Low energy consumption continuous process for production of SUV, LUV and GUV monolayer liposomes

Abstract

The present invention relates to a process for the production of SUV, LUV and / or GUV monolayer liposomes using both chemical synthesis and SDR (Spinning Disk Reactor: Rotating Disk Reactor) equipment, which together allows the production of SUV, LUV and GUV monolayer liposomes, preferably of controlled sizes, with low energy consumption, without the need for further post-treatment steps.

Description

Technical Field

[0001] The technical field of the present invention relates to the production of SUV, LUV and GUV unilamellar liposomes, preferably with a controlled or controllable size, ie a size that depends on the synthesis parameters at the start of the reaction, with the aid of specific apparatuses, such as SDR (Spinning Disk Reactor) apparatuses. Background Art

[0002] Liposomes are hollow spherical vesicles characterized by an inner aqueous core and an outer lipid bilayer, whose structure makes them similar to the human cell barrier (Patil and Jadhav, 2014). For this reason, liposomes can be used as drug or molecular carriers for dermatological, cosmetic, biomedical, therapeutic and nutritional purposes (Lian and Ho, 2001).

[0003] Unilamellar liposomes consist of a single phospholipid bilayer encapsulating a hydrophilic core. Based on their size, unilamellar liposomes can be further classified as follows (Ortega et al., 2017):

[0004] Nanosized small unilamellar vesicles (SUV), whose diameter varies from 1nm to 100nm;

[0005] Submicron large unilamellar vesicles (LUVs), whose diameters vary from 100 nm to 1 μm;

[0006] Giant unilamellar vesicles (GUVs) are vesicles with a diameter greater than 1 μm.

[0007] The size of the unilamellar liposomes used is important for the possible effects. For example, in the field of dermatology, only SUV and LUV unilamellar liposomes with a diameter of up to 600 nm can easily pass through and exceed the stratum corneum (SC), the outermost layer of the epidermis, while larger unilamellar liposomes tend to remain in the SC (Bakonyi et al., 2018). On the other hand, smaller unilamellar liposomes with a diameter of 100 nm tend to pass through the entire epidermis and manage to reach the innermost layer as long as the cardiac circulatory system is dispersed in the human body (Verma et al., 2003).

[0008] The production of the SUV, LUV and GUV unilamellar liposomes can be carried out using different techniques, including thin film hydration, dialysis with detergents or reverse evaporation (Maja et al., 2020). However, these very widely used techniques do not allow continuous operation, making it difficult to achieve industrial-scale production. The cost is very high, and this usually results in the production of liposomes that are too large to be easily absorbed by cells, with a material capture efficiency of less than 30%. These problems lead to the need to add post-production steps to reduce the average size of the particles and improve the control of the particle size. This necessary choice usually leads to the loss of the carried drug, resulting in an increase in cost and production time. In addition, due to incompatibility with the human body and the presence of residual toxic solvents, discontinuous processes are not easy to reproduce (Wagner et al., 2002).

[0009] For these reasons, the above-mentioned techniques of the prior art may not be considered as low-cost, precise, rapid, efficient, clean and continuous methods for the production of said SUV, LUV and GUV unilamellar liposomes, preferably with controlled size.

[0010] Other techniques used include the use of micromixers and spray dryers in controlled environments or high-pressure systems. Although these methods are characterized by the continuous production of submicron unilamellar liposomes and their improved size control, the energy requirements of the devices used are very high - more than 30,000 GJ per kg of submicron liposomes produced, considering a lipid concentration of 2 mg per ml of ethanol (Trucello et al., 2020). It should be emphasized that the technology described here cannot produce nanounilamellar liposomes.

[0011] Due to their biodegradability, biocompatibility, and size, SUV, LUV, and GUV liposomes, preferably with controlled size, are increasingly used for topical administration of active dermatological agents and for improving the stability of formulations containing easily degradable substances, including, for example, antibiotics, proteins, DNA, vitamins, antitumor agents, stabilizers, and antioxidants (Xie et al., 2020).

