An assembly apparatus for a lipid-based gene delivery vehicle

Abstract

The application discloses an assembly device for a lipid gene delivery carrier, which comprises a ring cavity for accommodating a mixing process and enabling the water phase and the organic phase to perform a ring motion before mixing; a mixing plane, which is a cylindrical cavity arranged in the middle of the ring cavity and used for mixing the water phase and the organic phase, the periphery of the cylindrical cavity is communicated with the inner wall of the ring cavity and divides the ring cavity into upper and lower parts; a forming cavity, which is a cylindrical cavity or a circular truncated cone cavity and is communicated with the bottom of the mixing plane and the top of the forming cavity is communicated with the outside of the assembly device; a water phase feeding port, an organic phase feeding port and a carrier outlet. The application can realize efficient mixing of the water phase and the organic phase, and thus realizes efficient and stable preparation of the lipid gene delivery carrier.

Description

Technical Field

[0001] This invention belongs to the field of medical device technology, specifically relating to a device that can be used to assemble lipid gene delivery vectors. Background Technology

[0002] Currently, common lipid gene delivery vectors, namely solid lipid nanoparticles, are formed through the following process: First, phospholipids (the organic phase) are dissolved in an organic solvent. Then, after dilution with an aqueous phase, the lipids begin to precipitate due to the decreased solubility of lipids in the mixed solvent. During this precipitation process, the lipids spontaneously self-assemble. In this self-assembly process, cationic lipids encapsulate the gene drug through electrostatic interactions, assisting lipids and cholesterol in forming an internal hydrophobic structure, while PEG lipids form an outer hydrophilic shell. This self-assembly process has a significant impact on the gene delivery capability of the vector, and controlling the rate and extent of dilution between the organic and aqueous phases is a key method for controlling this self-assembly process.

[0003] T-tubes are used to achieve efficient mixing of aqueous and organic phases. To improve mixing uniformity, it is usually necessary to increase the flow rates of both phases to achieve mixing under turbulent conditions. However, under turbulent conditions, the high energy of the water flow and the intense mixing can lead to excessively rapid lipid precipitation, potentially resulting in decreased gene drug encapsulation efficiency. On the other hand, if the flow rate is reduced, the limited contact area between the two phases due to the characteristics of the T-tube may lead to uneven lipid precipitation at different sites. Therefore, addressing these potential problems with T-tubes has become a pressing challenge in this field. Summary of the Invention

[0004] The purpose of this invention is to provide an assembly device for lipid gene delivery vectors that enables efficient mixing of aqueous and organic phases, thereby achieving efficient and stable preparation of lipid gene delivery vectors.

[0005] The objective of this invention can be achieved through the following measures:

[0006] An assembly apparatus for a lipid gene delivery vector, comprising:

[0007] An annular cavity is a chamber used to contain the mixing process and to allow the aqueous and organic phases to move in an annular motion before mixing.

[0008] A mixing plane, a cylindrical chamber located in the middle of the annular chamber for mixing the aqueous phase and the organic phase, wherein the periphery of the cylindrical chamber is connected to the inner wall of the annular chamber and divides the annular chamber into upper and lower parts;

[0009] The molding cavity has a bottom that is connected to the mixing plane and a top that leads to a cylindrical or frustum-shaped cavity outside the assembly equipment.

[0010] Aqueous phase feed inlet, located at the upper or lower end of the annular cavity and leading into the interior of the annular cavity;

[0011] An organic phase inlet is provided at the lower or upper end of the annular cavity and leads into the interior of the annular cavity; the organic phase inlet and the aqueous phase inlet are respectively located at the upper and lower ends of the annular cavity; and

[0012] The carrier outlet is located at the outlet of the molding cavity.

[0013] In the present invention, the annular cavity is divided into upper and lower parts by a mixing plane in the middle, which allows the aqueous phase introduced from the aqueous phase inlet and the organic phase introduced from the organic phase inlet to move in annular motion before mixing; in a preferred embodiment, the ratio of the height of the upper annular cavity to the height of the lower annular cavity is 1:0.1-10.

[0014] The mixing plane of the present invention is located inside the annular cavity and is used to achieve mixing of the aqueous phase and the organic phase.

[0015] The internal height of the cylindrical chamber in this invention is lower than that of the annular chamber.

[0016] In a preferred embodiment, the circular cross-section of the cylindrical chamber is parallel to the annular cross-section of the annular chamber.

[0017] In a preferred embodiment, the cylindrical surface of the cylindrical chamber is a segment of the inner annular surface of the annular chamber.

[0018] In a preferred embodiment, the circular cross-section of the cylindrical chamber is concentric with the annular cross-section of the annular chamber.

