA under different treatments: no treatment (grey), vibrating mesh (red), and microfluidic platform (blue). (Figures C to F) Changes in LNP / mRNA after aerosolization via vibrating mesh (black) or microfluidic platform (blue): Figure 3 C) Dimensions, Figure 3D) Polydispersion index, ( Figure 3 E) zeta potential (mV), and ( Figure 3 F) mRNA encapsulation. Figure 3 G) Representative images of agarose gel electrophoresis analyses from various treatment conditions: 2) mRNA only, 3) untreated LNP / mRNA, 4) LNP / mRNA + Triton X-100, 5) LNP / mRNA + vibrating net, and 6) LNP / mRNA + microfluidic platform. Figure 3 H) Schematic diagram of FRET-based lipid membrane fusion assay. Figure 3 I) FRET-based assay results of lipid membrane fusion of LNP / mRNA exposed to different conditions.

[0118] Figure 4 A to Figure 4 F. The performance of LNP / mRNA aerosols generated by the microfluidic aerosolization platform is superior to that of vibrating mesh nebulizers. Figure 4 A) in solution, ( Figure 4 B) After aerosolization via a vibrating mesh atomizer, and ( Figure 4 C) Cryotransmission electron microscopy (cryoTEM) image of LNP / mRNA after aerosolization via a microfluidic platform. Scale bar indicates 50 nm. Figure 4 D) Fluorescence microscopy images of HeLa cells subjected to various conditions: untreated, treated with LNP / ZsGreen1 in solution, aerosolized via a vibrating mesh, and aerosolized via a microfluidic platform (from left to right), at a dose of 1 μg mRNA / chamber. Green represents ZsGreen1 protein, and blue represents the cell nucleus. Scale bar indicates 200 μm. Figure 4 E) Normalized luciferase expression levels measured after treatment of 293T / 17 cells with LNP / Fluc solution (light blue), LNP / Fluc aerosolized via a vibrating mesh nebulizer (red), or LNP / Fluc aerosolized via a microfluidic platform (blue) at various mRNA doses. (N = 4) (F) Normalized luciferase expression in various cells treated with LNP / Fluc aerosolized via a vibrating mesh nebulizer (red) or microfluidic platform (blue) at a dose of 50 ng mRNA / well. (n = 5) p < 0.05; p < 0.01; p < 0.0001.

[0119] Figure 5 A to Figure 5 I. Utilizing microfluidics-assisted aerosolization to efficiently deliver mRNA to the lungs. Figure 5 A) A schematic diagram of the whole-body rodent inhalation system, consisting of a 3 L container connected to a microfluidic platform. Figure 5 B and Figure 5 C) Inhalation using a microfluidic platform ( Figure 5 B) LNP / Nluc inhalation or ( Figure 5 C) Representative images of BALB / c mouse lung sections after sterile PBS analysis using RNAscope in situ hybridization. Nluc mRNA transcript (red), SCGB1A1 (green), and cell nuclei (blue). 20× magnification. Figure 5 D to Figure 5 G) In vitro luminescence images of lungs obtained after LNP / Nluc aerosolization and application to mice ( Figure 5 D and Figure 5 F) and quantitative ( Figure 5 E and Figure 5 G). ( Figure 5 D and Figure 5 E) Changes include alterations in the amount of mRNA to be aerosolized when images are taken 24 h after inhalation. Figure 5 D and Figure 5 E), or luminescence imaging at different time points when 2.0 mg mRNA is aerosolized (E), Figure 5 F and 5G). (n = 2) Figure 5 H and Figure 5 I) Histopathological analysis of mouse lungs collected 24 h after (H) inhalation of LNP / Nluc, in which 1.0 mg of mRNA was aerosolized, or ( Figure 5 I) Use a microfluidic platform to aspirate an equal volume of sterile PBS. The scale bar indicates 100 μm.

[0120] Each ejector can be fluidly connected to a separate fluid chamber. In some examples, the chip includes at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 960, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, or 1800 droplet ejectors, or within the range defined by any two of the foregoing values. In some examples, the array includes 800 to 1200 droplet ejectors. In some examples, the array includes 900 to 1000 droplet ejectors. In some examples, the array includes 960 droplet ejectors. The droplet injectors can each be individually addressable, enabling the generation of droplet plumes containing LNP / mRNA.

