Module and device for the continuous production of nanoliposomes

Abstract

Module for the continuous production of nanoliposomes, comprising: - a first supply branch (2) comprising at least one first volumetric pump (4) which can be connected to a first container (5) for an aqueous solution - a second feed branch (3) comprising at least one second volumetric pump (9) which can be connected to a second container (10) for a second solution containing a lipid fraction dissolved in organic solvent; - a third supply branch (8) operatively arranged after the first (2) and the second branch (3); - a mixing coupling (6) provided with: - a main channel (13) with a diameter of between 1.5 mm and 5 mm, which is provided with a first arm (13a) and a second arm (13b) which are connected by a curved section (13c) and extend along a first (A) and second direction (B) at an angle to one another, - an injection channel (14) having a diameter of less than 1 mm and extending along the second direction (B) to an injection zone opening into said second arm (13b) of the main channel (13), wherein: - the first branch (2) is brought into fluid communication with the first arm (13a) of the main channel (13) of the coupling (6) to supply the first solution; - the second branch (3) is brought into fluid communication with the injection channel (14) of the coupling (6) in order to supply the second solution and mix it with the first solution; - the third branch (8) is brought into fluid communication with the second arm (13b) of the main channel (13) of the coupling (6) in order to obtain a third solution resulting from the mixing of the first and second solutions and to direct it into a third container (17).

Description

[0001] The present invention relates to a module and a device for the continuous production of nanoliposomes.

[0002] This invention is therefore preferably used in the pharmaceutical, nutraceutical, cosmetic, plant protection or animal breeding sectors, in particular in the production of nano-liposomal vectors for the encapsulation of active ingredients, preferably vitamin D3, using a continuous process.

[0003] As is well known, the encapsulation of pharmaceutical, nutraceutical or cosmetic active ingredients in micro- or nanoparticles is a common method to protect active ingredient molecules from the external environment, increase their bioavailability and suppress any undesirable properties.

[0004] This is also done to determine their release in the tissue, where they exert their effect, as well as to control its timing or duration. These micro- or nanoparticles can in particular be polymers or lipids, the latter in turn including micro- or nanospheres, i.e. solid lipid particles, and liposomes, which also have a micrometric or nanometric size. In contrast to solid lipid micro- or nanoparticles, which consist exclusively of lipid materials containing a fat-soluble active ingredient, liposomes are closed vesicular structures consisting of one or more phospholipid bilayers that form when membrane phospholipids such as phosphatidylcholine or cholesterol are dispersed in an excess of water. Unlike micelles, both the external and internal environment of liposomes is aqueous.The process for preparing nanoliposomes is based on injecting a solution of membrane phospholipids in ethanol (phosphatidylcholine, cholesterol) into an aqueous stream, as described, for example, in patent publications EP3681480 and EP3752131 in the name of the same applicant.

[0005] Patent application EP3681480, for example, relates to a process for the preparation of nano-liposomal vectors that aims to exploit the biocompatible structure of liposomes to protect iron and its associated molecule by maintaining iron in the +2 oxidation state (Fe(II)) for the production of nutraceuticals and fortified foods.

[0006] Although the solution described in patent applications EP3681480 and EP3752131 was optimal from a procedural and laboratory point of view, it proved difficult to adapt to industrial production in practice, mainly due to the difficulty of injecting the ethanol stream of membrane phospholipids (phosphatidylcholine, cholesterol) into the aqueous solution with the precision required for the microfluidic scale.

[0007] The aim of the present invention is therefore to provide a plant for the continuous production of nanoliposomes which overcomes the disadvantages of the above-mentioned known technology.

[0008] The present invention aims in particular at providing a module and an apparatus for the continuous production of nanoliposomes capable of implementing, on an industrial scale and with the required precision, a process for the continuous production of nanoliposomes similar to that described in patent applications EP3681480 and EP3752131 in the name of the same applicant.

[0009] The stated purpose is fully achieved by a module for the continuous production of nanoliposomes, which comprises a first feed branch, a second feed branch, a third feed branch and a mixing coupling.

[0010] The first supply branch comprises at least a first volumetric pump and can be connected to a first container with an aqueous solution.