[0019] In this invention, the bottom circular cross-section of the cylindrical or frustum-shaped cavity of the molding cavity is smaller than the circular cross-section of the cylindrical cavity.

[0020] In a preferred embodiment, the bottom circular cross-section of the cylindrical or frustum-shaped chamber of the molding cavity is concentric with the circular cross-section of the cylindrical chamber.

[0021] In this invention, the aqueous phase inlet is a channel for inputting aqueous phase components, and the organic phase inlet is a channel for inputting organic phase components. In a preferred embodiment, the feeding direction of the aqueous phase inlet is tangent to the annular surface of the annular cavity, and the feeding direction of the organic phase inlet is tangent to the annular surface of the annular cavity.

[0022] The carrier outlet in this invention is used to output the assembled carrier from the device. Preferably, the carrier outlet is coaxial with the cylindrical chamber.

[0023] In one specific embodiment, taking the annular cross-section of the annular cavity as the horizontal plane, the cavity width of the annular cavity is 1-3 mm, the outer diameter of the cavity is 3-30 mm, and the cavity height is 3-15 mm; the diameter of the bottom of the molding cavity is 90%-20% of the inner diameter of the annular cavity.

[0024] The assembly equipment of the present invention may further include a housing, which is in the shape of a stainless steel cylinder, made of stainless steel 304 or 316, with a smooth inner wall Ra less than 0.3 and a diameter of 8-30 mm.

[0025] The present invention also includes a method for preparing a lipid gene delivery vector using the assembly equipment provided by the present invention, which includes the following steps: introducing an aqueous phase and an organic phase into the annular cavity through the aqueous phase inlet and the organic phase inlet respectively, and causing them to move in annular motion in the upper and lower parts of the annular cavity respectively before mixing, and converging and uniformly mixing at the mixing plane; after the lipid gene delivery vector is formed at the mixing plane, it converges into the forming cavity and is finally discharged from the vector outlet.

[0026] In the preparation of lipid gene delivery vectors, preferably, the inlet flow rates at the aqueous phase inlet and the organic phase inlet are 0.5 – 10 ml / min. The internal pressure of the mixer is usually higher than the external atmospheric pressure during operation; therefore, the preparation process using this device can be carried out directly at room temperature.

[0027] The design scheme of the lipid gene delivery vector assembly device provided by the present invention can effectively solve the problem of efficient mixing in the preparation process of lipid gene delivery vectors. It can efficiently mix the aqueous phase and organic phase in the preparation of lipid gene delivery vectors. The device can enable the aqueous phase and organic phase to complete the assembly process of lipid gene vectors efficiently and uniformly under near laminar flow conditions, and ensure the stability and uniformity of the vectors.

[0028] Compared with the prior art, the beneficial effects of the present invention include, but are not limited to:

[0029] 1. This device, through the design of the aqueous / organic phase inlet and the annular cavity, achieves annular flow of the organic / aqueous phases before mixing, thereby realizing uniform mixing of the organic and aqueous phases across the entire mixing plane. The liquid flow rate is uniformly distributed across the mixing plane, which is a prerequisite for uniform mixing; simultaneously, the larger mixing area effectively reduces the liquid mixing speed, avoiding the problem of excessively rapid lipid precipitation leading to a decrease in gene drug encapsulation efficiency.

[0030] 2. This equipment is a closed design except for the inlet and outlet, and has no moving parts. It is suitable for the production of sterile injection solutions, is simple to use, easy to maintain, and is energy-saving and environmentally friendly.

[0031] 3. This equipment guides the aqueous and organic phases to mix in a mixing plane. Therefore, by adjusting the size of the mixing plane, the production capacity can be easily scaled up with a small scaling-up effect. Attached Figure Description

[0032] Figure 1 This is a longitudinal cross-sectional schematic diagram of the lipid gene delivery vector assembly device of the present invention;

[0033] Figure 2 This is a design drawing of the lipid gene delivery vector assembly device of the present invention;

[0034] In the diagram, 1 is the aqueous phase inlet, 2 is the organic phase inlet, 3 is the carrier outlet, 4 is the annular cavity, and 5 is the mixing plane. Detailed Implementation

[0035] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0036] Example 1: Lipid gene delivery vector assembly equipment

[0037] like Figure 1 and 2 As shown, the assembly apparatus for the lipid gene delivery vector of the present invention includes an annular cavity, a mixing plane, a forming cavity, an aqueous phase inlet, an organic phase inlet, and a vector outlet. The annular cavity is a ring-shaped chamber used to accommodate the mixing process and to allow the aqueous and organic phases to move in annular motion before mixing. The annular cavity is divided into upper and lower parts by the mixing plane in the middle, allowing the aqueous phase introduced from the aqueous phase inlet and the organic phase introduced from the organic phase inlet to move in annular motion before mixing, respectively. Depending on actual needs and the flow rate difference between the aqueous and organic phases, the ratio of the height of the upper annular cavity to the height of the lower annular cavity is 1:0.1-10. The mixing plane, located within the annular cavity, is used to achieve the mixing of the aqueous and organic phases.