[0012] Therefore, there is a need to provide a method for the synthesis and production of SUV, LUV and GUV liposomes, preferably with controlled size, which overcomes the shortcomings or deficiencies of the prior art.

[0013] It is known to use spinning disk reactors (SDRs) for use in technical fields other than for the production of SUV, LUV and GUV liposomes forming the subject of the present patent application.

[0014] It has been found that SDR reactors offer advantages in the production of SUV, LUV and GUV liposomes, preferably with controlled size. Summary of the invention

[0015] A first subject of the present invention is a method for the production of SUV, LUV and GUV unilamellar liposomes, preferably with controlled size, performed using chemical synthesis and a SDR (Spinning Disk Reactor) device.

[0016] The obtained characteristic features allow the production of SUV, LUV and GUV unilamellar liposomes preferably with controlled size, compatible with their use in the dermatological, cosmetological, biomedical, therapeutic and nutritional fields.

[0017] The present invention relates to a method as described above, characterized in that it can be performed without the use of a post-processing step.

[0018] The present invention relates to a method as described above, wherein an SDR (rotating disk reactor) apparatus is used for continuous production.

[0019] The present invention relates to a method as described above, wherein a SDR (spinning disk reactor) apparatus is used for the production method with energy consumption limited to the range of 90 MJ-450 MJ for each kg of SUV, LUV and GUV unilamellar liposomes of controlled size produced.

[0020] The present invention relates to a method as described above, comprising the following steps:

[0021] preparing a first aqueous solution A and a second solution B, which contain liposomes, preferably dissolved in an organic solvent;

[0022] activating the SDR by rotating the rotating disk;

[0023] Reagent A is injected at the center of the disk to generate a liquid film on the rotating disk;

[0024] injecting solution B on the surface of the disk, at a radial distance from the center of the disk surface, in particular 1 cm to 10 cm, preferably 2 cm to 4 cm;

[0025] The disk is continued to rotate to cause mixing of the reagent solutions in the liquid film and continuous production of reaction products of SUV, LUV and GUV liposomes;

[0026] The reaction product is collected. The organic solvent mentioned in the first step should be understood as any organic solvent known in the art.

[0027] The present invention relates to a method as described above, wherein the SDR reactor has at least one first nozzle for injecting at the center of the disk surface, and at least one second nozzle for injecting on the disk surface at a radial distance from the center of the disk surface, wherein, preferably, the distance between the injector nozzle and the disk surface is 0.1mm-4mm; preferably 1mm-2mm; and / or the radial injection distance of one or more second injector nozzles is 1cm-10cm, preferably 2cm-4cm.

[0028] The present invention relates to the method as described above, wherein the rotation speed of the disk of the SDR device is set to 200 rpm-3000 rpm, preferably 300 rpm-1500 rpm.

[0029] The present invention relates to a method as described above, wherein the volumetric injection flow rate of the reaction solution is 10 ml / min-1000 ml / min, preferably 40 ml / min-250 ml / min; and / or wherein the method is carried out at ambient temperature and pressure.

[0030] The present invention relates to a method as described above, characterized in that the obtained SUV, LUV and GUV unilamellar liposomes, preferably with controlled size, are suitable for use in the fields of dermatology, cosmetology, biomedicine, therapy and nutrition; the obtained SUV, LUV and GUV unilamellar liposomes, preferably with controlled size, have:

[0031] The modal size of SUV, LUV and GUV liposomes is 1 nm-200000 nm, preferably 20 nm-6000 nm;

[0032] A narrow and uniform, preferably unimodal, distribution has a mean square deviation of 5% to 50% relative to the mean, preferably a mean square deviation of 5% to 20% relative to the mean.

[0033] The invention also relates to a method for the production of SUV, LUV and GUV unilamellar liposomes, preferably with controlled or controllable size, for use as carriers of biologically active substances in the fields of dermatology, cosmetology, biomedicine, therapy and nutrition.

[0034] The SUV, LUV and GUV unilamellar liposomes preferably having controlled size obtained using the method according to the present invention preferably have a modal size of 1 nm-200000 nm, preferably 20 nm-6000 nm; the modal size can be measured by a particle size analyzer using conventional techniques.

