Preparation method and application of CDNF / Rg3 loaded biomimetic lipid nanoparticles

Abstract

The application belongs to the technical field of nano drug delivery, and particularly relates to a preparation method and application of a CDNF / Rg3 biomimetic lipid nanoparticle. The method comprises the following steps: constructing nerve stem cells overexpressing ARC, extracting cell membranes of the nerve stem cells, and preparing ROS-responsive lipid nanoparticles loaded with CDNF circRNA and ginsenoside Rg3 by using a microfluidic technology, and then obtaining ARC-RCD-LNP by film extrusion. The nanoparticles can enter the brain after intranasal administration, improve the uptake of damaged neurons by means of the ARC-related neuron targeting property, and release Rg3 and CDNF circRNA under a ROS microenvironment; the CDNF circRNA is translated into CDNF protein, inhibits alpha-Syn aggregation, and cooperates with Rg3 to exert the effects of anti-inflammation, anti-oxidation, relieving endoplasmic reticulum stress and anti-apoptosis, so as to repair damaged neurons and improve motor dysfunction.

Description

Technical Field

[0001] This invention belongs to the field of nanomedicine delivery technology, specifically relating to a method for preparing and applying CDNF / Rg3-loaded biomimetic lipid nanoparticles. Background Technology

[0002] Lipid nanoparticles (LNPs), as one of the earliest nucleic acid delivery carriers used for cell transfection and mRNA vaccination, possess lipophilicity, good biocompatibility, and biodegradability. However, traditional liposomes without engineered optimization still face problems such as insufficient targeting efficiency and drug leakage. While attaching targeting ligands to the surface of liposomes through physical or chemical binding can enhance their targeting ability, it can also damage the liposome structure to some extent, affecting their function or stability.

[0003] For central neurodegenerative diseases such as Parkinson's disease, traditional liposomes or LNPs still suffer from limited brain delivery efficiency, insufficient uptake by lesion neurons, inadequate stability of the drug delivery system, and premature drug release at non-lesion sites. In particular, the development of Parkinson's disease involves multiple pathological processes, including abnormal α-synuclein aggregation, neuroinflammation, oxidative stress, endoplasmic reticulum stress, and dopaminergic neuron damage, making effective intervention difficult with a single drug or delivery function. To improve the targeting of liposomes, existing technologies typically employ two strategies. One is to attach recognition elements such as antibodies, peptides, nucleic acid aptamers, or small molecule ligands to the surface of liposomes through physical adsorption or chemical coupling to enhance the carrier's recognition and uptake of specific cells or tissues; however, this method usually increases the complexity of preparation steps and processes, and may affect the structural stability of liposomes, ligand activity, and batch-to-batch consistency. The other is a biomimetic delivery strategy using cell membrane-coated liposomes or nanoparticles, allowing the nanocarrier to inherit some biological characteristics of the source cell membrane, such as immune escape, inflammatory homing, homologous targeting, or tissue retention. Existing technologies have disclosed methods for coating nanoparticles or liposomes onto erythrocyte membranes, platelet membranes, tumor cell membranes, macrophage membranes, chondrocyte membranes, and neural stem cell membranes. However, existing cell membrane coatings with liposomes or nanoparticles still have the following shortcomings: First, existing cell membrane coating systems largely rely on the natural tropism or homology recognition of the source cell membrane. Their targeting ability is significantly affected by cell origin, membrane protein composition, and disease type, making it difficult to directly target damaged neurons in Parkinson's disease. Second, while existing neural stem cell membrane-coated liposomes can utilize the natural lesion tropism of neural stem cells, they mainly rely on the multi-molecular synergistic effects of natural membrane proteins and lack further engineered enhancements targeting the neuronal uptake process. Finally, existing biomimetic liposomes mostly focus on single-target or single-drug delivery, lacking integrated therapeutic designs that simultaneously combine the oxidative stress microenvironment of Parkinson's disease, neurotrophic factor supplementation, α-Syn pathological aggregation intervention, and anti-inflammatory and antioxidant protection. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for preparing and applying CDNF / Rg3-loaded biomimetic lipid nanoparticles. This invention utilizes neural stem cell membranes overexpressing ARC protein as a targeting module, DHA as a ROS-responsive release switch, ginsenoside Rg3 as an anti-inflammatory and antioxidant active ingredient, and circRNA-form CDNF as a source of highly stable and highly expressed neuroprotective protein to construct an integrated biomimetic nanodelivery system (ARC-RCD-LNP). This system can specifically target damaged dopaminergic neurons, responding to and releasing therapeutic drugs in the neuronal ROS microenvironment. CDNF directly inhibits the pathological aggregation of α-Syn and works in conjunction with Rg3 to exert anti-inflammatory and antioxidant effects, synergistically intervening in Parkinson's disease from multiple pathological aspects, thus overcoming the bottleneck problems of low brain delivery efficiency and limited efficacy of single therapies in existing treatments.

[0005] This invention provides the following technical solutions: A method for preparing CDNF / Rg3-loaded biomimetic lipid nanoparticles, characterized in that the method comprises: Neural stem cells overexpressing the ARC gene were constructed, and the cell membranes of the neural stem cells were extracted; lipid nanoparticles loaded with CDNF circRNA and ginsenoside Rg3 were synthesized using microfluidic technology. The cell membrane of the neural stem cells was coated onto the surface of the lipid nanoparticles using a thin-film extrusion technique to obtain CDNF / Rg3-loaded biomimetic lipid nanoparticles.

[0006] The ARC gene is a human activity-regulated cytoskeleton associated protein gene, with the species origin of Homo sapiens, NCBI Gene ID 23237, and GenBank / RefSeq accession number NM_015193.5.

[0007] Furthermore, the method for constructing neural stem cells overexpressing the ARC gene includes: transfecting neural stem cells with a lentiviral overexpression vector, obtaining a stable integrated ARC-NSC cell line through resistance selection, wherein the ARC-NSC cell line is capable of overexpressing the ARC gene.

[0008] Specifically, the method for extracting cell membranes is as follows: neural stem cells overexpressing the ARC gene are collected, washed, centrifuged, resuspended in a buffer containing protease inhibitors, the cells are disrupted by sonication, and cell membrane fragments are collected by differential centrifugation.