[0038] The mixing plane is a cylindrical chamber located in the center of the annular chamber, which mixes the aqueous and organic phases. The periphery of this cylindrical chamber is connected to the inner wall of the annular chamber, dividing it into upper and lower parts. The internal height of the cylindrical chamber constituting the mixing plane is lower than that of the annular chamber, and the circular cross-section of the cylindrical chamber is parallel to the annular cross-section of the annular chamber. In one specific embodiment, the cylindrical surface of the cylindrical chamber is a segment of the inner annular surface of the annular chamber, and the circular cross-section of the cylindrical chamber is concentric with the annular cross-section of the annular chamber.

[0039] The molding cavity is a cylindrical or frustum-shaped chamber whose bottom is connected to the mixing plane and whose top opens to the outside of the assembly equipment. The bottom circular cross-section of the cylindrical or frustum-shaped chamber constituting the molding cavity is smaller than the circular cross-section of the cylindrical chamber constituting the mixing plane. In one specific embodiment, the bottom circular cross-section of the cylindrical or frustum-shaped chamber is concentric with the circular cross-section of the cylindrical chamber.

[0040] The aqueous phase inlet is located at the upper or lower end of the annular cavity and leads into the interior of the annular cavity; the organic phase inlet is located at the lower or upper end of the annular cavity and leads into the interior of the annular cavity. The organic phase inlet and the aqueous phase inlet are located at the upper and lower ends of the annular cavity, respectively. The aqueous phase inlet is a channel for inputting the aqueous phase component, and the organic phase inlet is a channel for inputting the organic phase component. In one specific embodiment, the feeding direction of the aqueous phase inlet is tangent to the annular surface of the annular cavity, and the feeding direction of the organic phase inlet is tangent to the annular surface of the annular cavity.

[0041] The carrier outlet is located at the outlet of the molding cavity. The carrier outlet is used to output the assembled carrier from the equipment, and the carrier outlet is coaxial with the cylindrical cavity.

[0042] The assembly equipment of the present invention may further include a housing, which is in the shape of a stainless steel cylinder, made of stainless steel 304 or 316, and has a smooth inner wall with Ra less than 0.3.

[0043] In one specific design, taking the annular cross-section of the annular chamber as the horizontal plane, the dimensions of each component are as follows: the diameter of the shell can be 8-30 mm, the width of the annular chamber can be 1-3 mm, the outer diameter of the chamber can be 3-30 mm, and the height of the chamber can be 3-15 mm; the diameter of the bottom of the forming cavity is 90%-20% of the inner diameter of the annular chamber. When the forming cavity has a frustum-shaped structure, the diameter of the carrier outlet is smaller than the diameter of the bottom of the forming cavity. The height of the mixing plane inside the cavity can be adjusted appropriately according to actual needs and the feeding rates of the aqueous and organic phases.

[0044] In use Figure 2 In the assembly equipment shown, during the preparation of lipid gene delivery vectors, the aqueous phase and organic phase are first introduced into the annular cavity through the aqueous phase inlet and organic phase inlet, respectively. Before mixing, they move in a ring shape in the upper and lower parts of the annular cavity, converging and mixing uniformly at the mixing plane. After the lipid gene delivery vector is formed at the mixing plane, it converges into the forming cavity and is finally discharged from the vector outlet. Examples 2 and 3 below both employ this method. Figure 2 Operate the device shown.

[0045] Example 2: Assembly method of lipid gene delivery vector

[0046] Organic phase: 10 mg / mL MC3 ethanol solution, 10 mg / mL DSPC ethanol solution, 10 mg / mL CHOL ethanol solution, 10 mg / mL CHOL ethanol solution, and anhydrous ethanol were mixed evenly in the following proportions: 52.8%, 13.0%, 24.5%, 6.2%, and 3.6%.

[0047] Aqueous phase: Dilute mRNA to 0.133 mg / mL with 25 mM, pH 4.0 acetate-sodium acetate buffer.

[0048] Mixing rate: 0.5 mL / min for organic phase and 1.5 mL / min for aqueous phase, fed into the organic phase inlet and aqueous phase inlet respectively via a liquid phase pump (plunger).

[0049] Experimental results: The lipid gene delivery vector was successfully prepared with a particle size of 142.6 nm, a PDI of 0.42, a potential of -5.43 mV, an encapsulation efficiency of 73.04%, and a recovery rate of 70.26%.

