Nerve conduit from decellularized plant that can be used with or without any drug release potential

Abstract

The present invention relates to a nerve conduit obtained by plant decellularization, which does not require 3D printing, is multi-channel, low-cost and capable of drug release. This nerve conduit is functionalized with GelMA and has properties that promote nerve healing.

Description

[0001] NERVE CONDUIT FROM DECELLULARIZED PLANT THAT CAN BE USED WITH OR WITHOUT ANY DRUG RELEASE POTENTIAL

[0002] Technical Field of the Invention

[0003] The present invention relates to a multi-channel artificial nerve conduit functionalized with organic components capable of drug release, produced from material obtained by plant decellularization and does not require 3D printing technology.

[0004] State of the Art

[0005] In existing nerve defects that occur after peripheral nerve injuries, the best method to provide nerve regeneration is microsurgical repairs with nerve grafts taken from another part of the body. However, this method has complications such as leaving a scar elsewhere on the body, numbness in the area of the nerve graft taken, and the risk of neuroma development. For this purpose, studies on the production of artificial nerve conduits that are hollow or secrete various neurotrophic factors as an alternative to nerve graft applications are being carried out all over the world, but no nerve conduit has yet been shown to be functionally and histopathologically superior to nerve graft in terms of nerve healing

[0001] ,

[0006] The working mechanism of nerve conduits is that they are hollow lumen-shaped guidance tubes that guide the existing nerve endings from proximal to distal without any deviation and to ensure that they merge with the correct axonal orientation [2],

[0007] Patent applications numbered CN117618653A, CN114848901 A, CN1 16602790A in the state of the art describe methods for the production of nerve conduits. The abovementioned applications require 3D printing technology and software infrastructure, and high-cost molecules such as PCL / PLA and collagen are used in the produced conduits.

[0008] In addition, it has been determined that multi-channel nerve conduits show better results than single-channel ones in terms of peripheral nerve healing [3]. However, there are many nerve conduits mentioned in the literature that are non-organic and produced with 3D printing technology, especially in the production of multi-channel nerve conduits. For example, the patent application numbered CN109172036A “Multi-channel peripheral nerve conduit and preparation method thereof” in the state of the art requires spinning technology together with 3D printing technology for the multi-channel preparation of the artificial nerve conduit.

[0009] In the state of the art, artificial nerve conduits prepared as a result of plant decellularization are few in number, and there is no plant-based artificial nerve conduit capable of drug release in the art.

[0010] For example, the patent application numbered CN1 18059312A in the state of the art is a plant-based artificial nerve conduit and is made of a natural material that does not require 3D printing. However, in the application, the rootstock part of the plant, which remained underground, was decellularized and used, which increases the diameter discrepancy problem, but said nerve conduit does not have a therapeutic use feature. In addition, the technique we used during the production of our invention also supports the ability of the conduit to expand during nerve regeneration and extracellular nutrition from the environment with the help of its functionalization with Gel-MA.

[0011] Due to the inadequacies in plant-based studies on nerve conduits in the known state of the art, especially in terms of supporting elements for treatment, there is a need to produce a plant-based nerve conduit that is low cost, functionalized with organic components and capable of drug release.

[0012] Brief Description and Objects of the Invention

[0013] This invention covers a multi-channel artificial nerve conduit made from material obtained by plant decellularization, does not require 3D printing technology and is functionalized with organic components capable of drug release.

[0014] The main object of the present invention is to produce an organic nerve conduit that is low-cost and can be an alternative to nerve grafts, which are not yet proven in the literature. For this purpose, vascular tissues obtained by plant decellularization were preferred. Since vascular plant tissues have a naturally channeled structure, a multichannel nerve conduit can be naturally obtained using this tissue. Another object of the present invention is to create a nerve conduit that can release drugs and contribute to nerve healing and provide an alternative healing to nerve grafts. For this purpose, a hydrogel layer was formed by crossing GelMA and tissue polymer, and this layer was given the ability to provide controlled drug release. In addition, with the organic structure of GelMA, it has conduit, swelling and expansion properties and creates connection points for the placement of niosomes for drug release. In addition, with its ability to absorb water, it gains an elastic consistency and enables the canal to expand to a level that can allow the passage of the nerve during the existing nerve regeneration and makes it easier to remain stable during movement.