[0121] The diameter of the nozzle for each droplet ejector can be 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 8 μm, 19 μm, 20 μm, 21 μm, 22 μm, 23 μm, 24 μm, or 25 μm, or a diameter within any two of the foregoing values. In some examples, the diameter of each nozzle can be from 5 μm to 15 μm. In some examples, the diameter of each nozzle can be from 8 μm to 14 μm. In some examples, the diameter of each nozzle can be from 9 μm to 13 μm. In some examples, the diameter of each nozzle can be about 10 μm. In some examples, the diameter of each nozzle can be about 12 μm. In some examples, the diameter of the microfluidic chamber can be equal to or greater than the diameter of the nozzle. In some examples, the diameter of the microfluidic chamber can be larger than the diameter of the nozzle.

[0122] PEG molar concentration can affect RNA delivery in aerosolized LNPs / mRNA. Incorporating PEG into LNPs helps maintain nanoparticle stability during self-assembly. Furthermore, PEG molecules inhibit the interaction between LNPs and serum proteins, prolonging nanoparticle circulation time. However, they also inhibit the formation of biomolecular crowns on the nanoparticle surface, which delays LNP endocytosis and subsequent mRNA delivery. In the case of LNP nebulization, PEG molecules are thought to contribute to the recovery or stabilization of nebulized nanoparticles through steric effects. This complex interaction of PEG in LNP chemistry increases the complexity of formulation design for inhalation. Even with meticulous optimization, the nebulization process can compromise the integrity of LNPs, obscuring key properties necessary for effective lung transfection. MAP can help eliminate or minimize uncertainties in formulation discovery by preventing LNP deformation during nebulization.

[0123] Example portable atomizer

[0124] The following is combined with Figures 6 to 14 An embodiment of the portable handheld atomizer according to the present invention is discussed.

[0125] Reference Figure 6An example of a microfluidic pulmonary drug delivery dispenser 10 according to the present invention is shown. The dispenser 10 includes a housing 12 which may have a handle 14. An outlet 16 is carried by the housing 12 and is capable of removably accommodating a mouthpiece 18. The dispenser 10 can dispense one or more pulmonary drug doses into the pulmonary system via the mouthpiece 18 in response to activation of a discharge controller and / or a breathing trigger, such as a button 20, thereby initiating the discharge by the patient's inhalation, thus synchronizing the treatment with the patient's inhalation. Such respiratory-actuated inhalers (e.g., U.S. Patent No. US7219664B2) and respiratory-driven nebulizers (e.g., U.S. Patent No. US11266795B2) are known, and this technology is a novel application of respiratory-actuated microfluidic aerosolizers.

[0126] The housing 12 may be provided with a docking structure, such as a cavity 22, for removably accommodating a cartridge 24, which contains a volume of one or more pulmonary medications. The housing 12 may additionally be provided with a cover 26 for sealing the cartridge 24 within the cavity 22. The cartridge 24 includes at least one reservoir and at least one microfluidic chip, which will be discussed in more detail below. When the cartridge 24 is located within the cavity 22, the medication flow path extends from the reservoir, through the chip, outflows through an outlet 16, and reaches the patient through an opening 18. In the case of multifluid delivery applications, therapeutic agents can be dispensed sequentially or simultaneously from one or more reservoirs according to a preset configuration. Furthermore, the ratio of different therapeutic agents from different reservoirs, whether sequentially or simultaneously dispensed, can be configured to different concentrations necessary to obtain preferred clinical outcomes.

[0127] Box 24 can be stored at up to -4°C for up to 6 months and reused as an excretion dose while maintaining clinical efficacy.

[0128] The housing 12 may also be equipped with a display 28 for showing the progress and status of the dispenser 10. A microprocessor, power supply, and associated electronics may be housed within the housing 12. A charging connection (e.g., an induction coil) or a charging port 30 (e.g., a USB port) may be provided to charge the internal power supply. An audible signal generator, such as a tone generator or a buzzer, may be provided and can be activated at preset times during the operating cycle, such as at the start of dose dispensing and upon completion of dose delivery.