[0009] Further, the preparation method of the lipid nanoparticles includes: dissolving ginsenoside Rg3, DOTAP (1,2-dioleoyl-3-trimethylammonium propane), DOPE (dioleoylphosphatidylethanolamine), and DHA (docosahexaenoic acid) in ethanol as an organic phase, dissolving CDNF circRNA in citrate buffer as an aqueous phase, mixing the organic phase and aqueous phase for self-assembly using a microfluidic chip, and removing the organic solvent by ultrafiltration to obtain lipid nanoparticles loaded with CDNF circRNA and ginsenoside Rg3; wherein the nucleotide sequence of CDNF circRNA is shown in SEQ ID NO.1.

[0010] Furthermore, the molar percentage ratio of ginsenoside Rg3, DOTAP, DOPE, and DHA is as follows: ginsenoside Rg3 45–55 mol%, DOTAP 30–35 mol%, DOPE 10–13 mol%, and DHA 4–6 mol%; preferably, ginsenoside Rg3 50.3 mol%, DOTAP 33 mol%, DOPE 11.7 mol%, and DHA 5 mol%.

[0011] The mass ratio of total lipids to CDNF circRNA is (10-20):1; preferably 15:1. The total lipids refer to the sum of the masses of ginsenoside Rg3, DOTAP, DOPE, and DHA used in the preparation of lipid nanoparticles.

[0012] Further, the method for extruding the film is as follows: the extracted neural stem cell membrane overexpressing ARC is ultrasonically dispersed at a power of 100–300 W for 5–30 min; then it is extruded sequentially through polycarbonate membranes with pore sizes of 300–500 nm and 100–300 nm to obtain neural stem cell membrane vesicles; the neural stem cell membrane vesicles are mixed with lipid nanoparticles at a mass ratio of (0.5–5):1, and then extruded through a polycarbonate membrane with a pore size of 200–500 nm to obtain lipid nanoparticles loaded with CDNF circRNA and ginsenoside Rg3 coated with ARC neural stem cell membrane, namely ARC-RCD-LNP.

[0013] Furthermore, the neural stem cell membrane vesicles and lipid nanoparticles are mixed at a membrane protein mass to LNP mass ratio of (1.5-2.5):1, preferably 2:1; wherein, the membrane protein mass is the membrane protein mass of the neural stem cell membrane vesicles as determined by protein quantification, and the LNP mass is the mass of the lipid component in the lipid nanoparticles.

[0014] The present invention also provides a method for preparing CDNF / Rg3-loaded biomimetic lipid nanoparticles, wherein the surface of the CDNF / Rg3-loaded biomimetic lipid nanoparticles is coated with a neural stem cell membrane; the cell membrane carries ARC protein; and the interior of the CDNF / Rg3-loaded biomimetic lipid nanoparticles is loaded with CDNF circRNA and ginsenoside Rg3.

[0015] The CDNF / Rg3-loaded biomimetic lipid nanoparticles exhibit ROS-responsive drug release properties.

[0016] This invention also provides the application of CDNF / Rg3-loaded biomimetic lipid nanoparticles in drugs for the prevention or treatment of neurodegenerative diseases.

[0017] Furthermore, the CDNF / Rg3-loaded biomimetic lipid nanoparticles are delivered into the brain via nasal delivery. Under the action of the carried ARC protein, the CDNF / Rg3-loaded biomimetic lipid nanoparticles target and bind to damaged dopaminergic neurons and are taken up by endocytosis. In the ROS microenvironment, they release ginsenoside Rg3 and CDNF circRNA in response. CDNF circRNA is translated into functional CDNF protein, which directly binds to α-Syn to inhibit its pathological aggregation. Moreover, CDNF circRNA and ginsenoside Rg3 synergistically exert anti-inflammatory and neuronal repair effects.

[0018] Beneficial technical effects of the present invention: The present invention provides ARC-RCD-LNP biomimetic lipid nanoparticles loaded with CDNF / Rg3, coated with a neural stem cell membrane overexpressing ARC protein. These nanoparticles can be targeted and delivered to damaged dopaminergic neurons. In the ROS microenvironment, they release ginsenoside Rg3 and CDNF circRNA in response. The CDNF circRNA is efficiently translated into functional CDNF protein, which directly binds to α-Syn to inhibit its pathological aggregation. This synergistic effect helps to reduce inflammation and repair damaged neurons, thereby improving motor coordination and balance dysfunction.

[0019] The CDNF / Rg3-loaded biomimetic lipid nanoparticles provided by this invention can be used to prepare nanomedicines, which are suitable for neuronal damage diseases including Parkinson's disease. Attached Figure Description

