Method and device for microfluidic formulation of stable nanoparticles as drug / biomolecules carriers

Abstract

This invention involves a microfluidic chip used to create carrier nanoparticles for applications like cancer treatment and vaccine production. The chip is made of PDMS polymer and utilizes soft lithography techniques. The chip's design includes inlets for cationic water and oil phases, and an outlet with channels arranged perpendicular to each other to control nanoparticle size. The nanoparticles produced are 20-100 nm with a lipid coating which enhances cell absorption. They are also stable in vitro and in vivo, with a PDI index of 0. 07 indicating optimal uniformity and homogeneity.

Description

Method and Device for Microfluidic Formulation of Stable Nanoparticles as Drug / Biomolecules Carriers

[0001] Nanoparticles play a significant role in the treatment and diagnosis of diseases, cosmetics and health products, and electronics. So far, conventional methods such as emulsion, precipitation, hydrothermal, etc. have been used to produce nanoparticles, but due to disadvantages such as economic inefficiency and inability to produce uniform and homogeneous particles (particles with high size dispersion), these methods have proved less than ideal.

[0002] Hence, there is a need for new and suitable alternative methods. Using a microfluidic droplet-forming system is a suitable solution to solve the challenges and problems of conventional methods. In the presented patent, we discuss how using a PDMS-based microfluidic chip has many advantages. This innovative chipset has the ability to produce stable nanoparticles with the ability to carry various types of materials including drugs, genes, proteins, etc. This feature can be used to transfer cancer suppressor genes, proteins that bind to cancer cell surface markers, and also to produce vaccines. These applications are very vital and important, and are among the most urgent and important needs of society today.

[0003] B22F 1 / 054

[0004] KR102164022B1 South Korea

[0005] Method for Synthesizing Nanoparticles Using Mircrodroplet-based Microfluidic Device Integrated with In-situ Quenching Zone

[0006] In the present disclosure, a reaction zone for generating microdroplets in a microfluidic device and synthesizing nanoparticles in units of microdroplets, and an in-situ sol-gel quenching zone at the rear end of the reaction zone are provided. A method of manufacturing the provided nanoparticles and a device for implementing the same are described.

[0007] This mentioned invention shares common ground with our claimed one, in that they both involve microfluidic manufacturing of nanoparticles. However, the approach, process and mechanisms employed to achieve the results are somewhat different. For example, this patent emphasizes the in-situ quenching zone while ours focuses primarily on formulation of small size nanoparticles of measured dispersity via the DLS technique.

[0008] KR102043161B1 South Korea

[0009] Microfluidic Device for Merging Micro-droplets and Method for Merging Micro-droplets Using Same

[0010] The present invention relates to a microfluidic control device and a method for merging microdroplets using the same. According to the present invention, by using a simple and short microchannel structure, it is possible to efficiently control the flow of microdroplets and is possible to further simplify a microfluidic chip structure by treating merging of microdroplets and chemical reactions at once. Therefore, it is possible to apply on a lab-on-a-chip that can integrate a variety of pre-processing and analysis processes on a chip in a high speed and high efficiency. In addition, a particle size of a chemical reaction product can be controlled, thereby being able to be effectively applied in the field of fine chemistry that requires control of nano units.

[0011] While the above mentioned patent and our claimed one have similarities in microfluidic formulation of nanoparticles, their focus and overall purposes differ. For instance, this patent discusses a control device and suggests merging the microdroplets, whereas ours emphasizes size and dispersity and uses the DLS technique. Also, ours discussed the medical use of the produced nanoparticles in drug delivery and treatment methods but this one focuses on controlling the production process.

[0012] United States Patent Application 20240050908

[0013] MICROFLUIDIC PLATFORMS FOR LARGE SCALE NANOPARTICLE FORMULATIONS

[0014] Provided are scalable, parallelized microfluidic chips that include arrays of microfluidic mixing channels for large-scale production of lipid nanoparticles, among other products. The disclosed chips can operate with a single set of inlets and outlet, and achieve production rates in excess of those achieved by existing methods. The disclosed devices provide large-scale production of formulations while still maintaining the physical properties and potency typical of existing methods of producing such formulations. Also provided are related methods of using the disclosed devices.