[0035] Due to their size, controlled size SUV, LUV and GUV unilamellar liposomes are particularly advantageous for dermatological, biomedical, pharmaceutical, therapeutic, cosmetic and nutraceutical uses; by way of non-limiting example, it is contemplated to use controlled size SUV, LUV and GUV unilamellar liposomes as carriers for those substances with biological or pharmacological effects that are not very soluble in aqueous solution, in effect increasing their therapeutic availability, also by virtue of passing through biological membranes and fabrics. Such substances may include antibiotics, proteins, DNA, vitamins, anti-tumor agents, stabilizers and antioxidants.

[0036] Further preferred features of the SUV, LUV and GUV unilamellar liposomes, preferably with controlled (predefined) size, according to the present invention include: a narrow and uniform, preferably unimodal distribution, which has a mean square deviation of 5%-50% relative to the mean, preferably a mean square deviation of 5%-20% relative to the mean. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 shows a diagram of the SDR used.

[0038] FIG. 2 shows the size distribution of SUV, LUV and GUV liposomes produced by SDR operating at 576 rpm and an EtOH / H2O volume ratio of 0.5.

[0039] FIG3 shows the size distribution of SUV, LUV and GUV liposomes produced by SDR operating at 1440 rpm and an EtOH / H2O volume ratio of 0.2. DETAILED DESCRIPTION

[0040] The method proposed according to the invention for the production of SUV, LUV and GUV liposomes preferably with controlled size therefore has the following advantages over other systems: (1) it allows continuous production, compatible with industrial practice; (2) the energy consumption for obtaining the product (expressed in kJ of energy used per kg of synthesized product) is less than that of conventional methods; (3) the size of the obtained product is controlled in the nanometer to micrometer range; (4) the number of steps required to obtain the synthesized product is reduced. These characteristics of the method are overall different from those commonly used or prior art.

[0041] Experiments were at an advanced level in order to justify the invention, with preferred demonstration of the production of SUV, LUV and GUV liposomes with controlled size, documented by particle size analysis (Figures 2 and 3).

[0042] The production method proposed here involves the use of a rotating disk reactor (SDR). This type of reactor is known in the art for fields other than the production of various SUV, LUV and GUV materials and, in the basic configuration shown in Figure 1, comprises a rotating disk 1 having an upper surface suitable for forming a liquid film when the disk 1 is rotated by suitable operating means. The disk 1 is particularly inserted into a chamber 2, the wall 3 of which is preferably cooled.

[0043] A plurality of injector nozzles 4 and 5 are arranged to supply liquid, in particular distilled water and / or a reagent solution, onto the surface of the disc. In particular, at least one first nozzle 4 is present at the center of the disc; and at least one second nozzle 5 is present on the surface of the disc at a given radial distance from the center of the disc.

[0044] During use, the disc 1 is rotated at a processing speed and, by injection of reagents, a liquid film is generated on the upper surface of the rotating disc 1. One or more reagent solutions are then injected onto the rotating disc 1 through the injector nozzles 4 and 5; in particular, at least a first reagent solution is injected into the centre of the disc through a respective first nozzle 4 and at least a second reagent solution is injected into a radial distance from the centre of the disc through at least one respective second injector nozzle 5.

[0045] The rotation of the disk creates the necessary micromixing conditions in the liquid film for the continuous production of SUV, LUV and GUV unilamellar liposomes, preferably of controlled size.

[0046] The precipitated product is collected in a container 6 arranged below the surface of the disc. In particular, the product can precipitate tangentially relative to the disc, hit the walls of the chamber and drip along the walls towards the bottom, where a collection outlet is provided in the container 6.

[0047] The disk may have a diameter of 5-100 cm, preferably 5-50 cm, for example 40 cm, and may be made of metal, ceramic or polymer material, with or without a coating on the surface of the disk.

[0048] Suitable feeding means, such as syringe pumps, may be provided to feed the reagents to the nozzle; preferred volume flow rates of these feeding means are in the range of 10 ml / min to 1000 ml / min, preferably 40 ml / min to 250 ml / min.