[0020] Figure 1 This is the result of Western blot detection of ARC protein expression in an embodiment of the present invention; Figure 2 The particle size distributions of RC-LNP, RCD-LNP, and ARC-RCD-LNP in the embodiments of the present invention are shown below. Figure 3 The diagram shows the zeta potential of RC-LNP, RCD-LNP, and ARC-RCD-LNP in the embodiments of the present invention. Figure 4 The encapsulation efficiency and drug loading rate of Rg3 in ARC-RCD-LNP in this embodiment of the invention; Figure 5 This describes the CDNF circRNA loading status in ARC-RCD-LNP in this embodiment of the invention. Figure 6 The encapsulation efficiency and drug loading rate of CDNF circRNA in ARC-RCD-LNP in this embodiment of the invention; Figure 7 This is a diagram showing the cumulative release of CDNF circRNA in ARC-RCD-LNP in an embodiment of the present invention; Figure 8A This is a fluorescence image of SH-SY5Y cells taking up lipid nanoparticles in an embodiment of the present invention; Figure 8B This is a fluorescence quantitative PCR image of SH-SY5Y cells taking up lipid nanoparticles in an embodiment of the present invention; Figure 9 The expression level of CDNF in SH-SY5Y cells after treatment with different lipid nanoparticles in the embodiments of the present invention; Figure 10 This is a representative Western blot of TH after treatment with different lipid nanoparticles in the embodiments of the present invention; Figure 11A This is an immunofluorescence image of TNF-α in SH-SY5Y cells in an embodiment of the present invention; Figure 11B This is a quantitative analysis of TNF-α in SH-SY5Y cells in this embodiment of the invention; Figure 12A This is an immunofluorescence image of IL-10 in SH-SY5Y cells in an embodiment of the present invention; Figure 12B This is a quantitative analysis of IL-10 in SH-SY5Y cells in this embodiment of the invention; Figure 13 This is a real-time fluorescence image and quantitative analysis of PD model mice in an embodiment of the present invention, where A is the fluorescence image and B is the quantitative analysis image; Figure 14 The behavioral tests for PD mice in this embodiment of the invention are as follows: A is the suspension test, which is used to evaluate the limb grasping ability, muscle strength and exercise tolerance of PD mice. The higher the score, the better the motor function; B is the rotating rod test, which is used to evaluate the motor coordination and balance of mice. The longer the time spent on the rotating rod, the better the motor coordination and balance; C is the pole test, which is used to evaluate the bradykinesia and motor coordination of mice. The shorter the time required to complete the pole climb, the better the recovery of motor function. Figure 15A This is a Western blot analysis of representative TH in the striatum of PD mice after treatment with different lipid nanoparticles in this embodiment of the invention. Figure 15B This invention provides a quantitative analysis of TH in the striatum of PD mice after treatment with different lipid nanoparticles in this embodiment. Figure 16A Immunofluorescence analysis of α-Syn in the striatum of PD mice after treatment with different lipid nanoparticles in this embodiment of the invention; Figure 16B This invention provides a quantitative analysis of α-Syn in the striatum of PD mice after treatment with different lipid nanoparticles in this embodiment. Figure 17 In this embodiment of the invention, ARC-RCD-LNP restores neurotrophic factor and dopaminergic levels in PD mice. A represents the dopamine (DA) content detection results in the striatum of PD mice, reflecting the recovery of dopaminergic neuronal function. B represents the brain dopamine neurotrophic factor (CDNF) content detection results in the striatum of PD mice, used to evaluate neurotrophic factor levels and the in vivo function of CDNF circRNA after delivery. Figure 18A Immunofluorescence of Neun in the striatum of PD mice in this embodiment of the invention; Figure 18B This invention provides a quantitative analysis of Neun in the striatum of PD mice. Figure 19 The expression level of IL-6 in the striatum of the brain of PD mice in this embodiment of the invention; Figure 20 The images show H&E staining of the major organs of mice in each group in this invention embodiment. Detailed Implementation

[0021] The technical solution of the present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0022] This invention does not simply replicate existing cell membrane-coated liposome technology, but rather further engineers and modifies a cell membrane-based biomimetic delivery strategy. Specifically, this invention constructs neural stem cells overexpressing the cytoskeleton-related protein ARC, extracts their cell membranes, and coats them onto the surface of lipid nanoparticles loaded with CDNF circRNA and ginsenoside Rg3, forming CDNF / Rg3-loaded biomimetic lipid nanoparticles (ARC-RCD-LNP). Using the ARC-overexpressing neural stem cell membrane as a biomimetic shell helps improve the interaction and uptake efficiency between the nanoparticles and neuronal-related cells. This invention addresses the problems of insufficient brain delivery efficiency, limited uptake by damaged neurons, lack of lesion-related ROS response release, and difficulty in achieving synergistic delivery of CDNF circRNA and ginsenoside Rg3 in the treatment of Parkinson's disease using existing cell membrane-coated liposomes.

[0023] Recombinant Activity Regulated Cytoskeleton Associated Protein (ARC) is a protein expressed on neurons and a major regulator of synaptic plasticity. ARC proteins can self-assemble into a virus-like capsid to encapsulate mRNA and then transfer the mRNA to receptor neurons via receptor-mediated endocytosis, exhibiting neuronal targeting capabilities. Editing stem cells to overexpress ARC proteins reduces the need for physical or chemical coupling, making in vivo application safer. Similarly, nanoparticles coated with ARC-overexpressing cell membranes also possess targeting properties.

[0024] circRNA, due to its unique circular structure, can resist degradation by exonucleases and exhibits a longer in vivo half-life and higher expression compared to linear mRNA, making it a promising form of nucleic acid drug. Docosahexaenoic acid (DHA) is an ω-3 polyunsaturated fatty acid whose carbon-carbon double bonds can break under ROS conditions, enabling controlled drug release and showing potential for preparing ROS-responsive drug delivery carriers. Ginsenoside Rg3 is a bioactive compound derived from ginseng that promotes apoptosis in various malignant tumor cells, inhibits tumor proliferation and angiogenesis, reduces cancer metastasis and recurrence, and possesses antioxidant and anti-inflammatory neuroprotective effects. Its amphiphilic structure makes it a good substitute for cholesterol in liposomes. Cerebral dopamine neurotrophic factor (CDNF) is an endoplasmic reticulum-resident protein that can directly interact with α-Syn to inhibit its pathological accumulation, but it suffers from low blood-brain barrier penetration. Therefore, the above functional modules can be integrated to construct a biomimetic nanodelivery system that combines targeted, responsive drug release, and efficient protein expression. Furthermore, existing technologies include neural stem cell exosomes carrying ARC proteins, which primarily utilize the natural vesicle structure of exosomes and the neuronal targeting characteristics related to ARC for delivery. This invention extracts the membrane of neural stem cells overexpressing ARC and coats it onto the surface of ROS-responsive lipid nanoparticles loaded with CDNF circRNA and ginsenoside Rg3. This retains the neuronal targeting advantage while improving the controllability of drug loading composition and release behavior, achieving synergistic intervention on multiple pathological aspects of Parkinson's disease. The following are specific embodiments: Example 1: A method for preparing CDNF / Rg3-loaded biomimetic lipid nanoparticles, comprising the following steps: (1) Construct an engineered ARC-NSCs cell line, and use the purified ARC as the target gene to construct a lentiviral overexpression vector to transfect neural stem cells, so that the neural stem cells express ARC protein; (2) Extraction of neural stem cell membranes overexpressing ARC protein; (3) Using microfluidic technology, ginsenoside Rg3, DOTAP, DOPE and DHA were used as lipid components, and CDNF circRNA was used as nucleic acid drug to synthesize ROS-responsive lipid nanoparticles (RCD-LNP) loaded with CDNF circRNA and ginsenoside Rg3. (4) By means of thin film extrusion, neural stem cell membranes overexpressing ARC protein are coated on the surface of RCD-LNP to obtain CDNF / Rg3-loaded biomimetic lipid nanoparticles (ARC-RCD-LNP).