[0129] Reference Figure 8 The plasma heat sterilization element 32 can be configured to communicate with the flow path, downstream of the microfluidic chip and upstream of the outlet 16. For the purpose of preventing cross-contamination, the plasma heat sterilization element can be activated between uses or between uses by different patients; the plasma heat sterilization element is capable of rapidly heating to inactivate any viruses or bacteria that may be present. (See reference...) Figure 10The user interface 28 may include power controls (e.g., button 40) and an on / off indicator 42 (e.g., an LED). An optional error indicator 44, which may also be an LED, may also be provided. The mode selection control 46 allows the user to switch between different dispensing settings, such as extended time dispensing or dispensing from multiple repositories for combined doses. One, two, or more mode indicators 48 confirm the selected mode.

[0130] Reference Figure 11 The opening 18 may be provided with a one-way valve 50 (e.g., a louvered door, a flap valve, a duckbill valve, or other valve structure), which can open in response to the forward velocity of the steam plume 52.

[0131] Figure 12A An exploded side view of the housing and cartridge is shown, including a front view of the cartridge and an enlarged view of the microfluidic chip 54. The chip 54 has a digitally addressable nozzle array 56, which may include at least about 100 or 200 nozzles, and in some embodiments may include at least about 300 or 400 or more individual nozzles 58. The microfluidic chip 54 may have a width ranging from about 1 mm to about 10 mm, and in some embodiments may have a width ranging from about 3 mm to about 5 mm, and in one embodiment may have a width of about 4 mm.

[0132] Reference Figure 12B and Figure 13 Each droplet ejector 58 has a nozzle 60 communicating with a microfluidic chamber 62 for discharging a liquid drug solution from the chamber and through the nozzle 60. The diameter of the opening can range from about 10 micrometers to about 100 micrometers, depending on the desired performance. The droplet size can typically be less than about 30 micrometers, and in some embodiments range from about 7 micrometers to about 17 micrometers. A single nozzle ejection can operate at a repetition rate of at least about 5,000 droplets or 10,000 droplets or 15,000 droplets or more per second to produce a flux of at least about 20 microliters or 25 microliters or more per second, with an initial velocity of about 8 m / s to about 12 m / s. Measured at 2 inches from the distal opening of the orifice, the velocity does not exceed about 1 m / s, and typically does not exceed about 0.5 m / s or about 0.1 m / s, thus producing a soft, low-velocity plume 52.

[0133] The thin film layer 64 is in thermal and / or mechanical communication with the microfluidic chamber 62 and is supported by a substrate such as a silicon layer 66. The thin film layer 64 can be ejected from the microfluidic chamber 62 and aspirated through a droplet ejector 58, for example, by applying thermal or mechanical energy. In the illustrated embodiment, the thin film layer 64 may include a heater layer to provide resistance heating in response to the application of an electric current. Alternatively, mechanical energy may be provided using piezoelectric or ultrasonic transducers, as understood in the art.

[0134] Figure 14 A side-view sequence diagram of droplet nucleation via jetting is shown. Droplet nucleation begins when heat is applied to a drug-loaded microfluidic chamber. Further application of heat induces droplet growth and jetting, resulting in final droplet detachment after exiting the droplet ejector 58, which facilitates an aerosol plume containing liquid nanoparticles loaded with mRNA.

[0135] Figure 15 This is an image sequence of aerosols generated from a microfluidic device.

[0136] Figure 16 The representative size distribution of liposomes was depicted.

[0137] Figure 17 The size distribution of aerosol droplets at different distances from the device is depicted. Laser scattering was measured at different distances from the nozzle plate: 20 mm (red), 30 mm (blue), 40 mm (green), and 60 mm (magenta). Aerosolization was performed under the following conditions: (A) all nozzles at 15 kHz (full), (B) half of the total nozzles at 15 kHz (space), or (C) all nozzles at 7.5 kHz (time).