[0011] The second feed branch comprises at least a second volumetric pump and can be connected to a container with a second solution containing a lipid fraction dissolved in organic solvent.

[0012] The third branch of care is operationally arranged after the first and second branches.

[0013] The mixing coupling is equipped with a main channel with a diameter between 1.5 mm and 5 mm.

[0014] Preferably, the main channel is provided with a first arm and a second arm connected by a curved portion and extending along a first and a second mutually angular direction, respectively.

[0015] The mixing coupling is also equipped with an injection channel having a diameter of less than 1 mm and extending along the second direction to an injection area which opens into the said second arm of the main channel.

[0016] Preferably, the first branch is fluidly connected to the first arm of the main channel of the coupling for supplying the first solution. Preferably, the second branch is fluidly connected to the injection channel of the coupling for supplying the second solution and mixing it with the first solution.

[0017] Preferably, the third branch is fluidly connected to the second arm of the main channel of the coupling to obtain a third solution resulting from the mixing of the first and second solutions and to direct it into a third container.

[0018] Preferably, the module further comprises a support plate with at least one anchoring section to which the first branch, the second branch, the third branch, and the mixing coupling are attached. The present invention also relates to a device for the continuous production of nanoliposomes.

[0019] Preferably, the system comprises a first container with a first aqueous solution.

[0020] Preferably, the system includes a second container with a second solution containing a lipid fraction dissolved in an organic solvent.

[0021] Preferably, the first and / or the second container are also mixed with an active ingredient.

[0022] Preferably, the system contains a third collection container for a final product containing nano-liposomal vectors encapsulating said active ingredient.

[0023] Preferably, the plant comprises a support frame and a plurality of modules for the continuous production of nanoliposomes having one or more of the properties described so far.

[0024] Preferably, each support plate is anchored to the frame via the anchoring section.

[0025] Preferably, each first branch is connected to the first container, each second branch is connected to the second container and each third branch () is connected to the third container.

[0026] These and other characteristics will become clearer from the following description of a preferred embodiment of the module and of a device for the continuous production of nanoliposomes, shown as a non-limiting example in the accompanying drawings, in which: - Fig. Figure 1 shows a schematic representation of a module for the continuous production of nanoliposomes according to the present invention; - Fig. 2 and Fig. 3 each shows a perspective cross-sectional view of a component of the module from Fig. 1 show; - Fig. 4 shows a perspective view of an embodiment of an apparatus for the continuous production of nanoliposomes according to the present invention; - Fig. 5 and Fig. 6 shows two alternative configurations of an apparatus for the continuous production of nanoliposomes according to the present invention.

[0027] With reference to the figures in the appendix, a module for the continuous production of nanoliposomes according to the present invention is designated 1. This module 1 is preferably, but not exclusively, used in a device 100 for the continuous production of nanoliposomes, which is also the subject of the present invention. The module and the device according to the invention are particularly suitable for the encapsulation of vitamin D3 using liposomal technology. Preferably, the module 1 comprises a first supply branch 2 and a second supply branch 3.

[0028] The first 2 and the second branch 3 open into the mixing coupling 6, from which a third supply branch 8 branches off, which is operatively arranged after the first 2 and the second branch 3.

[0029] The first branch 2 comprises at least a first volumetric pump 4 and can be connected to a first container 5 with a first aqueous solution.

[0030] Preferably, the first pump 4 is a peristaltic pump. Preferably, the first pump 4 comprises the following: - Speed ​​range 0.1 to 410 rpm in steps of 0.1 rpm; - Certified dual-port EtherNet / IP™ communication for network control and monitoring; - Sealed mounting plate for easy installation and protection against liquid ingress; - Sensor for open lid and input for suction switch; - Supply voltage range 12 to 48 VDC

[0031] The first container 5 therefore contains, during operation, a first aqueous solution, preferably sterilized water or deionized water. Preferably, the first branch 2 also comprises a first pressure stabilization device 7 (or damper) in fluid communication with a pressure side of the first pump 4.

[0032] Along the first branch 2, the first pump 4 and the first pressure stabilizing device 7 are arranged one after the other.