[0015] This mentioned invention resembles our claimed one in method and technique, although there are differences to be considered between the two. For instance, while this patent focuses on large scale production, ours emphasizes the formulation of small (20-100nm) particles. Moreover, their designed chips are different to match each of their overall purpose and functions.

[0016] United States Patent 11583504

[0017] Stabilized formulations of lipid nanoparticles

[0018] The disclosure features a lipid nanoparticle (LNP) formulation comprising a plurality of LNPs and a stabilizing agent that mitigates the degradation of the LNPs or a subpopulation thereof. Lipid nanoparticles further including therapeutics and / or prophylactics such as RNA are useful in the delivery of therapeutics and / or prophylactics to mammalian cells or organs to, for example, regulate polypeptide, protein, or gene expression. Methods of manufacturing LNP formulations and screening for a stabilizing agent are also disclosed.

[0019] This mentioned invention shares certain aspects with our design in terms of manufacturing stable lipid nanoparticles for medical purposes. However, the techniques in their processing methods are dissimilar, for example unlike ours, this one does not utilize soft lithography. Furthermore, this one does not focus on dispersity and size of the particles as much as ours, so the produced nanoparticles in our device are smaller and have PDI of 0.07.

[0020] United States Patent Application 20210378980

[0021] PREPARATION OF LIPID NANOPARTICLES AND METHODS OF ADMINISTRATION THEREOF

[0022] The present disclosure provides methods of producing lipid nanoparticle (LNP) formulations and the produced LNP formulations thereof. The present disclosure also provides therapeutic and diagnostic uses related to the produced LNP formulations.

[0023] The above mentioned patent is similar to our claimed one in function and overall purpose. Although both patents aim to introduce LNP formulation methods and medical applications of the produced particles, their processes differ. Also, their results are different in size and dispersity as our design prioritizes these aspects.

[0024] This patent involves a microfluidic chip designed to produce nanoparticles capable of carrying genes such as DNA, RNA, and siRNA, making it useful for applications like transferring cancer suppressor genes or vaccine production. The chip is made using the PDMS polymer and soft lithography method, a common approach for working with cells due to PDMS's biocompatibility, low cost, and flexibility. The manufacturing process involves placing a photoresist film on a silicon wafer, adding PDMS, punching holes, and bonding PDMS to a glass slide.

[0025] The chip has inlets for cationic water and oil phases and an outlet, with the channels arranged perpendicular to each other to exert hydrodynamic force and control nanoparticle size. The dimensions of the chip are designed to produce 20-100 nm nanoparticles. The nanoparticles produced have a lipid coating, making them suitable for cell membrane absorption. These nanoparticles are stable both in vitro and in vivo, with a PDI index of 0. 07 indicating uniformity and homogeneity.

[0026] Today, nanotechnology has gained many commercial applications due to its unique and unprecedented potential. The potential impact of nanotechnology stems from materials and components that are engineered at the nanometer scale and actually act as intelligent carrier systems for transporting various materials. For years, conventional methods have been used to produce micro / nanoparticles, including emulsion, precipitation, hydrothermal, etc.

[0027] In general, nanoparticle synthesis methods are divided into three categories which include physical, chemical, and biological. Physical methods were less popular due to the production of large amounts of waste and, as a result, their economic unprofitability. In chemical methods, the average size of nanoparticles produced in these methods is usually 600-1800 nm. Biological methods also use viruses, bacteria, and plants to synthesize nanoparticles, which are less toxic to cells than physical and chemical methods.

[0028] Most of these methods are designed for large-scale production, but they have significant disadvantages, such as the inability to produce uniform and homogeneous particles, which create particles with a high dispersion in size, which is why the entry of nanoparticles into the cell is difficult. Therefore, researchers are looking for an alternative method to overcome the challenges of conventional methods. One of these alternative methods is the microfluidic technique.