[0049] If desired, process temperature and pressure may be monitored and / or controlled using methods known to those skilled in the art, preferably under ambient temperature and pressure conditions.

[0050] The distance between the syringe nozzle and the disk surface may be 0.1 mm-4 mm, preferably 1 mm-2 mm.

[0051] The point on the disk surface where the spraying is performed by the second injector nozzle or nozzles (ie vertical direction of the nozzle) is preferably at a radial distance of 1 cm - 10 cm, and preferably 2 cm - 5 cm, from the center of the disk.

[0052] For the synthesis reaction, the rotation speed of the disk is preferably set to 200 rpm to 3000 rpm, preferably 300 rpm to 1500 rpm.

[0053] For the examples of experimental testing described here, according to a preferred aspect, the SDR device was equipped with a pan having a diameter of 40 cm and was made of uncoated AISI 316 stainless steel.

[0054] Example 1

[0055] The disk is driven at a speed of 576 rpm by a motor connected thereto. The reagent solution consists of a distilled water stream (reagent A) and a 60% ethanol and egg yolk phosphatidylcholine solution (reagent B). These reagent streams are injected simultaneously onto the surface of the disk through a syringe nozzle, at its center point and at a distance of 3 cm therefrom. The product is collected in a container arranged below. The experiment injects reagents A and B at a rate of 100 ml / min. The obtained particles are analyzed by a Brookhaven 90 Plus particle size analyzer. The modal size of the controlled size SUV, LUV and GUV unilamellar liposomes is 361 nm, with a narrow and uniform distribution as shown in Figure 2.

[0056] Example 2

[0057] The disk is driven at a speed of 1440 rpm by a motor connected thereto. The reagent solution consists of a distilled water stream (reagent A) and a 60% ethanol and egg yolk phosphatidylcholine solution (reagent B). These reagent streams are injected simultaneously onto the surface of the disk through a syringe nozzle, at its center point and at a distance of 3 cm therefrom. The product is collected in a container arranged below. The experiment was injected with reagents A and B at a rate of 400 ml / min and 100 ml / min, respectively. The obtained particles are analyzed by a Brookhaven 90 Plus particle size analyzer. The modal size of the controlled-size SUV, LUV and GUV unilamellar liposomes is 95 nm, with a narrow and uniform distribution as shown in Figure 3.

[0058] Typically, the energy consumption for producing 12 g / h-60 g / h of SUV, LUV and GUV unilamellar liposomes of controlled size is equal to 450 MJ and 90 MJ per kg of SUV, LUV and GUV unilamellar liposomes preferably of controlled size, respectively, and is much lower than the energy consumption in other methods in the prior art.

[0059] References

[0060] Bakonyi, M., Gácsi, A., Berkó, S., Kovács, A., & Csányi, E. (2018). Stratum corneum lipid liposomes for investigating skin penetration enhancers. RSC advances, 8(48), 27464-27469.

[0061] Lian, T., & Ho, RJ (2001). Trends and developments in liposome drug delivery systems. Journal of pharmaceutical sciences, 90(6), 667-680.

[0062] Maja, L., K., & Mateja, P. (2020). Sustainable technologies for liposome preparation. Journal of Supercritical Fluids, 104984 (Maja, L., K.,&Mateja,P.(2020).Sustainable technologies for liposome preparation.The Journal of SupercriticalFluids,104984.)

[0063] Ortega, V., Giorgio, S., & de Paula, E. (2017). Liposomal formulations in the pharmacological treatment of leishmaniasis: a review. Journal of liposome research, 27(3), 234-248.

[0064] Patil, Y.P., & Jadhav, S. (2014). Novel methods for liposome preparation. Chemistry and physics of lipids, 177, 8-18.

[0065] Trucillo, P., Campardelli, R., Iuorio, S., De Stefanis, P., & Reverchon, E. (2020). Economic Analysis of a New Business for Liposome Manufacturing Using a High-Pressure System. Processes, 8(12), 1604.

[0066] Verma, D.D., Verma, S., Blume, G., & Fahr, A. (2003). Particle size of liposomes influences dermal delivery of substances into skin. International journal of pharmaceutics, 258(1-2), 141-151.