[0025] The ARC gene is a human activity-regulated cytoskeleton associated protein gene, with the species origin of Homo sapiens, NCBI Gene ID 23237, and GenBank / RefSeq accession number NM_015193.5.

[0026] Specifically, the steps for constructing neural stem cells overexpressing ARC protein in step (1) are as follows: construct a lentiviral overexpression vector with ARC as the target gene, transfect neural stem cells, establish a stable integrated ARC-NSC cell line through resistance screening, and verify the expression of exogenous ARC protein through Western blotting.

[0027] The specific steps for extracting the membrane of neural stem cells overexpressing ARC protein in step (2) are as follows: collect neural stem cells overexpressing ARC protein, wash with PBS and resuspend in buffer containing protease inhibitors, disrupt the cells with ultrasound, collect cell membrane fragments by differential centrifugation, and store at -80℃ for later use.

[0028] The specific steps of microfluidic synthesis of RCD-LNP in step (3) are as follows: Ginsenoside Rg3, DOTAP (1,2-dioleoyl-3-trimethylammonium propane), DOPE (dioleoylphosphatidylethanolamine) and DHA (docosahexaenoic acid) are dissolved in ethanol as the organic phase, and CDNF circRNA is dissolved in citrate buffer as the aqueous phase. The organic phase and aqueous phase are rapidly mixed and self-assembled at a flow rate ratio of 1:3 using a microfluidic chip. The organic solvent is removed by ultrafiltration to obtain lipid nanoparticles (RCD-LNP) loaded with CDNF circRNA and ginsenoside Rg3.

[0029] The nucleotide sequence of CDNF circRNA is shown in SEQ ID NO.1.

[0030] In this embodiment, the molar percentage ratio of ginsenoside Rg3, DOTAP, DOPE, and DHA is: ginsenoside Rg3 45–55 mol%, DOTAP 30–35 mol%, DOPE 10–13 mol%, and DHA 4–6 mol%; preferably 50.3 mol% : 33 mol% : 11.7 mol% : 5 mol%; The mass ratio of total lipids to CDNF circRNA is (10-20):1; wherein, the mass of the total lipids is the sum of the masses of ginsenoside Rg3, DOTAP, DOPE and DHA used to prepare lipid nanoparticles.

[0031] The specific steps of the film extrusion method in step (4) are as follows: the extracted neural stem cell membrane overexpressing ARC is ultrasonically dispersed, the ultrasonic dispersion power is 100-300 W, and the ultrasonic time is 5-30 min; then it is extruded sequentially through polycarbonate membranes with pore sizes of 300-500 nm (preferably 400 nm) and 100-300 nm (preferably 200 nm) to obtain neural stem cell membrane vesicles, preferably by reciprocating extrusion 20 times each to obtain uniform neural stem cell membrane vesicles; The neural stem cell membrane vesicles and lipid nanoparticles are mixed at a mass ratio of (0.5-5):1 (preferably 2:1), and then extruded through a polycarbonate membrane with a pore size of 200-500 nm (preferably 400 nm) (preferably extruded for 20 cycles) to obtain lipid nanoparticles overexpressing ARC neural stem cell membrane-coated and loaded with CDNF circRNA and ginsenoside Rg3, namely ARC-RCD-LNP.

[0032] In this embodiment, the specific implementation steps of the preparation method include: S1 cell culture includes: (A) Cell thawing: Preheat the water bath to 36-38℃, preferably 37℃. Rapidly transfer the frozen human neuroblastoma cell lines SH-SY5Y, NSCs, and ARC-NSCs from liquid nitrogen to a 36-38℃ water bath, gently agitating until completely thawed. Then transfer to a biosafety cabinet, add the cell suspension to preheated complete culture medium, centrifuge at 800-1200 rpm for 3-8 min, preferably 1000 rpm for 5 min, discard the supernatant; resuspend in 2-5 mL of complete culture medium, preferably 3 mL, transfer to a T25 culture flask, and incubate in a cell culture incubator. (B) Cell passage: When cell confluence reaches 70-90%, preferably 80-90%, discard the culture medium, wash gently with PBS, add trypsin, and incubate at 36-38℃ for 1-5 min, preferably 37℃ for 2-3 min. After observing under a microscope that the cells have become rounded and begun to detach, immediately add complete culture medium to stop the digestion. Then centrifuge at 800-1200 rpm for 3-8 min, preferably 1000 rpm for 5 min, and discard the supernatant. Resuspend the cells and aliquot them into bottles at a ratio of 1:2-1:5, preferably 1:3, and continue culturing. (C) The cell digestion and centrifugation steps are the same as the cell passage steps. After discarding the supernatant, resuspend the cells in cell cryopreservation solution and aliquot 0.5-2 mL / tube into cryovials, preferably 1 mL / tube. After sealing, place in a pre-cooled gradient cooling box and freeze at -80℃ for 12-24 h, preferably overnight, then transfer to a liquid nitrogen tank for long-term storage.

[0033] Preparation of S2LNP: Lipid nanoparticles were prepared using a microfluidic method.