[0029] In fact, microfluidic science is the science of using very small amounts of liquid flow in microchannels. The use of microfluidic chips to produce fine particles is a very suitable solution to solve the challenges and problems of conventional methods and is useful in many ways, including increasing the accuracy and efficiency of processes, cost-effectiveness and reducing the consumption of reagents and materials, saving time in the process, safer and environmentally friendly efficiency because the consumption of chemicals is minimized. In this innovation, the designed microfluidic chip is capable of producing stable, homogeneous, and uniform nanoparticles with very small dimensions of 20-100 nanometers, and in addition to drugs, it is capable of carrying genes such as DNA, RNA, and more unstable molecules such as siRNA.Solution of Problem

[0030] In this research, the designed microfluidic chip is capable of producing nanoparticles with dimensions of 20-100 nm and, in addition to drugs, it is capable of carrying genes such as DNA, RNA and more unstable molecules such as siRNA. This feature can be used for the transfer of cancer suppressor genes, proteins that bind to cancer cell surface markers, and also for vaccine production. This technology and this microfluidic system are very practical and have the ability to produce carrier nanoparticles on a large scale.

[0031] In this study, the device is made with PDMS polymer and soft lithography method. Among the different approaches to producing microfluidic chips, soft lithography with polydimethylsiloxane (PDMS) polymer is most commonly used for working with cells. In fact, soft lithography is a technique using elastomeric polymers and gels, and PDMS is one of the most routinely used materials in soft lithography due to its low cost, biocompatibility, low toxicity, chemical neutrality, flexibility, and high durability.

[0032] The steps for manufacturing a microfluidic chip are as follows: 1- Placing a photoresist film on a silicon wafer and forming the desired pattern. 2- Adding PDMS on the pattern. 3- Punching holes with a biopsy punch. 4- Bonding PDMS to a glass slide.

[0033] This chip consists of two inlets for the cationic water and oil phases and one outlet. The arrangement of the channels is perpendicular to each other so that the water phase from the middle channel and the other phase from the surrounding channels exerts hydrodynamic force on the middle channel. The proportion of the dimensions of the chip channels and the precise determination of the orifice size play a vital role in the size of the nanoparticles.

[0034] In this project, the channel dimensions are designed in such a way that 20-100 nm nanoparticles can be successfully produced. The speed applied for the water phase is 1-3 ml / h and the speed of the oil phase is 10-20 ml / h. In this way, nanoparticles with a lipid coating are produced, which is suitable and compatible for entering the phospholipid layer of the cell membrane and facilitates absorption by the cell due to the presence of a positive charge in the lipid. These nanoparticles are stable so that they are able to maintain their shape and structure for about a month.

[0035] In addition, the nanoparticles produced in this microfluidic system are also stable in vivo. The uniformity of the produced nanoparticles is an important parameter, as in previous conventional methods, the size dispersion of the nanoparticles was high. The PDI (Poly dispersity index) index expresses the uniformity and homogeneity of the nanoparticles, which in this designed chipset, the PDI index is 0.07, which further expresses the homogeneity and uniformity of the produced nanoparticles. In fact, this index is evaluated by the Dynamic light scattering (DLS) technique.Advantage Effects of the Invention

[0036] • Ability to produce homogeneous and uniform nanoparticles with PDI= 0.07

[0037] • Use of cost-effective, non-chemical materials

[0038] • Production of very small (20-100 nm) nanoparticles that can be absorbed by cells

[0039] • Enhanced cellular absorption due to cationic lipid coating

[0040] • Affordable, user-friendly and accessible

[0041] • Ideal for use in cosmetic, health products and vaccines

[0042] • Production of nanoparticles in high volume with high efficiency and short time

[0043] • Eco-friendly and highly durable

[0044] • High stability of nanoparticles in vitro and in vivo without chemical surfactants

[0045] Shows a general view of the device.

[0046] Declares a flowchart of the production process.

[0047] Shows a schematic view of the microfluidic system and its dimensions:

[0048] 1- First phase inlet channel: 150 μm

[0049] 2- Second phase inlet channel: 180 μm

[0050] 3- Channel width: 350 μm

[0051] 4- Channel outlet: 150 μm

[0052] 5- Microfluidic system length: 10 mm

[0053] 6- Orifice: 150 μm

[0054] Displays a flowchart of the microfluidic formulation of the nanoparticles.Examples

[0055] This microfluidic chip does not require any special equipment and to launch, since the procedure is done simply via a syringe pump required to inject the aqueous and lipid phases into the chip.