[0015] Another important object of the present invention is that this drug release feature can work with all kinds of drugs. GelMA cross-links to the conduit structure, allowing drugs such as IGF-LR3 to easily bind to every point of the conduit via niosomes. In this way, controlled drug release can be achieved from every point during nerve healing.

[0016] Description of the Figures

[0017] Figure 1. Transverse section image of the natural channel structure of the decellularized nerve conduit under the microscope with H / E dye.

[0018] Figure 2. Stages of producing pure acellular nerve conduit after decellularization of plant stem with natural channels

[0019] Figure 3. Image of empty niosomes produced under STEM (transmission and scanning microscope) (a), image of IGF-1 LR3 drug-loaded niosomes under STEM (b)

[0020] Figure 4. Schematic natural multi-channel image of the nerve conduit we produced

[0021] Detailed Description of the Invention

[0022] The present invention relates to a low-cost and multi-channel nerve conduit based on decellularized plant tissue that does not require 3D printing technology for peripheral nerve repair. This nerve conduit obtained from decellularized plant tissue also has properties that support nerve healing with its ability to release drugs. The invention is an artificial nerve conduit obtained from decellularized plant tissue, which is a multi-channel nerve conduit comprising drug-loaded niosomes functionalized by organic components. In one embodiment, the organic component used for functionalization is Gel-MA. In another embodiment, the decellularized plant tissue is the stems of the Alstroemeria plant. Moreover, in another embodiment, the invention comprises IGF-1 LR3 loaded into niosomes.

[0023] The production method of the nerve conduit of the invention consists of the following steps respectively; i. Decellularizing plant vascular tissues and preparing them for use as natural nerve conduits ii. Confirming that nerve conduits are cell-free by performing mechanical and porosity analyzes iii. Functionalizing nerve conduit with organic compounds iv. Performing niosome synthesis by thin film formation method with ultra sonication for drug loading process, v. Confirming the encapsulation efficiency of the drug vi. Placing the produced niosome into functionalized conduit with organic components.

[0024] In one embodiment of the invention, in step i of the method, the plant stems are decellularized in PBS solution and streptomycin-comprising solution.

[0025] In one embodiment of the invention, DAPI staining and cell culture and ISO 10993-5 Cell Viability (MTT) cytotoxicity studies are performed in step ii of the method.

[0026] In one embodiment of the invention, the organic compound of step iii of the method is Gel-MA.

[0027] In one embodiment of the invention, in step iv of the method, a mixture of 20 mM Tween61 , cholesterol and DSPE-PEG(2000) (1 :1 :0.1 ) is mixed in chloroform and methanol (2:1 mL) solvents in a round bottom flask to form a thin film layer by ultrasonication and a thin film layer is formed with the help of a rotary evaporator. The resulting thin film was kept overnight in a vacuum desiccator, then poured onto the drug solution comprising different ratios of PBS, vortexed and finally sonicated at 130W and 20 kHz for 1 minute.

[0028] In another embodiment of the invention, the drug specified in Step iv of the method is selected as IGF-1 LR3.

[0029] In one embodiment of the invention, the organic compound of step v. of the method is Gel-MA.

[0030] In one embodiment of the invention, the niosomes of step vi. of the method are inserted into the conduits by impregnation.

[0031] The nerve conduit of the present invention has been produced based on the above methods, and said nerve conduit can be used by loading any drug.

[0032] The nerve conduit of the present invention has channels in the natural structure of the plant vascular tissue and has a multi-channel structure that mimics the same nerve axons and directs their progression without requiring an additional process after the decellularization process. This nerve conduit obtained from the decellularized plant does not show cytotoxic properties.

[0033] In addition, the functional organic component Gel-MA used in the invention gives the nerve conduit swelling and expansion properties and also creates connection points for the placement of niosomes. With the effect of Gel-MA absorbing water, the nerve conduit is easily integrated into the tissue, gains elasticity and prevents the conduit from moving during movement. In addition, with Gel-MA, said conduit can remain transparent even after it is implanted in the tissue with water absorption and thus can show how far the nerve endings have progressed in the conduit with the help of ultrasound.

[0034] As a result of the preliminary study conducted with the nerve conduit, it was observed that the nerve healed towards its distal end within its existing channeled structure in 8 weeks.