[0034] Rg3, DOTAP, and DOPE are dissolved in ethanol as the organic phase of RC-LNP; wherein, by total molar amount, the molar percentage of Rg3 is 50-60%, the molar percentage of DOTAP is 25-40%, the molar percentage of DOPE is 8-18%, and the sum of the molar percentages of the three is 100%; preferably, the molar ratio of Rg3, DOTAP, and DOPE is 55.3%:33%:11.7%. Rg3, DOTAP, DOPE, and DHA are dissolved in ethanol as the organic phase of RCD-LNP; wherein, by total molar amount, the molar percentage of Rg3 is 45-55%, the molar percentage of DOTAP is 25-40%, the molar percentage of DOPE is 8-18%, the molar percentage of DHA is 1-10%, and the sum of the molar percentages of the four is 100%; preferably, the molar ratio of Rg3, DOTAP, DOPE, and DHA is 50.3%:33%:11.7%:5%. The circRNA was dissolved in 10-100 mM citrate buffer as the aqueous phase, preferably in 50 mM citrate buffer. The total lipid to circRNA mass ratio was 5:1-30:1, preferably 10:1-20:1, and more preferably 15:1. The microfluidic chip was cleaned before synthesis. The chip was first cleaned with pure water, then the organic phase channel and the aqueous phase channel were cleaned with anhydrous ethanol and citrate buffer, respectively. The syringe pump parameters were set to mix the organic and aqueous phases at a flow rate of 1:2-1:10, preferably 1:3, 1:6, or 1:9 for synthesis screening, and more preferably at a flow rate of 1:3 for synthesizing RC-LNP and RCD-LNP. After synthesis, purification was performed using an ultrafiltration tube with a molecular weight cutoff of 50-150 KD, preferably a 100 KD ultrafiltration tube. Centrifuge at 2000-5000 g for 5-20 min, preferably at 3000 g for 10 min; then add 1×PBS to the original volume and ultrafilter again for 5-20 min, preferably 10 min. Repeat the above replenishment and ultrafiltration steps 2-5 times, preferably 3 times, to reduce the ethanol content to below 1%, preferably below 0.5%. Collect the ultrafiltered liquid and store at 2-8°C, preferably 4°C.

[0035] Extraction of S3-overexpressing ARC neural stem cell membrane: Cells were collected when the ARC-NSC confluence reached 70-90%, preferably 80%. The cells were washed 2-5 times with PBS, preferably 3 times. After centrifugation, the cells were resuspended in Hepes B buffer containing 0.5-2% protease inhibitor, preferably in Hepes B buffer containing 1% protease inhibitor. Cells were disrupted using an ultrasonic cell disruptor, with a single homogenization time of 1-5 min, a stop time of 0.5-2 min, and 10-30 cycles; preferably, homogenization for 2 min, stop time for 1 min, and 20 cycles. Subsequently, the cells were centrifuged at 8000-12000 rpm for 15-30 min in a refrigerated centrifuge at 0-8℃, preferably at 10000 rpm for 20 min at 4℃, and the supernatant was collected. The cell pellet was subjected to the same disruption process. All collected supernatants were centrifuged in a high-speed centrifuge at 0-8°C and 80,000-120,000 rpm for 20-60 min, preferably at 4°C and 100,000 rpm for 30 min. After discarding the supernatant, the cell membrane fragments were resuspended in Hepes C buffer, aliquoted, and stored at -80°C. The extraction of NSC cell membranes can also be performed using the above method.

[0036] Preparation of S4ARC-RCD-LNP: A neural stem cell membrane overexpressing ARC was placed in an ice-water bath and sonicated for 3-10 minutes at a frequency of 30-50 kHz; preferably, sonicated for 5 minutes at a frequency of 40 kHz, to uniformly disperse the cell membrane sheet in PBS. The cell membrane suspension was then extruded sequentially through polycarbonate membranes with pore sizes of 300-500 nm and 100-300 nm, preferably sequentially through polycarbonate membranes with pore sizes of 400 nm and 200 nm. Each type of polycarbonate membrane was extruded 10-30 times, preferably 20 times, to obtain neural stem cell membrane vesicles of uniform size. Next, the neural stem cell membrane vesicles were mixed with pre-prepared RCD-LNP at a protein-to-LNP mass ratio of 0.5:1-5:1, preferably 1:1-3:1, and more preferably 2:1. After mixing, the mixture is extruded through a polycarbonate membrane with a pore size of 200-500 nm for 10-30 cycles, preferably through a 400 nm polycarbonate membrane for 20 cycles, to obtain ARC-RCD-LNP.

[0037] This invention incorporates ginsenoside Rg3 as a cholesterol substitute into the lipid bilayer, which exerts anti-inflammatory and antioxidant neuroprotective activities while maintaining the stability of the lipid membrane structure. DHA is used as a ROS-responsive element; it maintains structural stability under normal physiological conditions. In the oxidative stress microenvironment of PD lesions, its carbon-carbon double bonds break in response to high concentrations of ROS, disrupting lipid membrane integrity and triggering the site-specific release of CDNF circRNA and Rg3, achieving on-demand drug release.

[0038] The ARC-RCD-LNP provided by this invention has three functions: the neural stem cell membrane overexpressing ARC protein in the outer coating endows it with the ability to specifically recognize and target damaged dopaminergic neurons; the DHA in the lipid core provides ROS-responsive drug release characteristics, ensuring that the drug remains stable before reaching the lesion and is accurately released at the target site; the released CDNF circRNA continuously and efficiently expresses CDNF protein in neurons through strong stability and high expression, directly binds to α-Syn to inhibit its pathological aggregation, and synergistically exerts anti-inflammatory and neuronal damage repair effects with ginsenoside Rg3.

[0039] Example 2: A CDNF / Rg3-loaded biomimetic lipid nanoparticle was prepared using the method described in Example 1. The surface of the CDNF / Rg3-loaded biomimetic lipid nanoparticle is coated with a neural stem cell membrane; the cell membrane carries ARC protein; the interior of the CDNF / Rg3-loaded biomimetic lipid nanoparticle is loaded with CDNF circRNA and ginsenoside Rg3. The CDNF / Rg3-loaded biomimetic lipid nanoparticles exhibit ROS-responsive drug release properties.

[0040] Example 3: An application of the CDNF / Rg3-loaded biomimetic lipid nanoparticles described in Example 2 in the prevention or treatment of Parkinson's disease.

[0041] Specifically, the CDNF / Rg3-loaded biomimetic lipid nanoparticles are delivered into the brain via nasal delivery. Under the action of the carried ARC protein, the CDNF / Rg3-loaded biomimetic lipid nanoparticles target and bind to damaged dopaminergic neurons and are taken up by endocytosis. In response to the elevated ROS microenvironment in the cell, they release ginsenoside Rg3 and CDNF circRNA. CDNF circRNA is translated into functional CDNF protein, which directly binds to α-Syn to inhibit its pathological aggregation. Furthermore, CDNF circRNA and ginsenoside Rg3 synergistically exert anti-inflammatory effects and repair damaged neurons.

