Method for aerosolizing nanoparticle formulations

Abstract

To deliver nanoparticles into the atmosphere, a liquid formulation containing the nanoparticles is provided and pressurized to a high operating pressure p. The liquid formulation is delivered at the high operating pressure through at least one spray nozzle orifice that expels the liquid formulation as a jet of continuous droplets containing at least one nanoparticle of the plurality of nanoparticles. The plurality of nanoparticles has a length λ and a maximum length before breakage λ when stretched. max and the liquid formulation has a wall shear rate γ wall [per second]. According to the present invention, the liquid formulation is subjected to λ max / (λ γ wall The wall shear rate is experienced within the spray nozzle orifice for a limited shear time, t, of less than 100 s.

Description

Technical Field

[0001] The present invention relates to a method for aerosolizing nanoparticle formulations, including formulations containing proteins, peptides, DNA, RNA, vesicles, liposomes, and antibodies.

Background Art

[0002] Examples of current aqueous aerosolization devices include nebulizers and soft mist inhalers. Nebulizers disperse the formulation by generating an aerosol using either compressed air, an ultrasonic source, or a vibrating mesh / membrane. Soft mist inhalers generate a mist consisting of fine water droplets by the very high-speed collision of microjets.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0004] Patent Document 1 discloses an example of such a soft mist inhaler or the like, and in particular, uses a small-diameter nozzle that is used for spraying non-Newtonian fluids such as suspensions or emulsions or liposome fluids. This known nebulizer comprises a fluid pump for pressurizing the fluid to an operating pressure of 5 to 250 MPa and forcing the pressurized fluid through two mutually inclined flow channels having a hydraulic diameter in the range of 3 to 20 microns. Thereby, two fluid jets that cross at a very high speed are generated, and when these collide with each other, a mist composed of fine water droplets is generated. This high-speed collision of the two fluid jets has a great impact on the fluid and the particles contained therein. Fragile and delicate nanoparticles tend to be damaged under such conditions and in many cases have been found to be unable to withstand the aerosolization process.

[0005] Patent Document 2 discloses an apparatus and method for generating particles from a fluid flow, and in this technique, a vibrator is used to change the fluid flow into individual droplets. The fluid is charged, and the vibrator comprises an ultrasonic piezoelectric transducer (converter) that can act on the charged fluid emerging from a nozzle channel having a diameter of at least 100 microns. This known method and apparatus apply a significant mechanical stress to the fluid due to the ultrasonic source or the vibrating mesh / membrane. The molecules contained in the fluid are exposed to that stress which causes shear stress, and the decomposition of these molecules is induced. Therefore, delicate and fragile molecules are likely to be decomposed when using this known apparatus and method. Furthermore, this atomization mechanism, together with a nozzle diameter of at least 100 microns, makes this known apparatus suitable only for applications at relatively large dimensions. For example, when the nozzle diameter is reduced from 100 microns to 5 microns, the damage to the encapsulated compound increases by several times. Therefore, this known method and apparatus cannot be used in the submicron range that requires smaller nozzles, and reducing the nozzle diameter further increases the wall shear stress, which may cause even greater damage to the compound encapsulated in the fluid.

[0006] The object of the present invention is to provide a method for aerosolizing a fluid composition containing multiple nanoparticles, which maintains molecular integrity when aerosolizing stress-sensitive nanoparticles, such as formulations containing complex proteins, peptides, long-chain DNA and RNA, large vesicles, liposomes and / or antibodies. [Means for solving the problem]

[0007] For this purpose, the present invention provides a method for delivering nanoparticles into the atmosphere. This method is: Producing a liquid by supplying multiple nanoparticles having a particle length (λ) into a liquid; The aforementioned liquid is pressurized to an appropriate operating pressure (p) to prepare the pressurized liquid; The pressurized liquid is supplied through a spray nozzle orifice having a channel length (L) between the inlet and outlet of the orifice, and an average channel diameter (H) between the inlet and outlet, thereby generating a velocity-driven liquid flow of the liquid; Includes, The appropriate operating pressure is less than 10 MPa, and the speed is less than 100 m / s. The orifice has a channel length (L) that is shorter than the average channel diameter (H), The liquid flow is collected at the outlet as a jet consisting of continuous droplets, each containing at least one nanoparticle from the plurality of nanoparticles.