[0138] Figure 18 The size distribution of aerosol droplets was plotted by varying the aerosolization frequency or the number of nozzles. Aerosolization was performed under the following conditions: all nozzles at 15 kHz (red; full), half of the total nozzles at 15 kHz (blue; space), or all nozzles at 7.5 kHz (green; time). Laser scattering was measured at different distances from the nozzle plate: (A) 20 mm, (B) 30 mm, (C) 40 mm, and (D) 60 mm.

[0139] Figure 19The size distribution of aerosol droplets over multiple time elapsed stages of aerosolization was depicted. Laser scattering was measured at a distance of 20 mm from the nozzle plate (A, C, E) or 60 mm from the nozzle plate (B, D, F). Measurements were recorded at the start (red), middle (blue), or end (green) of the distribution. Aerosolization was performed under the following conditions: (A, B) all available nozzles at 15 kHz (full), (C, D) half of the total nozzles at 15 kHz (space), or (E, F) all nozzles at 7.5 kHz (time).

[0140] Figure 20 Images are taken from agarose gel electrophoresis analysis of LNPs encapsulated in mRNA after aerosolization using a mesh nebulizer or microfluidic device. (AC) Reproduced samples: 1) mRNA only, 2) LNPs before treatment, 3) LNPs treated with Triton X-100, 4) LNPs after aerosolization with a vibrating mesh, and 5) LNPs after microfluidic device treatment. Yellow dashed lines indicate... Figure 3 Gel images used in G.

[0141] Figure 21 These are images showing the visual changes in the opacity of LNP solutions after atomization or aerosolization. LNP solutions before aerosolization (1), after vibrating mesh aerosolization (2), or after microfluidic aerosolization (3) were imaged to show the changes in the opacity of these solutions.

[0142] Figure 22 Cell viability was depicted during LNP transfection using nebulized / aerosolized LNP. Cell viability was measured after treatment of 293T / 17 cells with different mRNA doses using LNP / Fluc solution (grey), LNP / Fluc aerosolized via a vibrating mesh nebulizer (red), or LNP / Fluc aerosolized via a microfluidic platform (blue). (n = 4).

[0143] Figure 23 The effects of nebulized / aerosolized LNP delivery to relevant lung cell lines were depicted. (A) Normalized luciferase expression and (B) Cell viability of human bronchial epithelial cells (16HBE14o-) treated with LNP / Fluc solution (grey) and LNP / Fluc aerosolized at various mRNA doses via a vibrating mesh nebulizer (red) or microfluidic platform (blue). (n = 5). p < 0.05; p < 0.001; p < 0.0001.

[0144] Figure 24Cell viability was depicted for several cell lines treated with nebulized / aerosolized LNP. Cell viability (n = 5) was shown for multiple cell lines treated with PBS (grey), LNP / Fluc aerosolized via a vibrating mesh nebulizer (red), or LNP / Fluc aerosolized via a microfluidic platform (blue) at a dose of 50 ng FLuc mRNA per well. CFBE: CFBE41o-human bronchial epithelial cells, J774: mouse macrophages, and HBE: human bronchial epithelial cell line 16HBE140-.

[0145] Figure 25 Images showing the delivery of aerosolized LNP / Nluc to mouse lungs via spontaneous inhalation. (AC) Delivery consistency of the microfluidic device in the whole-body rodent inhalation system in all four mice. (A) Bioluminescent signal and (B) Photographs of the collected lungs. (C) Quantified bioluminescent signal in the captured images.

[0146] Figure 26A and Figure 26B It involves collecting histopathological images of mouse lungs over 24 hours. Figure 26A The inhalation of LNP / Nluc was shown when 1 mg of mRNA was aerosolized. Figure 26B The image shows an equal volume of sterile PBS aspirated using a microfluidic platform. The inset indicates the target region. Arrows indicate a small increase in lymphocytes in bronchial-associated lymphoid tissue (BALT). The scale bar indicates 100 μm.

[0147] the term

[0148] “CF” refers to cystic fibrosis.

[0149] "CMOS" refers to Complementary Metal-Oxide-Semiconductor.

[0150] COPD stands for Chronic Obstructive Lung Disease.

[0151] “CryoTEM” refers to a low-temperature transmission electron microscope.