[0050] Example 3: Assembly method of lipid gene delivery vector

[0051] Organic phase: 10 mg / mL TT3 ethanol solution, 10 mg / mL DSPC ethanol solution, 10 mg / mL CHOL ethanol solution, 10 mg / mL CHOL ethanol solution, and anhydrous ethanol were mixed evenly in the following proportions: 52.8%, 13.0%, 24.5%, 6.2%, and 3.6%.

[0052] Aqueous phase: Dilute mRNA to 0.133 mg / mL with 25 mM, pH 4.0 acetate-sodium acetate buffer.

[0053] Mixing rate: 0.5 mL / min for organic phase and 1.5 mL / min for aqueous phase.

[0054] Experimental results: The lipid gene delivery vector was successfully prepared with a particle size of 156.9 nm, a PDI of 0.35, a potential of -7.25 mV, an encapsulation efficiency of 63.51%, and a recovery rate of 94.11%.

[0055] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications may still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions may be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An assembly device for a lipid gene delivery vector, characterized in that... It includes: An annular cavity is a chamber used to accommodate the mixing process and to allow the aqueous and organic phases to move in annular motion before mixing, wherein the aqueous and organic phases move in annular motion in the upper and lower parts of the annular cavity, respectively, before mixing. A mixing plane is a cylindrical chamber located in the middle of the annular chamber, which allows the aqueous phase and the organic phase to converge and mix uniformly. The periphery of the cylindrical chamber is connected to the inner wall of the annular chamber and divides the annular chamber into upper and lower parts. The molding cavity has a bottom that is connected to the mixing plane and a top that leads to a cylindrical or frustum-shaped cavity outside the assembly equipment. Aqueous phase feed inlet, located at the upper or lower end of the annular cavity and leading into the interior of the annular cavity; An organic phase inlet is provided at the lower or upper end of the annular cavity and leads into the interior of the annular cavity; the organic phase inlet and the aqueous phase inlet are respectively located at the upper and lower ends of the annular cavity; and The carrier outlet is located at the outlet of the molding cavity.

2. The assembly apparatus for the lipid gene delivery vector according to claim 1, characterized in that... The annular cavity is divided into upper and lower parts by the mixing plane, which respectively allow the aqueous phase introduced from the aqueous phase inlet and the organic phase introduced from the organic phase inlet to move in annular motion before mixing; the ratio of the height inside the upper annular cavity to the height inside the lower annular cavity is 1:0.1-10.

3. The assembly apparatus for the lipid gene delivery vector according to claim 1, characterized in that... The internal height of the cylindrical chamber is lower than that of the annular chamber, and the circular cross-section of the cylindrical chamber is parallel to the annular cross-section of the annular chamber.

4. The assembly apparatus for the lipid gene delivery vector according to claim 1, characterized in that... The cylindrical surface of the cylindrical chamber is a segment of the inner annular surface of the annular chamber; the circular cross-section of the cylindrical chamber and the annular cross-section of the annular chamber are concentric.

5. The assembly apparatus for the lipid gene delivery vector according to claim 1, characterized in that... The bottom circular cross-section of the cylindrical or frustum-shaped chamber of the molding cavity is smaller than the circular cross-section of the cylindrical chamber; the bottom circular cross-section of the cylindrical or frustum-shaped chamber of the molding cavity is concentric with the circular cross-section of the cylindrical chamber.

6. The assembly apparatus for the lipid gene delivery vector according to claim 1, characterized in that... The feeding direction of the aqueous phase inlet is tangent to the annular surface of the annular cavity, and the feeding direction of the organic phase inlet is tangent to the annular surface of the annular cavity.

7. The assembly apparatus for the lipid gene delivery vector according to claim 1, characterized in that... The annular cavity has a cavity width of 1-3 mm, an outer diameter of 3-30 mm, and a cavity height of 3-15 mm; the diameter of the bottom of the molding cavity is 90%-20% of the inner diameter of the annular cavity.

8. The assembly apparatus for the lipid gene delivery vector according to claim 1, characterized in that... It also includes a shell, which is in the shape of a stainless steel cylinder, made of stainless steel 304 or stainless steel 316, with a smooth inner wall Ra less than 0.3 and a diameter of 8-30 mm.

9. A method for preparing a lipid gene delivery vector using the assembly equipment described in claim 1, characterized in that... It includes the following steps: Aqueous and organic phases are introduced into the annular cavity through the aqueous phase inlet and the organic phase inlet, respectively, and they move in annular motion in the upper and lower parts of the annular cavity before mixing. They then converge and mix evenly at the mixing plane. After the lipid gene delivery vector is formed, it converges into the forming cavity and is finally discharged from the vector outlet.

10. The method according to claim 9, characterized in that... The inlet flow rates at the aqueous phase inlet and the organic phase inlet are 0.5 – 10 mL / min.

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