[0008] In certain embodiments, the method according to the present invention comprises shear stress-sensitive nanoparticles selected from the group including complex proteins, large biomolecules, long-chain DNA and RNA, viruses, large vesicles, liposomes, bacteriophages, and antibodies, wherein the orifice has a channel length (L) shorter than half of the average channel diameter (H), and preferably the orifice has a channel length (L) that is at most one-quarter of the average channel diameter (H). In practice, it can be seen that even such delicate and fragile molecules and nanoparticles can maintain their integrity if the above conditions are met.

[0009] In one embodiment of the method according to the present invention, the liquid formulation comprises protein and / or antibody molecules having a molecular weight exceeding 100,000 g / mol, particularly nucleotide compounds such as DNA molecules or RNA molecules.

[0010] In a further embodiment of the method according to the present invention, the liquid agent comprises bacteriophages having an average size greater than 20 nanometers.

[0011] In a further embodiment of the method according to the present invention, the liquid formulation comprises lipid nanoparticles or liposomes, particularly lung surfactant, having a length greater than 20 nanometers. Herein, the liquid formulation comprises a vesicle having contents comprising nanoparticles selected from the group including proteins, biomolecules, DNA, RNA, vaccines, viruses, bacteriophages, and antibodies with a molecular weight greater than 100,000 Da.

[0012] In a further embodiment of the method according to the present invention, the nozzle orifice has a substantially constant diameter (H) of 1 to 10 microns.

[0013] In a further embodiment of the method according to the present invention, the nozzle orifice has an average diameter (H) of 1 to 10 microns, and the orifice is tapered over at least a portion of its length from inlet to outlet. In particular, the nozzle orifice has a positive taper that narrows substantially between 5° and 45° from inlet to outlet.

[0014] In a further embodiment of the method according to the present invention, the inner wall of the nozzle orifice is provided with a coating that enables hydrophobic slip flow.

[0015] In a particular embodiment, the method according to the present invention relates to the maximum particle length λ before the nanoparticle breaks during elongation. max The liquid agent has a wall shear rate γ as it passes through the spray nozzle orifice. wall [Per second] The liquid agent is supplied, λ max / ( λγ wall The spray nozzle orifice is characterized by being exposed to the wall shear rate for a shear time (t) of less than ) seconds.

[0016] For lipid nanoparticles, vesicles, liposomes, and other spherical core-shell particles, λ max The / λ ratio is typically in a small range between 3 and 5, with an average of 4, and has been experimentally found to be largely independent of particle sizes greater than 20 nm. For linear, coiled, and rigid molecules, or nanoparticles, such as DNA, RNA, and charged macromolecular electrolytes, the ratio λ max Experiments have shown that / λ is in the range of 4 to 6 when the molecular weight or weight of the nanoparticles exceeds 100,000.

[0017] Shorter molecules exhibit smaller maximum elongation but are more rigid and less susceptible to fracture due to shear forces. Larger molecules (over 500,000 Da) show greater maximum elongation but are more susceptible to fracture due to shear forces. These hydrodynamic effects tend to complement each other in terms of susceptibility to fracture.

[0018] For flexible molecules such as proteins and large antibodies, the ratio λ max / λ has been experimentally found to be 2 - 5 for molecules with a molecular weight exceeding 100,000. For semi - rigid nanoparticles such as bacteriophages and viruses, the ratio λ max / λ has been experimentally found to be in a wide range between 2 and 6 for nanoparticle weights exceeding 100,000. For the sake of safety, in a preferred embodiment of the method according to the present invention, the ratio λ max / λ is set to 4.

[0019] For very stress - sensitive, very complex, large nanoparticles (>100 nm) such as pulmonary surfactant, the ratio λ max / λ has been experimentally found to be in a typical range between 2 and 6. Also, the nanoparticle size effect also tends to compensate for each other's sensitivity to breakage. However, for these very complex types of nanoparticles, including the corrective effect, the ratio λ max / λ may optimally be set to 2 rather than 4.

[0020] Volume - average breakdown is usually determined by a γ wall having a value smaller than the wall - shear rate γ part . This means that according to this embodiment, a general criterion for avoiding the breakdown of nanoparticle formulations, namely, γ wall t = λ max / λ < 4, is set on the safe side to protect nanoparticles with a size λ exceeding 20 nm and / or having stress sensitivity with a molecular weight exceeding 100,000 Da. For very complex and large nanoparticles (λ > 100 nm), a safer margin can be set at γ wall t = λ max / λ < 2.

