Apparatus and methods for making nanosomes loaded with nucleic acid
Inventors
Assignees
Publication Number
US-9981238-B2
Publication Date
2018-05-29
Expiration Date
2031-08-23
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Abstract
Embodiments of the present invention are directed to an apparatus and methods for making nucleic acid loaded nanosomes. One embodiment of the present invention directed to an apparatus comprises a first containment means for containing a mixture of an aqueous solution of nucleic acid and a phospholipid solution with a supercritical, critical or near critical fluid. The apparatus further comprises injection means in fluid communication with said first containment means for receiving the mixture and releasing the mixture as a stream into a decompression liquid. The apparatus further comprises a decompression vessel in fluid communication with the injection means for holding a decompression liquid and receiving the mixture as a stream. The stream forms one or more nanosomes loaded with a nucleic acid in the decompression liquid.
Core Innovation
The invention pertains to an apparatus and methods for producing nanosomes loaded with nucleic acids, such as siRNA, for improved delivery to targeted cells or organs. The apparatus comprises a first containment means for mixing an aqueous solution of nucleic acid with a phospholipid solution in the presence of a supercritical, critical, or near critical fluid. This mixture is subsequently directed to an injection means, which releases it as a stream into a decompression liquid within a decompression vessel, resulting in the formation of nanosomes encapsulating the nucleic acid.
The invention addresses the challenge of delivering small interfering RNAs (siRNAs) to cells. Existing methods face difficulties in producing liposomes of suitable nanoscale size for effective delivery and achieving consistent, high loading of siRNA. Additionally, prior processes often fail to allow for recycling siRNA not incorporated into liposomes, leading to wastage and increased manufacturing costs.
A further aspect of the invention is the use of a circulation loop that enhances the preparation of the phospholipid solution with a supercritical, critical, or near critical fluid by recirculating the mixture to increase the phospholipid concentration. Key process parameters such as buffer pH and co-injection ratios are optimized to achieve high encapsulation efficiencies and nanosome sizes suitable for efficient cellular absorption. Methods can be applied specifically to siRNA, and the resulting nanosomes may include trace amounts of a low ionic strength buffer.
Claims Coverage
There are two independent claims disclosing inventive features related to the method of forming siRNA loaded nanosomes and the configuration of the apparatus used.
Method for forming siRNA loaded nanosomes with controlled encapsulation efficiency
A method is described involving: 1. Forming a mixture of an aqueous solution of siRNA and a phospholipid solution with a supercritical, critical, or near critical fluid in a first containment means that includes an inline mixer. 2. Directing this mixture to an injector that releases it as a stream into a decompression liquid within a decompression vessel. 3. Forming nanosomes loaded with siRNA in the decompression liquid, where encapsulation efficiency is 95–100% by maintaining the decompression liquid as about 10% sucrose buffer, adjusting co-injection ratios of the phospholipid solution to the aqueous siRNA between 1.0 and 9.0, and setting the pH of the aqueous siRNA to about 4.0.
Method involving device with first containment means, circulation loop, injector, and decompression vessel
A method is outlined that includes: - Providing a device having a first containment means, a circulation loop (with mixing chamber and solids vessel) in fluid communication with the first containment means, an injector, and a decompression vessel holding a decompression liquid. - Forming a phospholipid solution with a supercritical, critical, or near critical fluid in the circulation loop, where the concentration of phospholipid increases until it reaches a final value. - Mixing an aqueous siRNA solution with the phospholipid solution at final concentration in the first containment means. - Injecting this mixture as a stream into the decompression liquid in the decompression vessel. - Forming nanosomes loaded with siRNA in the decompression liquid, achieving total siRNA recovery efficiency of 95–100% under the specified co-injection ratio and pH conditions.
The inventive features cover specific methods and apparatus configurations for the efficient formation of siRNA-loaded nanosomes utilizing supercritical, critical, or near critical fluids, precise mixing, and optimized process parameters to maximize encapsulation and recovery efficiency.
Stated Advantages
The process achieves high siRNA encapsulation efficiencies, with values reported between 40% and 100%, and total siRNA recovery efficiencies up to 95–100%.
Particle sizes in the nanometer range (100–200 nm) are achieved, which are suitable for effective delivery to cells.
The method allows for optimization of critical process variables, such as feed and formulation pH, to further improve encapsulation and recovery efficiencies.
Documented Applications
Nanoencapsulation of siRNA and other biologies in phospholipid nanosomes for improved delivery to targeted human or animal organs and cells.
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