[0042] Validation of NSC cell membranes overexpressing ARC and ARC-RCD-LNP, and functional validation in in vitro cell models and in vivo PD mouse models: The successfully constructed NSC cells overexpressing ARC were validated by Western blot. The prepared RC-LNP, RCD-LNP, and ARC-RCD-LNP were validated. The encapsulation status, encapsulation efficiency, and drug loading rate of CDNF circRNA were verified by agarose gel electrophoresis and the RiboGreen assay. The encapsulation efficiency and drug loading rate of Rg3 were verified by HPLC. The drug release characteristics of ARC-RCD-LNP under ROS conditions were verified. Simultaneously, the uptake capacity of ARC-RCD-LNP in the MPTP-induced SH-SY5Y cell model was verified by laser confocal microscopy. The effects of ARC-RCD-LNP on neurotrophic factors, TH, DA, and inflammatory factors in MPTP-induced SH-SY5Y cells were assessed by immunofluorescence staining and ELISA. The effect of ARC-RCD-LNP on neuronal damage was assessed by immunofluorescence staining. Furthermore, in vivo imaging experiments in small animals were used to verify the level of ARC-RCD-LNP entering the brain of PD mice after intranasal administration and its distribution in brain tissue. Behavioral experiments (rotating bar, extreme point, and suspension tests) were used to assess the recovery of motor function in PD mice. Western blotting was used to detect TH levels in the substantia nigra, and immunofluorescence staining was used to detect striatal α-Syn clearance and neuronal damage repair. ELISA was used to detect striatal DA, CDNF, and inflammatory factor levels; H&E staining was used to assess the biocompatibility of the nanoparticles.

[0043] Results of Western blot detection of ARC protein expression: In this invention, genetic engineering technology was used to assemble a recombinant ARC plasmid that can target and recognize neurons; subsequently, it was transfected into NSC cells, and a stable integrated ARC-NSC cell line was established through resistance selection. Figure 1 Western blot data showed that, compared with NSC cells and negative controls, the ARC-NSC group showed a clear band at approximately 60 kDa, confirming the efficient expression of exogenous ARC protein and suggesting the successful construction of a neuron-specific targeted ARC-NSC cell model.

[0044] Particle size and Zeta potential of RC-LNP, RCD-LNP, and ARC-RCD-LNP: Three different groups of lipid nanoparticles were synthesized at an organic phase to aqueous phase flow ratio of 1:3. Figures 2-3As shown, DLS characterization revealed that the particle size increased with the increase of liposome components. Specifically, the average hydrodynamic diameters of ginsenoside Rg3 liposomes loaded with CDNF circRNA (RC-LNP), RC-LNPs with added DHA (RCD-LNP), and RCD-LNPs encapsulating neural stem cell membranes overexpressing ARC (ARC-RCD-LNP) were approximately 108.3 nm, 115.7 nm, and 132.2 nm, respectively. Due to the loading of CDNF circRNA, the zeta potentials of the three groups of liposome nanoparticles were negative, approximately -6.32 mV, -5.23 mV, and -5.58 mV, respectively.

[0045] Preparation and characterization of ARC-RCD-LNP: The drug loading and encapsulation efficiency of Rg3 in ARC-RCD-LNP were determined by high performance liquid chromatography (HPLC). The results are as follows: Figure 4 As shown, the encapsulation efficiency was approximately 85.6%, and the drug loading rate was approximately 23.8%. The encapsulation of CDNF circRNA in ARC-RCD-LNP was detected by agarose gel electrophoresis, and the results are as follows: Figure 5 As shown, free CDNF circRNA (Lane 1) exhibited a distinct band, while CDNF circRNA encapsulated in liposomes (Lanes 2, 3, and 4) showed no distinct band. Furthermore, the drug loading and encapsulation efficiency of CDNF circRNA in ARC-RCD-LNP were detected using the RiboGreen method, and the results are as follows. Figure 6 As shown, the encapsulation efficiency was approximately 77.9%, and the drug loading rate was approximately 1.1%. These results indicate that the drug was successfully loaded into liposome nanoparticles.

[0046] Cumulative release of CDNF circRNA from ARC-RCD-LNP under 1mM H2O2 conditions: To investigate the release of CDNF circRNA from ARC-RCD-LNP under ROS conditions, PBS (pH 7.4) and 1mM H2O2 were used as the release medium, and drug release curves were constructed using the RiboGreen method. The results are as follows: Figure 7As shown, after 2 h of H2O2 treatment, the C-carbon double bond breakage of DHA in ARC-RCD-LNP exhibits ROS-controlled drug release characteristics. After 24 h of H2O2 treatment, 81.56% of CDNF circRNA was released from ARC-RCD-LNP. In contrast, under PBS conditions, only 16.58% of CDNF circRNA was released from ARC-RCD-LNP after 24 h. This ROS-responsive drug release ensures the stability of ARC-RCD-LNP during its entry into the bloodstream and its precise release upon reaching the brain.

[0047] Fluorescence images and quantitative data of lipid nanoparticle uptake by SH-SY5Y cells: ARC proteins self-assemble into virus-like capsids to encapsulate mRNAs, which are then translocated to receptor neurons via receptor-mediated endocytosis. Therefore, ARC-RCD-LNPs were prepared using the neuronal targeting properties of ARC proteins. The uptake of different lipid nanoparticles by SH-SY5Y cells was detected using laser confocal microscopy. The results are shown below. Figure 8A - Figure 8B As shown, compared with RC-LNP and RCD-LNP, SH-SY5Y cells had the highest uptake efficiency of ARC-RCD-LNP, which is a lipid nanoparticle coated with an overexpression of ARC neural stem cells. This indicates that ARC-RCD-LNP has good neuronal targeting.