[0021] The interrelationship between wall shear rate and shear time surprisingly suggests that it is possible to maintain the molecular integrity of both long-chain molecules and vesicles during the aerosolization process to produce droplets between 2 and 20 microns, using spray nozzles with small diameters between 1 and 10 microns and lengths not exceeding 2 microns. Surprisingly, it was found that even with wall shear rates at least 10 to 100 times higher than those of conventional sprayers, the molecular integrity of nanoparticle formulations can still be preserved for very short periods of time.

[0022] According to insights from the present invention, these conditions are particularly satisfied by using a spray orifice having a small diameter between 1 and 10 microns and a nozzle length of less than 2 microns. This allows the nozzle to withstand the wall shear rate γ wall Even if the wall shear rate γ is high, nanoparticles in the fluid further away from the nozzle wall will have a high wall shear rate γ wall It is designed to be less affected by the wall shear rate γ. wall This is the difference in velocity near the wall and perpendicular to the wall, divided by the distance to the wall, and its unit is per second. The shear stress on nanoparticles originates from the local velocity gradient along the length of the particle, and here, the particle shear velocity γ is obtained by dividing the velocity difference on the nanoparticle by the size of the nanoparticle, with its unit being per second. part It is called that.

[0023] Further insights into the present invention suggest that the drag force acting on the nanoparticles can induce tension in the nanoparticles and subsequent fracture, primarily due to the particle shear rate γ part It is proportional to the wall shear rate γ, and its value is essentially equal to the wall shear rate γ wall It is less than. In a long pipe with a parabolic velocity distribution, the wall shear velocity γ wall and particle shear velocity γ near the wall part It is 8V / H. However, the particle shear rate γ in the center of the pipe is part γ is zero. Furthermore, the liquid flows through the center of the pipe at a velocity of 2V, but there is virtually no flow near the pipe walls. Therefore, the volume-average particle shear velocity γ inside the pipe is zero. partThe volume-average particle shear rate γ varies between approximately 4V / H for long and medium-length pipes, and to substantially small values ​​for short pipes or orifices, depending on the type of flow and pipe length. part To reduce this, according to the present invention, it is preferable to use a relatively short pipe or orifice, so an orifice that is relatively short compared to its diameter is used.

[0024] According to the insights of the present invention, large macromolecules or large vesicles have a particle shear rate γ part It does not have sufficient rigidity or resistivity to withstand the deformation force due to the hydrodynamic shear force proportional to γ. Further insights that form the basis of the present invention indicate that the volume-average particle shear rate γ part This is the wall shear rate γ near the wall. wall A substantially smaller and safe state for maintaining the integrity of nanoparticles is when the shear time is preferably λ max / (λ·γ wall ) is less than λ. Next, λ max / (λ·γ wall During a shear time of ) seconds, the nanoparticles near the wall are λ × λ max / (λ·γ wall )=λ max It is stretched or deformed by a length λ, approximately γ times. The aerosolizing device is λ max / (λ·γ wall During an ultrashort shear time t of less than ) seconds, the wall shear rate γ per second wall It must be possible to impose conditions that set this condition. As a result, not only nanoparticles near the nozzle wall, but also nanoparticles further away from the nozzle wall will maintain their integrity.

[0025] In a further embodiment of the method according to the present invention, the wall shear rate γ wall This number far exceeds 100,000, and in particular, it exceeds 1,000,000 per second.

[0026] In a further embodiment of the method according to the invention, the nanoparticles comprise macromolecules having a molecular weight greater than 100,000 g / mol. Here, the macromolecule or nanoparticle has a ratio λ max / λ of at least 2, preferably a ratio λ max / λ of at least 4.

[0027] In a further embodiment of the method according to the invention, the nozzle orifice produces a jet having an average velocity V that exits from an orifice having the following shear conditions with a diameter H and a length L: γ part <γ wall = 8V / H, t = L / V = λ max / (λ·γ wall ) < λ max / (λ·γ part ) or γ part ·t < γ wall ·t = 8L / H