[0033] In the preferred embodiment, the pressure stabilizing device 7 is defined by a pulsation damper with the following properties: - Frequency 0 to 2000 Hz, - Internal volume 11 mL, - Maximum pressure 0.18 mpa, - Flow rate 0 to 1200 mL / min.

[0034] Preferably, therefore, the first branch 2 comprises a first line 2a which runs in several sections between the first container 5 and the first pump 4, between the first pump 4 and the pressure stabilizing device 7 and between the latter and the coupling 6.

[0035] Preferably, the first line 2a has an inner diameter between 2 mm and 6 mm, preferably of approximately 3 mm.

[0036] The first line 2a is preferably made of food-safe silicone hoses.

[0037] The second branch 3 comprises at least one second volumetric pump 9 and can be connected to a second container 10 containing a second solution containing a lipid fraction dissolved in an organic solvent and preferably the active ingredient to be encapsulated. Therefore, in use, the second container 10 contains a second solution containing a lipid fraction dissolved in an organic solvent. Preferably, the second solution contains an ethanolic solution enriched with the active ingredient to be encapsulated.

[0038] More preferably, the ethanolic solution comprises soy lecithin (fostatidylcholine) and cholesterol, preferably in a ratio of 5:1.

[0039] The active ingredient to be encapsulated is preferably vitamin D3.

[0040] In the preferred embodiment, vitamin D3 is added to 10% of the ethanolic solution.

[0041] Preferably, the second pump 9 is also a peristaltic pump. Preferably, the second pump 9 comprises the following: - Speed ​​range 0.1 to 410 rpm in steps of 0.1 rpm; - Certified dual-port EtherNet / IP™ communication for network control and monitoring; - Sealed mounting plate for easy installation and protection against liquid ingress; - Sensor for open lid and input for suction switch; - Supply voltage range 12 to 48 VDC.

[0042] Preferably, the second branch 3 further comprises a second pressure stabilizing device 11 (or damper) which is in fluid communication with a pressure side of the second pump 9.

[0043] Thus, along the second branch 3, the second pump 9 and the second pressure stabilizing device 11 are arranged one after the other.

[0044] In the preferred embodiment, the second pressure stabilizing device 11 is defined by a pulsation damper, comprising: - Frequency 0 to 2000 Hz, - Internal volume 11 mL, - Maximum pressure 0.18 mpa, - Flow rate 0 to 1200 mL / min.

[0045] Preferably, the second branch 3 therefore comprises a second line 3a which runs in several sections between the second container 10 and the second pump 9, between the second pump 9 and the second pressure stabilizing device 11 and between the latter and the coupling 6.

[0046] Preferably, the second line 3a has an inner diameter between 2 mm and 6 mm, preferably of approximately 3 mm.

[0047] The second line 3a is preferably made of food-safe silicone hoses.

[0048] The mixing coupling 6 is a component operatively arranged after and in connection with the first 2 and second branch 3.

[0049] Preferably, the coupling 6 comprises a main channel 13 and an injection channel 14.

[0050] The main channel 13 is provided with a first arm 13a and a second arm 13b, which each extend at an angle to each other in a first direction “A” and a second direction “B”.

[0051] Preferably, the main channel 13 has two end portions 16a which are tapered and designed to correspond to the respective end portions of the first 2 and third branches 8.

[0052] Preferably, the first arm 13a and the second arm 13b are connected to each other by a curved portion 13c.

[0053] The main channel 13 therefore has the shape of an “L”, with the two arms connected to each other in order to minimize the turbulence in the flow of the first solution flowing through it.

[0054] Preferably, the main channel 13 has an (inner) diameter between 1.5 mm and 5 mm, more preferably between 2.2 mm and 2.8 mm.

[0055] Preferably, the curved portion 13c of the main channel 13 has a radius of curvature in a ratio of approximately 10:1 to the inner diameter of the main channel 13.

[0056] Preferably, the injection channel 14 has an (inner) diameter of less than 1 mm, preferably between 0.6 mm and 0.9 mm. The injection channel 14 extends along the second direction "B" to an injection region 15, which opens into the second arm 13b of the main channel 13.