[0035] A series of comparative tests regarding the invention were carried out and the technical features of the invention were verified in the laboratory environment.

[0036] After the plant stems were cut to the desired size, they were kept in 10% sodium dodecyl sulfate (SDS) solution for 5 days. After this process, the stems were removed from the solution and kept in distilled water for 1 day. After the incubation period, the plant stems were kept in distilled water comprising 1 % non-ionic surfactant and 10% hydrochloride until their color turned white, and during this period, the solution was replaced with a new one every 24 hours. At the end of the period, the plant samples were washed with distilled water in a shaking water bath to remove solvents and solutions. Then, the samples were tested with total DNA quantification to clarify decellularization and were then lyophilized and stored. Before use, the samples were sterilized by UV and soaking in a solution ccomprising 2% penicillin streptomycin for 2 hours, and then the antibiotic was removed by washing with PBS. Thus, decellularized plant stem was obtained. Then, it was confirmed that the plant stems had sufficient strength by mechanical tension-tensile tests and porosity analysis under STEM.

[0037] The resulting conduit was impregnated with 5% Gel-MA hydrogel and distributed on all surfaces, and its permeability levels were examined with a spectrophotometer and functionalized with an organic compound. Next, the drug called IGF-1 LR3 (which has not been used in any nerve conduit production studies before and its anabolic effects on growth are much more than IGF-1 ) was loaded into the niosomes.

[0038] Niosomes were prepared by ultrasonication and thin film formation method. The mixture of 20 mM Tween61 , cholesterol and DSPE-PEG(2000) (1 :1 :0.1 ) was mixed in chloroform and methanol (2:1 mL) solvents in a round bottom flask and the solvent was removed by rotary evaporator to form a thin film layer. The resulting thin film was kept in a vacuum desiccator overnight to completely remove the solvent. The next day, the solution comprising the drug dissolved in PBS at different rates was poured onto the thin film layer and vortexed. To reduce the macro and micro vesicles in the resulting solution to the nano level, the solution was sonicated at 130W and 20 kHz for 1 minute. Finally, the resulting niosomes were dialyzed against PBS to remove nonencapsulated conjugates and / or polymers.

[0039] The process of calculating the encapsulation efficiency of the loaded drug has begun. To release the niosome content, encapsulated niosomes were blasted with the help of chloroform; samples were added to 96 wells and absorbance was scanned with a thermo plate reader and the data obtained were calculated with the curve equation prepared in advance. Additionally, size analysis of drug-loaded niosomes under STEM was performed and confirmed to be 200-300 nm. Next, niosomes were loaded into conduits and cytotoxicity tests were completed using DAPI staining and MTT assays.

[0040] The nerve conduit of the present invention was applied in an animal experimental model (sciatic nerve defect) involving 30 rats and compared with nerve grafts and Neuragen commercial nerve conduit in terms of gait analysis (functional), histopathologic and electrophysiologic, and was found to provide significant improvement compared to Neuragen without any significant functional difference with nerve grafts. In the study, the nerve conduit releases IGF-1 LR3 through the niosome. This drug, with its anabolic effects, provides elongation of axon endings and directing Schwann cells through the receptor. The study with the IGF-1 LR3 drug is an example of the ability of the neural conduit of the present invention to release any drug or growth factor by easily adding any drug or growth factor as desired without restriction without interacting with any drug or molecule.

[0041] The produced nerve conduit was applied as a preliminary study on the 1 cm defect model in the left rat sciatic nerve. In this study, 6 groups with 5 animals in each group were designed and the nerve conduit produced in the specified groups was compared with nerve graft application and commercial conduit (Neuragen). Gait analysis (Functional), histopathologic (number of axons) and electrophysiologic evaluations showed that the improvement provided as an alternative to nerve grafts was significantly superior to that of commercial nerve conduit.

[0042] Table 1 . Experiment and Control Groups

[0043] In the table, there will be 5 animals in each group, nerve conduit application will be done once in each group, and a total of 30 rats will be used in 6 groups. The groups with the group names written in the table are stated in detail below.