[0028] A specific embodiment according to the invention is that during aerosolization of a nanoparticle formulation that produces aerosol droplets of a size of 2 to 20 microns through a small nozzle with a diameter H of 1 to 10 microns and a length L of less than 2 microns, the nanoparticle formulation is substantially subjected to a wall shear rate γ -6 for a period of less than 4×10 max seconds (when the maximum nanoparticle elongation λ wall / λ is 4, γ wall ·t < 4 is obtained), which exceeds 1,000,000 / second. When aerosolizing a formulation containing shear-sensitive nanoparticles such as proteins, peptides, DNA and RNA, vesicles, liposomes, lipid nanoparticles and antibodies, the condition γ[[ID=4​​​​If the nozzle length is further shortened, the total area of the nozzle wall surface decreases, and the contribution of the wall shear to the nanoparticles decreases. Therefore, the nozzle length L should not exceed one-fourth of the nozzle diameter, that is, it is desirable that L < H / 4. This is particularly applicable to very complex and stress-sensitive nanoparticles typically having sizes exceeding 100 nm. Preferably, according to the present invention, the nozzle is used with a short-length orifice that exhibits a plug flow and slip flow state that reduces the shear rate of the nanoparticles during operation. In the plug flow state, the particle shear rate away from the nozzle wall becomes considerably small due to a more uniform velocity distribution, generating slip flow conditions (e.g., using a hydrophobic nozzle wall) and also reducing the particle shear rate near the nozzle wall. Preferably, the nozzle combines both plug flow and slip flow simultaneously to reduce the volume-averaged particle shear rate.

[0030] A further embodiment of the method according to the present invention is characterized in that the device generates a jet of average velocity V exiting from a nozzle with a diameter H of 1 to 10 microns, a length L of L < H / 2, and a nozzle pressure of 10 to 100 bar. It has also been found that increasing the velocity V of the jet beyond 10 m / s by increasing the nozzle pressure reduces the decomposition amount, and an optimum operating pressure of about 30 to 80 bar has been found to reduce the risk of decomposition of the nanoparticle formulation.

[0031] An average flow velocity of approximately V = 100 m / s or higher at operating pressures above 100 bar has been found to be detrimental to the integrity of nanoparticles. This is likely because the high operating pressures exceeding 100 bar required to achieve such velocities induce severe turbulence in the formulation, potentially leading to nanoparticle breakdown. Furthermore, at such high nozzle pressures, the liquid jet exiting the nozzle carries so much kinetic energy that the jet immediately collapses before it can generate a jet consisting of continuous droplets approximately twice the jet diameter. This undesirable turbulence and collapse of the jet also results in a significant increase in volume-average particle shear rate and an increased level of nanoparticle breakdown. This undesirable transition to turbulence and collapse can occur whenever the ratio of jet mass density ρ × jet velocity V × nozzle diameter H divided by jet viscosity η is greater than 2,500 (ρ·V·H / η > 2500). Therefore, a more preferred embodiment of the method according to the present invention is characterized in that the product of the mass density of the fluid (ρ), the fluid velocity in the orifice (V), and the nozzle diameter (H), divided by the viscosity of the fluid (η), expressed as ρ·V·H / η, is maintained at less than 2,500.

[0032] In a further preferred embodiment, the method according to the present invention is characterized in that the nozzle orifice is part of a substantially identical collection of multiple nozzle orifices extending through a common film layer supported by a substrate, wherein the substrate has at least one cavity extending to the nozzle orifice of the collection of multiple orifices, and the liquid is introduced into the cavity together with the operating pressure and supplied to the nozzle orifice of the collection of multiple orifices.

[0033] Furthermore, satisfactory results have been obtained even with short, tapered orifices that have a minimum diameter H of 1 to 10 microns, a total length L of less than 2 microns, and a taper angle of 5° to 45°, slightly tapering in either positive or negative direction. [Brief explanation of the drawing]

[0034] [Figure 1]Figure 1 shows the fracture of macromolecules in a shear flow. [Figure 2] Figure 2 shows the failure of a vesicle in a shear flow. [Figure 3] Figure 3 shows vesicle failure in strong shear flow. [Figure 4] Figure 4 shows how the vesicle passes through a long pipe. [Figure 5] Figure 5 shows how the vesicle passes through the orifice. [Figure 6] Figure 6 shows the vesicle passing through a tapering orifice. [Figure 7] Figure 7 shows the behavior of vesicles passing through a negatively tapering orifice. [Modes for carrying out the invention]

[0035] Figure 1 shows the decomposition of a macromolecule in a shear flow. Figure 1A shows a normal spherical macromolecule (1) with a rotational diameter λ in the absence of a shear flow. Figure 1B shows the initial elongation of the spherical macromolecule in the presence of a shear flow indicated by arrows (2,3). Figure 1C shows the maximum length λ of the macromolecule. max Further elongation is observed, and the upper part of the macromolecule, moving at a higher speed, begins to separate from the lower part, which is moving at a lower speed. The upper (4) and lower (5) are separated by only one or two remaining covalent bonds. Figure 1D shows the macromolecule after hydrodynamic shear forces acting on the upper and lower parts of the macromolecule cause the covalent bonds in the central part of the macromolecule to break, splitting it into two parts.