[0067] Wagner, A., Vorauer-Uhl, K., & Katinger, H. (2002). Liposomes produced in a pilot scale: production, purification and efficiency aspects. European journal of pharmaceutics and biopharmaceutics, 54(2), 213-219.

[0068] Xie, P., Dolovo, D.M., Jia, S., Cheng, X., Salcido, J., Galiano, R.D., ... & Mustoe, T.A. (2020). Liposome-encapsulated statins reduce hypertrophic scars by topical administration. Wound Repair and Regeneration, 28(4), 460-469 (Xie, P., Dolivo, D.M., Jia, S., Cheng, X., Salcido, J., Galiano, R.D., ... & Mustoe, T.A. (2020). statinsreduce hypertrophic scarringthrough topical application.Wound Repair andRegeneration,28(4),460-469.)

Claims

1. A method for the production of nano small unilamellar liposomes SUV, submicron large unilamellar liposomes LUV and micron giant unilamellar liposomes GUV unilamellar liposomes preferably with controlled size, which is performed using chemical synthesis and a spinning disk reactor SDR device.

2. The method according to claim 1, characterized in that The method is performed without using a post-treatment process.

3. The method according to claim 1 or 2, wherein: The rotating disk reactor SDR device is used for continuous production.

4. The method according to claim 1 or 2, wherein: The energy consumption of the production process used in the spinning disk reactor SDR device is defined in the range of 90 MJ to 450 MJ per kg of SUV, LUV and GUV unilamellar liposomes of controlled size produced.

5. The method according to any one of the preceding claims, comprising the steps of: preparing a first aqueous solution A and a second solution B, which contain liposomes, preferably dissolved in an organic solvent; activating the SDR by rotating the rotating disk; injecting reagent A at the center of the disk to generate a liquid film on the rotating disk; injecting the solution B on the surface of the disc, at a radial distance from the center of the disc surface, in particular between 1 cm and 10 cm, preferably between 2 cm and 4 cm; continuing to rotate the disk to cause mixing of the reagent solutions in the liquid film and continuous production of reaction products of SUV, LUV and GUV liposomes; The reaction product is collected.

6. A method according to any one of the preceding claims, wherein: The SDR device has at least one first nozzle for injecting at the center of the surface of the disk, and at least one second nozzle for injecting on the disk surface at a certain radial distance from the center of the surface of the disk, wherein preferably, the distance between the injector nozzle and the disk surface is between 0.1mm-4mm, preferably between 1mm-2mm; and / or the radial injection distance of one or more second injector nozzles is between 1cm-10cm, preferably between 2cm-4cm.

7. A method according to any one of the preceding claims, wherein: The rotation speed of the disk of the SDR device is set to between 200 rpm and 3000 rpm, preferably between 300 rpm and 1500 rpm.

8. The method according to any one of claims 4 to 7, wherein: The volume injection flow rate of the reagent solution is between 10 ml / min and 1000 ml / min, preferably between 40 ml / min and 250 ml / min; and / or wherein, The process is carried out at ambient temperature and pressure.

9. A method for the production of SUV, LUV and GUV unilamellar liposomes, preferably with controlled size, according to any of the preceding claims, characterized in that The obtained SUV, LUV and GUV unilamellar liposomes preferably having controlled sizes are suitable for use in the fields of dermatology, cosmetology, biomedicine, therapy and nutrition; the obtained SUV, LUV and GUV unilamellar liposomes having controlled sizes have: The modal size of the SUV, LUV and GUV liposomes is between 1 nm and 200,000 nm, preferably between 20 nm and 6,000 nm; A narrow and uniform, preferably unimodal, distribution has a mean square deviation of between 5% and 50% relative to the mean, preferably a mean square deviation of between 5% and 20% relative to the mean.

10. Process for the production of SUV, LUV and GUV liposomes, preferably of controlled or controllable size, according to the preceding claims, for use as carriers of biologically active substances in the fields of dermatology, cosmetology, biomedicine, therapy and nutrition.

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