[0048] ARC-RCD-LNP restores neurotrophic factor and TH levels in PD cell models: CDNF expression levels were detected by ELISA, and the results are as follows. Figure 9 As shown, compared with the control group, the expression level of CDNF was significantly decreased after MPTP modeling. After treatment with different lipid nanoparticles, the content of CDNF in cells gradually recovered. Notably, the CDNF expression level in the ARC-RCD-LNP group was basically consistent with that in the control group. TH level represents the degree of dopaminergic neuron damage. TH level was detected by Western blot, and the results are as follows... Figure 10 As shown, compared to the control group, neurons were damaged and TH levels were significantly lower after MPTP modeling. Compared to the model group, TH levels were reversed after treatment with different lipid nanoparticles, with the ARC-RCD-LNP group showing the highest TH expression level.

[0049] Effects of ARC-RCD-LNP on inflammatory factors in a PD cell model: The expression levels of pro-inflammatory cytokine TNF-α and anti-inflammatory cytokine IL-10 were detected by immunofluorescence staining, and the results are as follows: Figures 11A-11B , Figures 12A-12BAs shown, compared with the control group, the model group had higher fluorescence intensity of the pro-inflammatory factor TNF-α and lower fluorescence intensity of the anti-inflammatory factor IL-10. Compared with the model group, the fluorescence intensity of the pro-inflammatory factor TNF-α was significantly reduced and the fluorescence intensity of the anti-inflammatory factor IL-10 was significantly increased after treatment with different lipid nanoparticles. Notably, ARC-RCD-LNP showed the best therapeutic effect.

[0050] Real-time fluorescence and quantitative analysis in PD model mice: Since specific targeting of the brain is a prerequisite for the treatment of central nervous system diseases, this study investigated the brain targeting ability of different drug-loaded nanoparticles in MPTP-induced PD model mice. Three groups of drug-loaded nanoparticles (RC-LNP, RCD-LNP, and ARC-RCD-LNP) were labeled with Cy7 dye, and fluorescence at key time points (0-48 h) after intranasal instillation was monitored using a small animal in vivo imaging system. Results are as follows: Figure 13 As shown, all groups reached their fluorescence signal peak at 6 h, and then the fluorescence signal gradually decreased over time. Notably, compared with the RC-LNP and RCD-LNP groups, the ARC-RCD-LNP group had the highest fluorescence signal at 6 h, indicating that the liposomes encapsulated by neural stem cells overexpressing ARC have good neuronal targeting in the brain.

[0051] Behavioral testing in PD mice: The main clinical symptom of Parkinson's disease is motor dysfunction. Therefore, suspension, rotating bar, and pole climbing tests were performed to analyze the spontaneous movement and motor coordination abilities of PD model mice. The results are as follows: Figure 14 As shown. The suspension test was designed to assess the muscle endurance of mice. Compared with the control group, the suspension score of the model group was significantly lower, and the suspension score increased after treatment with different nanoparticles. The rotating bar test was designed to quantitatively assess the motor coordination and balance of mice. The fall latency on the accelerated rotating bar was used to objectively quantify the motor deficits caused by damage to dopaminergic neurons in the substantia nigra. Compared with the control group, the motor coordination of mice after MPTP modeling decreased, as evidenced by a significant reduction in the time spent on the rotating bar. The motor function of mice treated with different nanoparticles was significantly better than that of the model group. Similarly, the pole climbing test, a classic and simple behavioral test, was also used to quantitatively assess the motor coordination and balance of PD model mice. Mice in the MPTP modeling group took about 20 seconds to climb to the bottom of the pole, and this time was significantly reduced after treatment. In summary, these results indicate that the motor coordination of PD mice was improved after treatment. Consistent with the in vitro findings of neuroprotection, the ARC-RCD-LNP group showed the best treatment effect.

[0052] ARC-RCD-LNP restored TH levels and neuronal damage in PD mice: Furthermore, the relative expression level of TH in the mouse striatum was detected by Western blotting. Results are as follows... Figures 15A-15B As shown, MPTP induced a decrease in TH expression in the striatum of mice, and the TH expression level was reversed after treatment with different lipid nanoparticles. To evaluate the effect of ARC-RCD-LNP on α-Syn in PD mice, α-Syn in the striatum region of the brain of each group of mice was labeled by immunofluorescence staining. The results are as follows: Figures 16A-16B As shown, compared to the control group, mice modeled with MPTP exhibited a large amount of abnormal α-Syn aggregation, and the α-Syn content was reversed after treatment with different lipid nanoparticles. Notably, the ARC-RCD-LNP group showed the most significant decrease in α-Syn levels. This is attributed to the release of CDNF circRNA under oxidative stress conditions after ARC-RCD-LNP precisely targets neurons. CDNF can reduce the uptake of pre-formed α-Syn by neurons and block α-Syn dimerization.

[0053] ARC-RCD-LNP restores neurotrophic factor and dopaminergic levels in PD mice: Figure 17 As shown, key downstream products and intuitive quantitative indicators (DA) for evaluating TH function were further investigated. Compared to the control group, dopamine levels in the striatum of mice were significantly reduced after MPTP modeling, and these reductions were reversed after treatment with different lipid nanoparticles. Furthermore, the expression levels of CDNF in the striatum of mice from different groups were examined. The model group had the lowest CDNF levels among all groups. Treatment with RC-LNP, RCD-LNP, and ARC-RCD-LNP significantly promoted CDNF expression, and the CDNF level in the ARC-RCD-LNP group approached that of the control group after treatment.

[0054] Immunofluorescence and quantitative analysis of Neun in the striatum of PD mice: To further evaluate the damage to dopamine neurons after the removal of the abnormal aggregate protein α-Syn in the ARC-RCD-LNP group, Neun in the striatum region of each group of mice was labeled by immunofluorescence staining. Results are as follows: Figures 18A-18B As shown, treatment with RC-LNP, RCD-LNP, and ARC-RCD-LNP groups can repair neuronal damage after MPTP modeling.

[0055] Expression levels of IL-6 in the striatum of PD mice after treatment with different lipid nanoparticles: Figure 19 The study showed that treatment with different lipid nanoparticles significantly reduced IL-6 expression levels and inhibited the cascade effect of neuroinflammation in neuronal cell death events.

[0056] H&E staining images of major organs in mice of each group: To evaluate the safety of different lipid nanoparticles, H&E staining was performed on the heart, liver, spleen, lung, and kidney tissues of mice in each group. Results are as follows: Figure 20 As shown, the heart, liver, spleen, lung, and kidney tissues of mice in each group were similar to those in the normal group; the cells were uniform in size, with visible nuclei, and no signs of inflammatory cell infiltration or necrosis were observed. These results demonstrate that different lipid nanoparticles have good safety profiles in vivo.