[0036] Long, heavy macromolecules with molecular weights exceeding 100,000 g / mol, such as large proteins, long RNAs and DNAs, and antibodies, are susceptible to shear-induced degradation. Table 1 below summarizes some results regarding the shear degradation of immunoglobulins (proteins / antibodies) of various molecular weights in aerosol form.

[0037] [Table 1]

[0038] Using a vibrating membrane nebulizer with a 1 ml formulation reservoir, aerosol droplets ranging from 2 to 10 microns were generated during an 8-minute atomization time. Diluted formulations containing proteins / antibodies with larger molecular weights (>100,000 g / mol) clearly undergo shear degradation. According to the present invention, aerosol droplets with an average size of 4 microns were also produced by passing the same formulation containing antibodies through an orifice with a diameter H=2 microns and a length L=1 micron at an average flow rate V=5 m / s, with a passage time t of approximately 200 nanoseconds. This short shear time according to the present invention resulted in substantially less degradation of macromolecules present in the droplets. Using an orifice with a 25° positive taper shape, antibodies underwent almost no degradation, even with molecular weights far exceeding 1,000,000 g / mol. At an average flow rate V=20 m / s, the passage time t through a non-tapered orifice was approximately 50 nanoseconds, showing less degradation than at an average flow rate of 5 m / s. However, increasing the average flow rate to V=100 m / s (t=10 ns) results in substantial decomposition of the formulation. Reducing the nozzle length L to 0.5 microns (H=2 microns) typically reduces decomposition loss by a relative 15% compared to a 1-micron nozzle.

[0039] Macromolecules such as DNA and RNA nucleic acids are frequently used in gene therapy, particularly in non-viral gene transfer vectors containing messenger RNA (mRNA) or mini-DNA vectors. These macromolecules, with corresponding pathway lengths ranging from 30 nanometers to 3 microns and containing 100 to 10,000 nucleotides or base pairs, are susceptible to shear degradation. Table 2 below summarizes some results regarding the shear degradation of several of these vector molecules.

[0040] [Table 2]

[0041] Using a vibrating membrane nebulizer with a 1 ml formulation reservoir, aerosol droplets ranging from 2 to 10 microns were generated with an 8-minute atomization time. Formulations containing DNA or RNA vectors (5 μg / ml) with larger molecular weights (>200,000 g / mol) were clearly degraded. According to the present invention, aerosol droplets with an average size of 4 microns were also produced by passing the same formulation containing the vector molecules through an orifice with a diameter H=2 microns and a length L=1 micron at an average flow rate V=5 m / s. Due to the plug flow condition, the volume-average wall shear rate is 4V / H=10 × 10⁻¹⁰ 6 Although significantly lower than per second, the transit time t is approximately 200 nanoseconds. Due to this short shear time according to the present invention, the vector molecules present in the droplet are substantially undegraded. At an average flow velocity V = 20 m / s, the transit time t through a non-tapered orifice is approximately 50 nanoseconds, showing less degradation than at an average velocity of 5 m / s. However, when the average velocity is increased to V = 100 m / s (t = 10 ns), the formulation degrades significantly. Using an orifice with a length of 1 micron and a 25° negative taper shape, the antibody is hardly degraded even at a velocity V = 50 m / s and a molecular weight far exceeding 1,000,000 g / mol. Reducing the nozzle length L to 0.5 microns (H = 2 microns, non-tapered) reduces the degradation loss by an average of 10% compared to a 1-micron non-tapered nozzle. The results for nozzles with a hydrophobic fluorine coating with a water contact angle of 112° also show a relative reduction in decomposition loss of typically 5–15% compared to uncoated nozzles.

[0042] Figure 2 shows the disruption of vesicles or liposomes in shear flow. Figure 2A shows a spherical vesicle (11) with size λ in the absence of shear flow. Figure 2B shows the initial extension of the vesicle in the presence of shear flow, indicated by arrows (12,13). Figure 2C shows the upper part of the vesicle moving at a higher velocity than the lower part, and the longest length of the vesicle λ. maxThis shows how it has further extended. Figure 2D shows the vesicle after fracture has occurred in the central part of the vesicle due to hydrodynamic shear forces acting on the upper and lower parts of the vesicle, causing it to break into two parts (14,15).