[0044] Group 1 : Rat group that was explored and was not subjected to nerve damage (Control group)

[0045] Group 2: Rat group in which sciatic nerve defect was created but not repaired (Sham group)

[0046] Group 3: Group in which sciatic nerve defect will be created and repaired with autologous nerve graft

[0047] Group 4: Group in which a sciatic nerve defect will be created and repaired with a standard commercial nerve conduit

[0048] Group 5: Group in which a sciatic nerve defect was created and repaired with a GelMA- functionalized decellularized nerve conduit that does not release drugs. Group 6: Group in which a sciatic nerve defect was created and repaired with a decellularized nerve conduit functionalized with GelMA and providing IGF-1 LR3 controlled drug release. Table 2. Results of nerve conduit applied in the rat sciatic defect model

[0049] As seen in Table 2, our nerve conduits with and without IGF-1 LR3 release provided significant improvement compared to the negative control group and the commercial conduit, with no significant difference in gait analysis and function compared to the nerve graft group. Functional analysis results are shown in the table with ANOVA (Oneway analysis of variance) and the significance level is accepted as p<0.05. When the table is analyzed, p<0.05 was found between nerve graft and commercial conduit and significant and meaningful improvement was achieved by nerve graft compared to commercial conduit. However, when the conduit with drug produced within the scope of our project was compared with the nerve conduit, p=1 and no significant difference was detected; with this result, the decellularized plant stem conduit produced provided functional improvement at a level that could be an alternative to nerve grafts. In addition, when the produced conduit and the commercial conduit were examined, pcO.001 was found and significant functional improvement was provided by the decellularized plant stem nerve conduit within the scope of our invention compared to the commercial conduit.

[0050] These results show that the nerve conduit subject to the invention shows superior performance both in terms of production method and treatment efficiency compared to the conduits known in the art.

[0051] REFERENCES

[0052]

[0001] "A novel decellularized nerve graft for repairing peripheral nerve long gap injury in the rat, " Cell and Tissue Research.

[0053] [2] Belkas JS, Shoichet MS, Midha R. "Peripheral nerve regeneration through guidance tubes, " Neurol Res, 2004.

[0054] [3] "Typical and atypical properties of peripheral nerve allografts enable novel strategies to repair segmental-loss injuries, " Journal of Neuroinflammation.

[0055] [4] "Fundamentals and Current Strategies for Peripheral Nerve Repair and Regeneration, " SpringerLink.

Claims

CLAIMS1. An artificial neural conduit derived from decellularized plant tissue, wherein it is a multichannel nerve conduit comprising drug-loaded niosomes functionalized by organic components.

2. A nerve conduit according to claim 1 , wherein the organic compound used for functionalization is Gel-MA.

3. A nerve conduit according to claim 1 or 2, wherein the decellularized plant tissue is the stems of the plant Alstroemeria.

4. A nerve conduit according to any one of claims 1 -3, wherein the drug loaded into the niosomes is IGF-1 LR3.

5. Production method of the nerve conduit, comprising respectively the process steps of; i. decellularizing plant vascular tissues and preparing them for use as natural nerve conduits ii. Confirming that nerve conduits are cell-free by performing mechanical and porosity analyzes iii. Functionalizing nerve conduit with organic compounds iv. Performing niosome synthesis by thin film formation method with ultra sonication for drug loading process, v. Confirming the encapsulation efficiency of the drug vi. Placing the produced niosome into functionalized conduit with organic components.

6. A method according to claim 5, wherein the organic compounds of step iii of said method are Gel-MA.

7. A method according to claim 5 or 6, wherein the drug mentioned in step iv. is IGF-1 LR3.

8. A method according to any one of claims 5-7, wherein in step iv. of the method, a mixture of 20 mM Tween61 , cholesterol and DSPE-PEG(2000) in 1 :1 :0.1 ratios in chloroform and methanol (2:1 mL) solvents is mixed in a round bottom flask to form a thin film layer by ultrasonication using a rotary evaporator; the thin film obtained is kept overnight in a vacuum desiccator, then poured ontothe drug solution comprising different ratios of PBS, vortexed and finally sonicated at 130W and 20 kHz for 1 minute.

9. A method according to any one of claims 5-8, wherein in step vi. of the method, the niosomes are placed in the conduits by impregnation method.

10. An artificial neural conduit, wherein it is produced by a method according to any one of claims 5-9.

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