[0152] "DLS" refers to Dynamic Light Scattering.

[0153] "DMF" refers to dimethylformamide.

[0154] "DOPE" refers to 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine.

[0155] “DSPC” refers to 1,2-distearyl-sn-glycerol-3-phosphocholine.

[0156] “DSPE” refers to 1,2-distearate-sn-glycerol-3-phosphate ethanolamine.

[0157] "FFPE" refers to formalin-fixed paraffin embedding.

[0158] "Fluc" refers to firefly luciferase.

[0159] "FRET" refers to fluorescence resonance energy transfer.

[0160] "H&E" refers to hematoxylin and eosin.

[0161] “HEPES” refers to 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid.

[0162] "ISH" refers to in situ hybridization; LNP stands for lipid nanoparticles.

[0163] “MAP” refers to a microfluidic aerosolization platform.

[0164] “NBD” refers to 7-nitrobenzo-2-oxa-1,3-diazole.

[0165] “Nluc” refers to nano-luciferase.

[0166] "PBS" refers to phosphate buffer solution.

[0167] "PDI" refers to the Polydispersity Index; "PEG" refers to polyethylene glycol.

[0168] "POPC" refers to 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine.

[0169] "Rho" refers to Rhodamine B.

[0170] The term “and / or” as used herein has its broadest and least restrictive meaning, meaning that this disclosure includes A alone, B alone, both A and B together, or either A or B, but not both A and B or only one of A or B. As used herein, the phrase “at least one of A, B, and C” should be interpreted as meaning logical A or B or C using a non-exclusive logical “or”.

[0171] The description herein is illustrative in nature and is in no way intended to limit this disclosure, its application, or its uses. For clarity, the same reference numerals will be used in the accompanying drawings to identify similar elements. It should be understood that the steps in the method may be performed in a different order without altering the principles of this disclosure.

[0172] Features, materials, characteristics, or groups described in connection with a particular aspect, embodiment, or example shall be understood to be applicable to any other aspect, embodiment, or example described herein, unless incompatible therewith. All features disclosed in this specification (including any appended claims, abstract, and drawings), or all steps of any method or process so disclosed, may be combined in any combination, except for combinations in which at least some such features or steps are mutually exclusive. Protection is not limited to the details of any of the foregoing embodiments. Protection extends to any novel one or any novel combination of features disclosed in this specification (including any appended claims, abstract, and drawings), or to any novel one or any novel combination of steps of any method or process so disclosed.

[0173] Although certain embodiments have been described, these embodiments are given by way of example only and are not intended to limit the scope of protection. In fact, the novel methods and systems described herein can be implemented in many other forms. Furthermore, various omissions, substitutions, and changes can be made to the form of the methods and systems described herein. Those skilled in the art will understand that in some embodiments, the actual steps taken in the illustrated or disclosed process may differ from those shown in the figures. Depending on the embodiment, some of the above-described steps may be removed, and others may be added. For example, the actual steps or the order of steps taken in the disclosed process may differ from those shown in the figures. Depending on the embodiment, some of the above-described steps may be removed, and others may be added. For example, the various components shown in the figures can be implemented as software or firmware on a processor, controller, ASIC, FPGA, or dedicated hardware. Hardware components (e.g., processors, ASICs, FPGAs, etc.) may include logic circuitry. Furthermore, the features and attributes of the specific embodiments disclosed above can be combined in different ways to form additional embodiments, all of which fall within the scope of this disclosure.

[0174] While this disclosure includes certain embodiments, examples, and applications, those skilled in the art will understand that this disclosure extends beyond the specific embodiments disclosed to other alternative embodiments or uses, as well as their obvious modifications and equivalents, including embodiments that do not provide all the features and advantages described herein. Therefore, the scope of this disclosure is not intended to be limited by the specific disclosure of preferred embodiments herein, but may be defined by the claims set forth herein or by any claims to be filed hereafter.