[0043] Figure 3 shows the degradation of vesicles or liposomes under strong shear flow. Figure 3A shows a vesicle (11) with size λ in the absence of shear flow. Figure 3B shows the initial elongation of the vesicle in the presence of shear flow, indicated by arrows (12,13). Figure 3C shows the longest length λ of the vesicle because the top of the vesicle moved at a much higher velocity than the bottom. max This shows an even more extreme stretching. Figure 3D shows the vesicle after a large hydrodynamic shear force has acted on it, causing it to fracture into three parts (14, 15, 16).

[0044] Figure 4 shows the degradation of vesicles or liposomes (21) in a long pipe (22) of length L and diameter H. Before the entrance to the pipe, the vesicles (21) are round due to the absence of a large shear flow. Upon entering the pipe, the vesicles are subjected to a shear flow (23) and, after a vesicle extension phase, break into three elongated parts before exiting the pipe. The three parts continue to move within the jet, and as the shear is removed, the three parts become spherical.

[0045] Figure 5 shows how a vesicle or liposome (21) passes through an orifice of length L and diameter H. Before the entrance to the orifice, the vesicle (21) is round because there is no large shear flow. As the vesicle passes through the orifice, it is exposed to the shear flow (23) and becomes elongated but does not break. The vesicle then enters the jet and becomes spherical again.

[0046] Figure 6 shows vesicles or liposomes (21) passing through a positively tapered orifice having length L and diameter H, which represents plug flow. The vesicles are rounded before the entrance to the tapering orifice, and because the shear during plug flow is small, the vesicles maintain an almost spherical shape. The vesicles then enter the jet and become spherical.

[0047] Figure 7 shows vesicles or liposomes (21) passing through a negatively tapered orifice having length L and diameter H, also known as a diffuser. The broadening of the orifice reduces shear on the nanoparticles in the flow.

[0048] Since the adverse effects on the integrity of nanoparticles are greatest at the narrowest portion of a tapered orifice having a ratio of narrow portion length L to narrow diameter H, as presented according to the present invention, it may be apparent that a tapered orifice having a wide tapering angle according to the present invention may be selected such that its overall length is substantially longer than its narrowest diameter.

[0049] Vesicles and liposomes are also commonly used in gene therapy, particularly to facilitate the delivery of encapsulated RNA and DNA vectors into the lungs. These vesicles can typically range in size from 0.02 to 2 microns, making them potentially susceptible to shear fracture during aerosolization. Table 3 below summarizes some results regarding the shear degradation of several of these vesicles.

[0050] [Table 3]

[0051] Using a vibrating membrane nebulizer with a 1 ml formulation reservoir, aerosol droplets ranging from 2 to 10 microns were generated during an 8-minute atomization time. All liposome formulations (15 μg / ml) containing hydrogenated soybean phosphatidylcholine (HSPC) and cholesterol (CH) underwent rupture, with larger vesicles being more prone to rupture than smaller ones. According to the present invention, aerosol droplets with an average size of 4 microns were also produced by passing the same formulation containing vesicles through an orifice with a diameter H=2 microns and a length L=1 micron at an average velocity V=5 m / s. Due to the plug flow condition, the volume-average wall shear rate was 4V / H=10 × 10⁻¹⁰. 6 Although significantly lower than / second, the transit time t is approximately 100 nanoseconds. This short shear time according to the present invention results in minimal degradation or destruction of vesicles present in the droplet. At an average velocity V = 20 m / s, the transit time t through the untapered orifice is approximately 50 nanoseconds, indicating less destruction than at an average velocity of 10 m / s. However, increasing the average velocity to V = 100 m / s (t = 10 ns) results in significant degradation of the formulation. Reducing the nozzle length L to 0.5 microns (H = 2 microns) reduces degradation loss by a relative 10-40% compared to a 1-micron nozzle. With the lung surfactant Curosurf, degradation was significantly reduced compared to a vibrating membrane nebulizer. Results using a 2-micron nozzle with a hydrophobic fluorine coating and a water contact angle of 112° also typically show a relative reduction of 5-20% in degradation loss.