[0057] Preferably, the injection channel 14 and the second arm 13b of the main channel 13 are coaxial, ie, a central axis of the injection channel 14 coincides with a central axis of the second arm 13b along the second direction “B”.

[0058] Preferably, therefore, the first branch 2 is brought into fluid communication with the first arm 13a of the main channel 13 of the coupling 6 in order to supply the first solution.

[0059] The second branch 3 is fluidly connected to the injection channel 14 of the coupling 6 in order to supply the second solution and mix it with the first solution.

[0060] The third branch 3a is fluidly connected to the second arm 13b of the main channel 13 of the coupling 6 to obtain a third solution resulting from the mixing of the first and second solutions and to direct it into a third container 17.

[0061] The third solution is preferably a finished product containing nano-liposomal vectors encapsulating the active ingredient.

[0062] More specifically, the third solution is a liposomal suspension with a concentration of 5 g / liter of liposomes containing 10% active ingredient. In the preferred embodiment, the injection channel 14 has a widened inlet portion 14b designed to allow coupling of an end portion of the second branch 3. Preferably, the inlet portion 14b has a cross-section (transverse to the second direction B) with a polygonal, preferably quadrangular, shape. The inlet portion 14b therefore has an end stop designed to abut the end portion of the second branch 3 (which may end with a special connection).

[0063] The third branch 8 is therefore coupled into the end section 16a of the second arm 13b of the main channel and extends to the third tank 17. Along the branches (first 2, second 3 or third branch 8) there are preferably one or more sensors designed to analyze quantitative (e.g. flow rate) and qualitative (e.g. turbidity) parameters of the third solution.

[0064] In the preferred embodiment, the module 1 comprises a turbidity meter 18 arranged along the third branch 8 and designed to generate a first signal representative of the turbidity of the third solution.

[0065] Preferably, the module 1 further comprises a flow meter 19 positioned along the first 2 and / or second 3 and / or third branch 8 and adapted to generate a second signal representative of a flow rate of the first and / or second and / or third solution along the respective branch.

[0066] In this context, the module 1 preferably comprises or is connected to a control device 21 which is connected to the pumps 4, 9, the turbidity meter 18 and / or the flow meter 19.

[0067] The control unit 21 is preferably defined by a microcontroller and more preferably a master control board capable of monitoring the process of continuous production of “nanoliposomes” by continuously controlling the flow rate of the mixture of ethyl alcohol and water mixed with specific substances characteristic of the production process.

[0068] The control unit 21 is therefore designed to control the volumetric pumps 4, 9 depending on the said first signal and / or the said second signal.

[0069] Advantageously, this makes it possible to control the quality of the third solution in real time and adapt it to the current conditions.

[0070] A module of this type also makes it possible to optimize the operating parameters depending on the type of active ingredient to be encapsulated.

[0071] In the preferred embodiment, the control unit 21 is designed to control the first volumetric pump 4, the second volumetric pump 9, and the coupling 6 are designed to maintain a ratio between 1:20 and 1:5, preferably 1:10, between the flow rates of the second solution and the first solution. In the preferred embodiment, the flow rate of the first solution is set at approximately 50 mL / min, while the concentration of the membrane phospholipids is selected to produce a concentration of approximately 5 g / L liposomes in the aqueous solution. Structurally, the module 1 preferably comprises a support plate 22 with at least one anchoring section 23.

[0072] The first branch 2, the second branch 3, the third branch 8, and the mixing coupling 6 are preferably anchored to the plate 22. Advantageously, the entire module 1 can thus be positioned and moved in its entirety in a simple and effective manner. In the preferred embodiment, the plate 22 is preferably made of metal or polymer material and has a thickness of less than 3 cm.

[0073] Furthermore, the presence of suitable alarm systems is provided to indicate any anomalies or obstructions, which are preferably designed to transmit the value and / or alarm signal to a remote server, for example, a cloud platform. The control unit 21 is therefore preferably designed to: - Identify the pump status (in or out of service); - Checking the status of sensors and collecting data in real time; - Collecting turbidity meter data; - Checking the fill levels of the containers before and after the module; - Generating audiovisual warnings related to operations.