[0056] In summary, the method of working with this 3D system is user-friendly and includes the following general steps:

[0057] 1) Design and manufacture of the microfluidic chip using a soft lithography routine method

[0058] 2) Preparation of the aqueous and cationic lipid phases and the desired transfer agent and transfer them into a 10 cc syringe

[0059] 3) Installation of the syringe on the pump and introduction of the two phases through the microfluidic channel inlets at set speeds

[0060] 4) Collection of nanoparticles at the end of the process from the microfluidic device outlet

[0061] The PDI (Poly dispersity index) index was evaluated by the Dynamic light scattering (DLS) technique to ensure the uniformity and homogeneity of the produced nanoparticles.

[0062] [Pic. 1] Shows a line graph of the evaluation of the efficiency of the innovative microfluidic system by the DLS technique wherein:

[0063] Z-Average(d nm):50.7 and PDI:0.07.

[0064] [Pic. 1]

[0065]

[0066] The production of carrier nanoparticles in the microfluidic droplet-forming system is new and innovative in design, and unlike similar examples, no external force (such as electrodes and magnets) is used. On the other hand, the cationic lipid phase increases the rate of biomolecule entry into the cell phospholipid layer and, due to its biocompatibility, it can also be used in clinical trials.

[0067] The application of this invention is as follows:

[0068] 1) Production of stable nanoparticles with the ability to carry various types of materials, including drugs (drug delivery), genes, vaccines, etc.

[0069] 2) Application in the production of vaccines, drug carriers and cosmetic products using cost-effective materials

[0070] 3) Production of nanoparticles with very small dimensions of 20-100 nanometers suitable for absorbing various types of cells to transfer cancer suppressor genes, proteins that bind to cancer cell surface markers, etc.

[0071] 4) Ability to produce nanoparticles carrying biomolecules on a large scale and in a short time and use in research and clinical trials to treat a wide range of diseases

[0072] 5) Contribution to the production cycle and self-sufficiency in the field of cell therapy, gene therapy, drug delivery and the production of health cosmetic products to reduce the import of expensive devices.

Claims

A method and device is introduced for microfluidic formulation of stable and homogeneous nanoparticles (20-100nm) used as biomolecule or drug carriers.According to claim 1, the device is a microfluidic chip manufactured as follows: Placing a photoresist film on a silicon wafer and forming the desired pattern. Adding PDMS on the pattern. Punching holes with a biopsy punch. Bonding PDMS to a glass slide.According to claim 1, the microfluidic chip includes the following components and dimensions:1) First phase inlet channel: 150 μm2) Second phase inlet channel: 180 μm3) Channel width: 350 μm4) Channel outlet: 150 μm5) Microfluidic system length: 10 mm6) Orifice: 150 μmAccording to claim 1, after manufacturing the chip, preparation of the aqueous, cationic lipid phases and the desired transfer agent is done and they are transferred into a 10 cc syringe.According to claim 1, after preparing the ingredients, the syringe is installed on the pump and the two phases are introduced through the microfluidic channel inlets at set speed.According to claim 1, the produced nanoparticles are collected at the end of the process from the microfluidic device outlet.According to claim 1, the channel or chip dimensions are designed in such a way that 20-100 nm nanoparticles can be successfully produced.According to claim 1, the speed applied for the water phase is 1-3 ml / h and the speed of the oil phase is 10-20 ml / h, therefore nanoparticles with a lipid coating are produced, which enhances cellular absorption.According to claim 1, the produced nanoparticles are stable in vitro and in vivo, and able to maintain their shape and structure for about a month.According to claim 1, the DLS technique is used to evaluate the poly dispersity index of the produced nanoparticles at 0.07, which further expresses their homogeneity and uniformity.According to claim 1, the produced nanoparticles can carry drugs, DNA, RNA and even siRNA; which can be useful in cancer treatment and vaccine production.

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