[0052] Preferably, a formulation is provided comprising lipid nanoparticles (LNPs) or polymer-containing hybrid nanoparticles (HNPs) having a length or size λ greater than 20 nanometers, wherein the LNPs or HNPs contain other nanoparticles selected from the group including proteins, biomolecules, DNA, RNA, mRNA, and antibodies. In this method, large nanoparticles that are susceptible to shear degradation are more safely protected.

[0053] Since the adverse effects on the integrity of nanoparticles are greatest in the narrowest portion of a tapered orifice having a ratio of narrow portion length L to narrow diameter H, as presented according to the present invention, it will be apparent that a tapered orifice having a wide tapering angle according to the present invention may be selected to have an overall length substantially longer than the narrowest diameter. This application includes the following configuration . [Configuration 1] A method for delivering nanoparticles into the atmosphere: Producing a liquid by supplying multiple nanoparticles having a particle length (λ) into a liquid; The aforementioned liquid is pressurized to an appropriate operating pressure (p) to prepare the pressurized liquid; The pressurized liquid is supplied through a spray nozzle orifice having a channel length (L) between the inlet and outlet of the orifice, and an average channel diameter (H) between the inlet and outlet, thereby generating a velocity-driven liquid flow of the liquid; Includes, The appropriate operating pressure is less than 10 MPa, and the speed is less than 100 m / s. The orifice has a channel length (L) that is shorter than the average channel diameter (H), The liquid flow is collected at the outlet as a jet consisting of continuous droplets, each containing at least one nanoparticle from the plurality of nanoparticles. method . [Configuration 2] The liquid preparation comprises shear stress-sensitive nanoparticles selected from the group including complex proteins, large biomolecules, long-chain DNA and RNA, viruses, large vesicles, liposomes, bacteriophages, and antibodies. The orifice has a channel length (L) that is shorter than half of the average channel diameter (H). Method described in Configuration 1 . [Configuration 3] The orifice has a channel length (L) that is at most one-quarter of the average channel diameter (H). Method of configuration 2 . [Structure 4] The liquid preparation comprises protein and / or antibody molecules having a molecular weight exceeding 100,000 g / mol, and / or nucleotide compounds such as DNA or RNA molecules. Method of configuration 2 or 3 . [Composition 5] The liquid agent comprises a bacteriophage having an average size greater than 20 nanometers. Method of configuration 2 or 3 . [Composition 6] The liquid preparation comprises lipid nanoparticles or liposomes, particularly lung surfactant. The aforementioned length λ is greater than 20 nanometers. Method of configuration 2 or 3 . [Composition 7] The liquid preparation comprises a vesicle having contents containing nanoparticles selected from the group including proteins, biomolecules, DNA, RNA, vaccines, viruses, bacteriophages, and antibodies, having a molecular weight exceeding 100,000 Da. Method of configuration 6 . [Structure 8] The nozzle orifice has a substantially constant diameter (H) of 1 micron to 10 microns. Method of configuration 2 or 3 。 [Composition 9] The nozzle orifice has an average diameter (H) of 1 to 10 microns. The orifice is tapered over at least a portion of its length from the inlet to the outlet. Method of configuration 2 or 3 。 [Configuration 10] The nozzle orifice has a positive taper that narrows from the inlet to the outlet, substantially between 5° and 45°. Method of configuration 9 。 [Composition 11] The inner wall of the nozzle orifice is provided with a coating that enables hydrophobic slip flow. Methods of any of the preceding configurations 。 [Composition 12] The product of the mass density of the fluid (ρ), the flow velocity in the orifice (V), and the nozzle diameter (H), expressed as ρ·V·H / η, divided by the viscosity of the fluid (η), is maintained at less than 2,500. Methods of any of the preceding configurations 。 [Composition 13] The aforementioned nanoparticles, when stretched, have a maximum particle length λ before breakage. max It has, The liquid agent, while passing through the spray nozzle orifice, undergoes a wall shear rate γ wall [Every second] The aforementioned liquid preparation is λ max / (λ·γ wall The spray nozzle orifice is exposed to the wall shear rate for a shear time (t) of less than ) seconds, Methods for any of the preceding configurations 。 [Composition 14] The wall shear rate γ wall This significantly exceeds 100,000, and especially exceeds 1,000,000 per second. Method described in configuration 13 。 [Composition 15] The aforementioned nanoparticles include macromolecules with a molecular weight exceeding 100,000 g / mol. The aforementioned macromolecule has a ratio of λ max / λ is 2 or greater, preferably a ratio of λ max / λ is 4 or greater. Method of any of the above configurations 。 [Composition 16] The nozzle orifice is part of an aggregate of substantially identical nozzle orifices extending through a common film layer supported by the substrate. The substrate has at least one cavity extending to the nozzle orifice among the aggregate of the plurality of orifices, The liquid is introduced into the cavity together with the operating pressure and supplied to the nozzle orifice in the orifice assembly. Methods of any of the preceding configurations 。