[0074] In a preferred embodiment, module 1 is located inside a device 100 for the continuous production of nanoliposomes, which is also the subject of the present invention. The device 1 is modular in design and comprises a plurality of modules 1 according to the above explanations.

[0075] These modules are fluidly connected to a common first container 5 containing the first solution and a common second container 10 containing the second solution.

[0076] Specifically, the first branches 2 are connected to the first container 5 and the second branches 3 of the modules 1 are connected to the second container 10.

[0077] A common third tank 17 is operatively arranged after the modules to collect the third solution exiting from each of the third branches 15.

[0078] In the preferred embodiment, the device comprises a support frame 101 to which each support plate 22 of the modules 1 is removably attached, preferably by means of its corresponding anchoring portion 23.

[0079] Preferably, the frame 101 has a lattice or tubular structure. Preferably, the frame 101 comprises two parallel cross members 102.

[0080] The anchoring portion 23 of each plate 22 is preferably defined by two hook-shaped portions 23a located on opposite sides of an upper half 22a of the plate and each suspended from a corresponding cross member 102.

[0081] Advantageously, adding or removing a module, whether for maintenance purposes or to change the system's production capacity, is quick and does not affect the other modules, increasing the versatility and productivity of the device.

[0082] In this regard, each cross member 102 preferably has a plurality of individual receptacles which are arranged one after the other along the direction of extension of the cross members and are designed such that they can each accommodate an anchoring section 23.

[0083] Advantageously, this makes it easier to position the individual modules.

[0084] Note also that in some embodiments the modules may be operatively arranged in series ( Fig.6), wherein the third branch 15 of a first module 1' opens into a second branch 3 of a second and / or third module 1" in order to carry out a coating with bioadhesive polymers

[0085] The invention achieves the set objectives and brings significant advantages.

[0086] The use of liposomal technology for the delivery of vitamin D3 represents a promising development in animal nutrition. By improving the bioavailability and stability of the vitamin, liposomes can optimize the absorption of vitamin D3 in ruminants, poultry, and other animal species, thus contributing to improved skeletal health, milk and egg quality, and increased productivity.

[0087] This form of administration offers significant advantages over conventional methods as it improves the stability and absorption of the vitamin.

[0088] One of the most important advantages of using liposomes is the increased bioavailability of vitamin D3. The bilayer structure of liposomes, which resembles the structure of cell membranes, facilitates the transport of vitamin D3 through the intestinal barrier, allowing for more efficient absorption than conventional forms. This is particularly useful for fat-soluble nutrients like vitamin D3, which normally require lipid carriers for effective absorption.

[0089] In animal nutrition, administering vitamin D3 via liposomes can improve bone health and growth. In cattle, for example, vitamin D3 is crucial for calcium and phosphorus metabolism, which impacts milk quality and bone health. In poultry, proper administration of vitamin D3 can reduce the risk of skeletal deformities, improve egg production, and promote optimal growth.

[0090] Furthermore, the use of liposomes reduces the risk of vitamin D3 degradation during transport through the gastrointestinal tract and protects it from stomach acids and enzymes. This improved stability results in more effective dosing, allowing for more precise delivery and reducing nutrient losses that can occur with other delivery methods. QUOTES CONTAINED IN THE DESCRIPTION

[0000] This list of documents submitted by the applicant was generated automatically and is included solely for the convenience of the reader. This list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions. Cited patent literature

[0000] EP 3681480 [0004, 0005, 0006, 0008] EP 3752131 [0004, 0006, 0008]