[0057] In summary, this invention comprehensively elucidates a novel biomimetic nanomedicine therapy strategy through material design and characterization, as well as in vitro and in vivo experimental verification. ARC-RCD-LNP, through targeted design and multi-mechanism synergy, enables systematic research in PD models from the molecular and cellular levels to the mouse behavioral level, providing new insights for developing PD nanomedicine combination therapy regimens.

[0058] The ARC-RCD-LNP of this invention can be further developed into a biomimetic nanoparticle formulation for nasal administration of Parkinson's disease, in dosage forms such as nasal sprays, nasal drops, or lyophilized and reconstituted formulations. Its lipid nanoparticle core can be continuously prepared using microfluidic technology, and the neural stem cell membrane overexpressing ARC can be obtained through standardized cell culture, membrane extraction, and co-extrusion processes, providing a foundation for further scale-up production and quality control. By establishing a quality evaluation system encompassing particle size, potential, membrane coating efficiency, ARC protein expression, drug loading, encapsulation efficiency, ROS-responsive drug release, and nasal administration stability, it is hoped that a naso-brain delivery product for Parkinson's disease can be developed, combining neuronal targeting, lesion-responsive release, and multi-mechanism synergistic therapeutic effects.

[0059] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, modifications or variations can still be made to the technical solutions described above, and these modifications and variations all fall within the protection scope of the present invention.

Claims

1. A method for preparing CDNF / Rg3-loaded biomimetic lipid nanoparticles, characterized in that, The method includes: Neural stem cells overexpressing the ARC gene were constructed, and the cell membranes of the neural stem cells were extracted; lipid nanoparticles loaded with CDNF circRNA and ginsenoside Rg3 were synthesized using microfluidic technology. The cell membrane of the neural stem cells was coated onto the surface of the lipid nanoparticles using a thin-film extrusion technique to obtain CDNF / Rg3-loaded biomimetic lipid nanoparticles.

2. The preparation method according to claim 1, characterized in that, The method for constructing neural stem cells overexpressing the ARC gene includes: transfecting neural stem cells with a lentiviral overexpression vector, obtaining a stable integrated ARC-NSC cell line through resistance selection, wherein the ARC-NSC cell line is capable of overexpressing the ARC gene.

3. The preparation method according to claim 1, characterized in that, The preparation method of the lipid nanoparticles includes: dissolving ginsenoside Rg3, DOTAP, DOPE and DHA in ethanol as the organic phase, dissolving CDNF circRNA in citrate buffer as the aqueous phase, mixing the organic phase and the aqueous phase for self-assembly using a microfluidic chip, and removing the organic solvent by ultrafiltration to obtain lipid nanoparticles loaded with CDNF circRNA and ginsenoside Rg3; wherein the nucleotide sequence of CDNF circRNA is shown in SEQ ID NO.

1.

4. The preparation method according to claim 3, characterized in that, The molar percentage ratios of ginsenoside Rg3, DOTAP, DOPE, and DHA are as follows: ginsenoside Rg3 45–55 mol%, DOTAP 30–35 mol%, DOPE 10–13 mol%, and DHA 4–6 mol%. The mass ratio of total lipids to CDNF circRNA is (10-20):1; wherein, the mass of the total lipids is the sum of the masses of ginsenoside Rg3, DOTAP, DOPE and DHA used to prepare lipid nanoparticles.

5. The preparation method according to claim 1, characterized in that, The method for extruding the film is as follows: the extracted neural stem cell membrane overexpressing ARC is ultrasonically dispersed at a power of 100–300 W for 5–30 min; then it is extruded sequentially through polycarbonate membranes with pore sizes of 300–500 nm and 100–300 nm to obtain neural stem cell membrane vesicles; the neural stem cell membrane vesicles are mixed with lipid nanoparticles at a mass ratio of (0.5–5):1, and then extruded through a polycarbonate membrane with a pore size of 200–500 nm to obtain lipid nanoparticles loaded with CDNF circRNA and ginsenoside Rg3 coated with ARC neural stem cell membrane, namely ARC-RCD-LNP.

6. The preparation method according to claim 5, characterized in that, The neural stem cell membrane vesicles and lipid nanoparticles are mixed at a membrane protein mass to LNP mass ratio of (1.5-2.5):1, preferably 2:1; wherein the membrane protein mass is the membrane protein mass of the neural stem cell membrane vesicles as determined by protein quantification, and the LNP mass is the mass of the lipid component in the lipid nanoparticles.

7. A CDNF / Rg3-loaded biomimetic lipid nanoparticle, prepared by the method according to any one of claims 1-6, characterized in that, The surface of the CDNF / Rg3-loaded biomimetic lipid nanoparticles is coated with the cell membrane of neural stem cells; the cell membrane carries ARC protein; the interior of the CDNF / Rg3-loaded biomimetic lipid nanoparticles is loaded with CDNF circRNA and ginsenoside Rg3; the CDNF / Rg3-loaded biomimetic lipid nanoparticles have ROS-responsive drug release characteristics.

8. The application of the CDNF / Rg3-loaded biomimetic lipid nanoparticles according to claim 7, characterized in that, Application of the CDNF / Rg3-loaded biomimetic lipid nanoparticles in drugs for the prevention or treatment of neurodegenerative diseases.

9. The application according to claim 8, characterized in that, The CDNF / Rg3-loaded biomimetic lipid nanoparticles were delivered into the brain via nasal delivery. Under the action of the carried ARC protein, the CDNF / Rg3-loaded biomimetic lipid nanoparticles targeted and bound to damaged dopaminergic neurons and were taken up by endocytosis. In the ROS microenvironment, they released ginsenoside Rg3 and CDNF circRNA in response. CDNF circRNA was translated into functional CDNF protein, which directly bound to α-Syn to inhibit its pathological aggregation. Furthermore, CDNF circRNA and ginsenoside Rg3 synergistically exerted anti-inflammatory effects and repaired damaged neurons.

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