A pPAM long-circulating liposome, a preparation method and application thereof

Abstract

The application discloses a pPAM long-circulating liposome, a preparation method and application thereof, and the long-circulating liposome takes a blank liposome as a carrier, internally carries a plasmid pPAM, and is decorated on the surface with a transferrin-penetratin complex Tf-Pen and an oxytocin receptor antibody OTR Ab； The plasmid pPAM carries a cDNA sequence of a peptide acylglycine alpha amidation monooxygenase PAM; and the blank liposome is composed of DSPC, PEG 2000 , cholesterol and lecithin. The application provides a new direction for development of endogenous polypeptide synthesis enzyme delivery technology and mental drugs, and improves the possibility of preparing new anti-anxiety and depression drugs, relieving mental stress and improving patient compliance.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to a pPAM long-circulating liposome, its preparation method and its application. Background Technology

[0002] Oxytocin is one of the first polypeptide hormones to be isolated and sequenced. It is a nonapeptide containing two cysteine ​​residues, forming a disulfide bond between positions 1 and 6. Its molecular formula is NH2-Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-CONH2, and it has various derivatives, such as carbetocin and arginine vasopressin. This polypeptide is synthesized via the hypothalamus, stored in the posterior pituitary gland, and released when needed. It stimulates certain smooth muscle cells, constricts certain blood vessels, and increases the sensitivity of other hormones and nerves—this is its permissive effect. This hormone has a short lifespan in the body and a rapid onset of action. Tissues in which it can act include, but are not limited to, the uterus, vagina, breast, seminal vesicles, and corpora cavernosa of the penis. Oxytocin has specific effects on promoting uterine muscle contractions during labor and orgasm, promoting vasoconstriction to reduce placental separation bleeding, and the milk-letting reflex in breastfeeding mothers upon hearing their baby cry. Currently, oxytocin is indicated for stimulating uterine contractions to induce labor, controlling postpartum hemorrhage after placental delivery, and stimulating lactation. Recent reports suggest that peptide oxytocin and its analogue arginine vasopressin, in addition to their effects on the reproductive system, may also affect the central nervous system and play a role in stress regulation. Therefore, oxytocin is effective in treating or preventing various diseases and controlling their conditions, and its applications can be expanded accordingly.

[0003] In the maturation, synthesis, and secretion of oxytocin, peptidylglycine α-amidating monooxygenase (PAM, enzyme code EC 1.14.17.3) plays a crucial catalytic role, primarily responsible for C-terminal amidation modification: Oxytocin is initially synthesized as a prepro-oxytocin, which is cleaved to generate a glycine-extended intermediate. At this point, PAM plays a crucial role, first hydroxylating the C-terminal glycine residue, followed by cleavage to remove glycine, thereby generating the α-amidated mature peptide, the final product being biologically active oxytocin. This is an essential step in the conversion of oxytocin to its active form. Amidation enhances the peptide's resistance to degradation and improves its stability. During its action, it enhances receptor affinity, promoting receptor binding and facilitating its function. It is also the rate-limiting enzyme in oxytocin synthesis in vivo, controlling the final synthesis step of oxytocin. Tf is a serum glycoprotein that is abundant in brain capillary endothelial cells. It can promote iron entry into the brain through receptor-mediated endocytosis. Therefore, modifying drug carriers with Tf is a promising brain-targeted delivery strategy.

[0004] Unfortunately, natural oxytocin has a short half-life and is rapidly eliminated in its original form. Therefore, it is necessary to develop drug formulations that can increase the duration of action of oxytocin to reduce the frequency of administration and improve patient compliance. However, to date, apart from short-acting aqueous solutions for intramuscular injection, intravenous infusion, or nasal spray, there are no clinically available controlled-release formulations of oxytocin or its analogues. The development of oxytocin synthase PAM as a prodrug sustained-release formulation will provide improved treatment options for treatment of its matching indications. The main routes of nasal administration to the brain are: (1) through the intraaxonal and transneuronal transport mechanism of the olfactory pathway, i.e., the balloon pathway, directly through the lymphatic fluid into the cerebrospinal fluid and brain; (2) after passing through the nasal mucosa wall, through peripheral blood flow and the blood-brain barrier (BBB); (3) through the peripheral nerves and other spaces along olfactory fibers and other intracranial nerves into the arachnoid space and the cerebrospinal fluid around the brain; (4) the trigeminal nerve pathway to the brainstem. Oxytocin administered via conventional intravenous injection has a half-life of only two minutes, while nasal administration increases its half-life in cerebrospinal fluid to 28 minutes, significantly prolonging its duration of action. Cell-penetrating peptides (Pen) are short peptides that mediate the entry of biomolecules or drug carriers into cells. Pen modification strategies can effectively improve the intracellular delivery efficiency of drugs. Studies have shown that Pen can significantly increase the permeability of nanocarriers in the olfactory epithelium during nasal administration to the brain, demonstrating great potential in promoting the nasal delivery of macromolecules to the brain. Liposomes show promising applications in nasal administration to the brain, as they can cross the blood-brain barrier well, and Pen modification can effectively improve their brain distribution. Transferrin (Tf), also known as serum transferrin, is a glycoprotein mainly produced by the liver. It has two iron-binding sites, each capable of binding to one iron ion, and acts as a ligand that promotes receptor-mediated endocytosis. When Pen and Tf combine, they can form a complex ligand Tf-Pen that effectively crosses the blood-brain barrier, allowing the formulation to successfully cross the blood-brain barrier and enter the central nervous system.