Claims

[1] Module for the continuous production of nanoliposomes, comprising: - a first supply branch (2) comprising at least one first volumetric pump (4) which can be connected to a first container (5) for an aqueous solution - a second feed branch (3) comprising at least one second volumetric pump (9) which can be connected to a second container (10) for a second solution containing a lipid fraction dissolved in organic solvent; - a third supply branch (8) operatively arranged after the first (2) and the second branch (3); - a mixing coupling (6) provided with: - a main channel (13) with a diameter of between 1.5 mm and 5 mm, which is provided with a first arm (13a) and a second arm (13b) which are connected by a curved section (13c) and extend along a first (A) and second direction (B) at an angle to one another, - an injection channel (14) having a diameter of less than 1 mm and extending along the second direction (B) to an injection zone opening into said second arm (13b) of the main channel (13), wherein: - the first branch (2) is brought into fluid communication with the first arm (13a) of the main channel (13) of the coupling (6) to supply the first solution; - the second branch (3) is brought into fluid communication with the injection channel (14) of the coupling (6) in order to supply the second solution and mix it with the first solution; - the third branch (8) is brought into fluid communication with the second arm (13b) of the main channel (13) of the coupling (6) in order to obtain a third solution resulting from the mixing of the first and second solutions and to direct it into a third container (17). [2] Module according to claim 1, wherein the main channel (13) has an inner diameter between 2.2 mm and 3.5 mm. [3] Module according to claim 1 or 2, wherein the injection channel (14) has an inner diameter between 0.6 mm and 0.9 mm. [4] Module according to any one of the preceding claims, wherein the injection channel (14) has a widened inlet portion (15) adapted to allow the coupling of an end portion of the second branch (3). [5] Module according to claim 4, wherein the injection channel (14) has an inlet section (15) with a polygonal, preferably quadrangular, cross-section. [6] A module according to any one of the preceding claims, wherein the main channel (13) has two tapered end portions (16a) adapted to correspond to the respective end portions of the first (2) and third (3) branches. [7] A module according to any one of the preceding claims, wherein the first (2) and second branches (3) each comprise a pressure stabilizing device (7, 11) in fluid communication with a pressure side of the respective first (4) or second volumetric pump (9). [8] A module according to any one of the preceding claims, wherein the curved portion (13c) of the main channel (13) has a radius of curvature in a ratio of approximately 10:1 to an inner diameter of the main channel (13). [9] Module according to any one of the preceding claims, wherein the first volumetric pump (4), the second volumetric pump (9) and the coupling (6) are designed to maintain a ratio of between 1:20 and 1:5, preferably 1:10, between the flow rates of the second solution and the first solution. [10] Module according to any one of the preceding claims, comprising at least one of the following components: - a turbidity meter (18) arranged along the third branch and designed to generate a first signal (S1) representative of the turbidity of the third solution; - a flow meter (19) arranged along the first (2) and / or second (3) and / or third branch (8) and designed to generate a second signal (S2) representative of a flow rate of the first and / or second and / or third solution along the respective branch. [11] Module according to claim 10, comprising a control device (21) connected to the volumetric pumps (4, 9), the turbidity meter (18) and / or the flow meter (19) and designed to control the volumetric pumps (4, 9) depending on said first signal (S1) and / or said second signal (S2). [12] Module according to any one of the preceding claims, comprising a support plate (22) provided with at least one anchoring portion (23) and to which the first branch (2), the second branch (3), the third branch (8) and the mixing coupling (6) are fixed. [13] Apparatus for the continuous production of nanoliposomes, comprising: - a first container (5) containing a first aqueous solution; - a second container (10) with a second solution containing a lipid fraction dissolved in an organic solvent, wherein said first and / or said second container is additionally admixed with an active ingredient; - a third container (17) for collecting a final product containing nano-liposomal vectors encapsulating the active ingredient; - a supporting frame (101); - a plurality of modules (1) for the continuous production of nanoliposomes according to claim 12, wherein each support plate (22) is anchored to the frame (101) by means of the anchoring section (23), wherein each first branch (2) is connected to the first container (5), each second branch (3) is connected to the second container (10) and each third branch (8) is connected to the third container (17). [14] Apparatus according to claim 13, wherein the frame (101) comprises two parallel cross members (102) and the anchoring portion (23) of each plate (22) is defined by two hook-shaped portions (23a) located on opposite sides of an upper half (22a) of the plate (22) and each suspended from a corresponding cross member (102). [15] Apparatus according to claim 13 or 14, comprising a plurality of modules (1) arranged in parallel one after the other along the frame (101) and selectively removable.

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