[0175] Conditional language, such as “can,” “could,” “might,” or “may,” unless explicitly stated otherwise or interpreted differently depending on the context of its use, is generally intended to convey that certain embodiments include certain features, elements, or steps, while other embodiments do not. Therefore, such conditional language is generally not intended to imply that a feature, element, or step is necessary in any way for one or more embodiments, or that one or more embodiments necessarily include logic for determining whether such features, elements, or steps are included or to be performed in any particular embodiment, with or without user input or prompting. The terms “comprising,” “including,” “having,” etc., are synonymous and used inclusively in an open-ended manner, without excluding additional elements, features, actions, operations, etc. Furthermore, the term “or” is used in its inclusive meaning (rather than its exclusive meaning) such that, when used, for example, to connect lists of elements, the term “or” means one, some, or all of the elements in the list. Additionally, the term “each,” as used herein, in addition to its usual meaning, can also refer to any subset of the set of elements to which the term “each” applies.

[0176] Unless otherwise specified, connective language such as the phrase "at least one of X, Y, and Z" is, depending on the context, also understood to generally convey that an item, term, etc., can be any one of X, Y, or Z. Therefore, such connective language is generally not intended to imply that some implementation requires the presence of at least one X, at least one Y, and at least one Z.

[0177] The terms of degree used herein, such as “approximately,” “about,” “generally,” and “substantially,” mean a value, quantity, or characteristic that is close to the stated value, quantity, or characteristic and still performs the desired function or achieves the desired result. For example, the terms “approximately,” “about,” “generally,” and “substantially” may refer to a quantity that is less than 10%, less than 5%, less than 1%, less than 0.1%, and less than 0.01%. As another example, in some embodiments, the terms “generally parallel” and “substantially parallel” refer to a value, quantity, or characteristic that deviates from exact parallelism by less than or equal to 15 degrees, 10 degrees, 5 degrees, 3 degrees, 1 degree, or 0.1 degrees.

[0178] The scope of this disclosure is not intended to be limited by the specific disclosure of preferred embodiments in this section or elsewhere in this specification, and may be defined by the claims set forth in this section or elsewhere in this specification or by any claims to be filed thereafter. The language of the claims will be interpreted broadly based on the language used in the claims, and not limited to the examples described in this specification or as interpreted during the execution of this application, and the examples will be interpreted as non-exclusive.

Claims

1. A microfluidic aerosolization system for delivering therapeutic mediators into the pulmonary airways, comprising: A microfluidic chip having multiple microfluidic chambers and corresponding pores; Multiple heat sources, each including a resistor in thermal communication with each of the microfluidic chambers, which are individually addressable for droplet ejection; A cartridge containing the microfluidic chip and a plurality of storage units holding the treatment medium, the plurality of storage units being configured to deliver the treatment medium into the microfluidic chamber; and The processor is configured to generate activation pulse heating modes; The microfluidic chamber discharges multiple droplets in response to the activated pulse heating mode.

2. The microfluidic aerosolization system according to claim 1, wherein the chip is monolithically fabricated on a silicon substrate.

3. The microfluidic aerosolization system according to claim 2, wherein the activation pulse heating mode has a pulse range of about 5 volts to about 40 volts and a timing range of about 0.1 microseconds to about 10 microseconds.

4. The microfluidic aerosolization system according to claim 1, wherein the heating mode has a frequency of at least about 10 kHz.

5. The microfluidic aerosolization system of claim 1, wherein the chamber discharges droplets at a rate of at least about 5 million drops per second.

6. The microfluidic aerosolization system according to claim 1, comprising at least about 500 microfluidic chambers and corresponding pores.

7. The microfluidic aerosolization system of claim 1, wherein at least some of the pores have a diameter of no more than about 15 micrometers.

8. The microfluidic aerosolization system of claim 1, wherein the cartridge contains a volume of the therapeutic medium in one or more reservoirs.

9. The microfluidic aerosolization system of claim 8, wherein the therapeutic medium comprises mRNA encapsulated within lipid nanoparticles.

10. The microfluidic aerosolization system of claim 8, wherein the processor is configured to deliver the volume of medium that is broken down into a plurality of individual patient doses.

11. The microfluidic aerosolization system of claim 1, wherein the cartridge comprises at least two reservoirs.

12. The microfluidic aerosolization system of claim 11, wherein the first reservoir has a first therapeutic medium and the second reservoir has a different second medium.