Claims

1. A method for delivering nanoparticles into the atmosphere: Producing a liquid by supplying multiple nanoparticles having a particle length (λ) into a liquid; The aforementioned liquid is pressurized to an appropriate operating pressure (p) to prepare the pressurized liquid; The pressurized liquid is supplied through a spray nozzle orifice having a channel length (L) between the inlet and outlet of the spray nozzle orifice, and an average channel diameter (H) between the inlet and outlet, thereby generating a velocity-based liquid flow of the liquid; Includes, The appropriate operating pressure is less than 10 MPa, and the speed is less than 100 m / s. The spray nozzle orifice has a channel length (L) that is shorter than the average channel diameter (H), The liquid flow is collected at the outlet as a jet consisting of continuous droplets, each containing at least one nanoparticle from the plurality of nanoparticles. method.

2. The liquid preparation comprises shear stress-sensitive nanoparticles selected from the group including complex proteins, biomolecules, long-chain DNA, long-chain RNA, viruses, vesicles, liposomes, bacteriophages, and antibodies. The spray nozzle orifice has a channel length (L) that is shorter than half of the average channel diameter (H). The method according to claim 1.

3. The spray nozzle orifice has a channel length (L) that is at most one-quarter of the average channel diameter (H). The method according to claim 2.

4. The liquid preparation comprises a protein and / or antibody molecule having a molecular weight exceeding 100,000 g / mol, and / or a nucleotide compound such as a DNA or RNA molecule. The method according to claim 2.

5. The liquid agent comprises a bacteriophage having an average size greater than 20 nanometers. The method according to claim 2.

6. The liquid preparation comprises lipid nanoparticles or liposomes, The aforementioned length λ is greater than 20 nanometers. The method according to claim 2.

7. The liquid agent comprises a pulmonary surfactant, The aforementioned length λ is greater than 20 nanometers. The method according to claim 6.

8. The liquid preparation comprises a vesicle having contents containing nanoparticles selected from the group including proteins, biomolecules, DNA, RNA, vaccines, viruses, bacteriophages, and antibodies, having a molecular weight exceeding 100,000 Da. The method according to claim 6.

9. The spray nozzle orifice has a diameter (H) of 1 to 10 microns. The method according to claim 2.

10. The spray nozzle orifice has an average diameter (H) of 1 to 10 microns. The spray nozzle orifice is tapered over at least a portion of its length from the inlet to the outlet. The method according to claim 2.

11. The spray nozzle orifice has a positive taper that narrows from the inlet to the outlet, with a taper between 5° and 45°. The method according to claim 10.

12. The inner wall of the spray nozzle orifice is provided with a coating that enables hydrophobic slip flow. The method according to claim 1.

13. The product of the mass density of the liquid flow (ρ), the flow velocity in the spray nozzle orifice (V), and the average channel diameter (H), expressed as ρ・V・H / η, divided by the viscosity of the liquid flow (η), is maintained at less than 2,500. The method according to claim 1.

14. The aforementioned nanoparticles, when stretched, have a maximum particle length λ before breakage. max It has, The liquid agent, while passing through the spray nozzle orifice, undergoes a wall shear rate γ wall [Every second] The aforementioned liquid preparation is λ max / (λ・γ wall The spray nozzle orifice is exposed to the wall shear rate for a shear time (t) of less than ) seconds, The method according to claim 1.

15. The wall shear rate γ wall This exceeds 100,000 per second. The method according to claim 14.

16. The aforementioned nanoparticles include macromolecules with a molecular weight exceeding 100,000 g / mol. The aforementioned macromolecule has a ratio of λ max / λ is 2 or greater, The method according to claim 1.

17. The aforementioned spray nozzle orifice is part of an aggregate of multiple spray nozzle orifices extending through a common film layer supported by the substrate. The substrate has at least one cavity extending to the spray nozzle orifice among the aggregate of the plurality of spray nozzle orifices, The liquid is introduced into the cavity together with the operating pressure and supplied to the spray nozzle orifice in the assembly of spray nozzle orifices. The method according to claim 1.

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