13. The microfluidic aerosolization system of claim 11, wherein each reservoir contains the same therapeutic medium.

14. The microfluidic aerosolization system of claim 11, wherein at least one reservoir contains a buffer.

15. The microfluidic aerosolization system of claim 1, wherein all pores are individually programmable using the activation pulse heating mode to control at least one of droplet size, droplet size composition, droplet ejection frequency, and droplet volume.

16. The microfluidic aerosolization system of claim 1, wherein the processor is configured to simultaneously or sequentially activate the plurality of microfluidic chambers and pores.

17. The microfluidic aerosolization system of claim 1, further comprising an orifice having a profile matching the chip size to influence the shape of the plume as it is ejected from the orifice.

18. The microfluidic aerosolization system of claim 1, wherein the therapeutic medium comprises a drug.

19. The microfluidic aerosolization system of claim 18, wherein the therapeutic medium comprises a small molecule drug.

20. The microfluidic aerosolization system of claim 1, wherein the therapeutic medium comprises a bioactive agent.

21. The microfluidic aerosolization system of claim 20, wherein the bioactive agent comprises mRNA.

22. The microfluidic aerosolization system of claim 21, wherein the bioactive agent comprises encapsulated mRNA.

23. The microfluidic aerosolization system of claim 1, wherein the applied voltage and pulse width cause the formation of microbubbles, which nucleate, expand, and collapse to eject droplets of fluid into a microchamber.

24. The microfluidic aerosolization system of claim 23, wherein the applied voltage is less than about 5V and the pulse width is less than about 10 μs.

25. The microfluidic aerosolization system of claim 23, wherein the applied voltage comprises a waveform, the waveform being at least one of square, sawtooth, or sinusoidal.

26. The microfluidic aerosolization system of claim 23, wherein the applied voltage and pulse width cause the microelectromechanical membrane to deflect, and the microelectromechanical membrane then ejects droplets of fluid into the microchamber.

27. The microfluidic aerosolization system of claim 1, wherein individual chamber addressing model and spray frequency It is adjustable to regulate the formation of the plume of the multiple droplets, the depth of the droplets penetrating into the lung path, and the aggregation of the droplets in the plume.

28. A method for delivering a therapeutic medium via the lungs, the method comprising the following steps: The therapeutic medium is introduced into at least one microfluidic chamber with pores; and The microfluidic chamber is heated to cause at least a portion of the medium to evaporate; and At least one droplet is expelled through the pore and into the lung airway. The hole has a diameter of no more than about 20 micrometers.

29. The method of claim 28, wherein the hole has a diameter of no more than about 15 micrometers.

30. The method of claim 28, wherein the droplet has a diameter of no more than about 20 micrometers.

31. The method of claim 28, wherein the droplet has a diameter of at least about 50 micrometers.

32. The method of claim 28, further comprising introducing the therapeutic medium into at least about 100 microfluidic chambers.

33. The method of claim 32, further comprising introducing the therapeutic medium into at least about 500 microfluidic chambers.

34. The method of claim 33, wherein at least about 1 million droplets are discharged per second.

35. The method of claim 34, comprising discharging at least about 5 million droplets per second.

36. The method of claim 28, wherein the therapeutic medium comprises an mRNA vaccine.

37. The method of claim 36, wherein the therapeutic medium comprises an mRNA vaccine encapsulated within lipid nanoparticles.

38. The method of claim 28, wherein the therapeutic medium comprises a drug.

39. The method of claim 38, wherein the therapeutic medium comprises a small molecule drug.

40. The method of claim 28, wherein the therapeutic medium comprises a bioactive agent.

41. The method of claim 40, wherein the bioactive agent comprises mRNA.

42. The method of claim 41, wherein the bioactive agent comprises encapsulated mRNA.

43. The method of claim 28, wherein the processor is configured to activate the plurality of microfluidic chambers and pores simultaneously or sequentially.

44. The method of claim 28, wherein all orifices are individually programmable using an activation pulse mode to control at least one of drop size, drop size composition, drop ejection frequency, and drop volume.

Images