[0005] Therefore, based on the above reasons, designing and constructing novel penetrant long-circulating liposomes to deliver oxytocin synthase PAM across the blood-brain barrier, enabling it to function in the central nervous system, regulate emotions, and achieve sustained-release and targeted drug delivery, has significant positive implications for modern society. Summary of the Invention

[0006] The purpose of this application is to provide a pPAM long-circulating liposome, its preparation method, and its application, in order to solve one or more technical problems existing in the prior art, or at least provide a beneficial option or create conditions.

[0007] To achieve the above-mentioned objectives, this application employs the following technical solution:

[0008] In a first aspect, this application provides a pPAM long-circulating liposome, wherein the long-circulating liposome uses a blank liposome as a carrier, internally encapsulates the plasmid pPAM, and is surface-modified with a transferrin-penetrin conjugate complex (Tf-Pen) and an oxytocin receptor antibody (OTR). Ab ;

[0009] The plasmid pPAM carries the cDNA sequence of peptidylglycine α-amidyl monooxygenase PAM.

[0010] The blank liposomes were composed of distearate phosphatidylcholine (DSPC) and polyethylene glycol 2000 (PEG). 2000 Cholesterol and lecithin.

[0011] Furthermore, the penetrant includes the unmodified subtype Pen1 and the capped analog subtype Pen2;

[0012] The amino acid sequence of the unmodified subtype Pen1 is SEQ ID: No. 1;

[0013] The amino acid sequence of the capped analog subtype Pen2 is SEQ ID: No. 2.

[0014] Furthermore, the oxytocin receptor antibody OTR Ab This includes natural polyclonal antibodies, artificially synthesized polyclonal antibodies, and monoclonal antibodies.

[0015] Furthermore, the long-circulating liposomes contain DSPC-PEG 2000 The molar ratio of cholesterol to total cholesterol is 0.5:1 to 2:1;

[0016] The DSPC-PEG 2000 The molar percentage in liposomes is 0.5–3 mol%;

[0017] Using maleimide (Mal) as the coupling medium, the DSPC-PEG 2000 -Mal and transferrin-penetrin conjugate complex Tf-Pen, oxytocin receptor antibody OTR Ab The coupling molar ratio is 1:1 to 1:10.

[0018] Secondly, this application provides a method for preparing pPAM long-circulating liposomes, the method comprising:

[0019] The liposome suspension was subjected to ultrasonic treatment under a set temperature condition to obtain an activated liposome suspension.

[0020] At room temperature, activated liposome suspension was reacted with thiol-modified oxytocin receptor antibody OTR. Ab Mix thoroughly and react;

[0021] Transferrin-penetin conjugate complex Tf-Pen, oxytocin receptor antibody OTR Ab Mixed with N-succinimide-3-(2-pyridyldithio)propionate SPDP;

[0022] Control of N-succinimide-3-(2-pyridyldithio)propionate SPDP and oxytocin receptor antibody OTR Ab The molar ratio is used to complete the coupling modification reaction;

[0023] The reaction system was separated and purified to remove free unreacted impurities, and pPAM long-circulating liposomes were prepared by plasmid encapsulation process, followed by freeze-drying and vacuum storage.

[0024] Furthermore, the liposome suspension was subjected to ultrasonic treatment at 60 times power for 5 min at 50–70 °C to obtain an activated liposome suspension.

[0025] Further, 1–10 μg of transferrin-penetrin conjugate complex (Tf-Pen) and 5–50 μg of oxytocin receptor antibody (OTR) were added. Ab Mix with 5-15 mg / mL of N-succinimide-3-(2-pyridyldithio)propionate.

[0026] Furthermore, controlling the interaction between N-succinimide-3-(2-pyridyldithio)propionate (SPDP) and oxytocin receptor antibody OTR Ab The molar ratio is 5:1 to 50:1.

[0027] Furthermore, the reaction system was separated and purified using a PD-10 chromatographic column equilibrated with distilled water.

[0028] Thirdly, this application also provides the application of the above-mentioned pPAM long-circulating liposomes in the preparation of nanomedicines to relieve mental stress in animals.

[0029] The beneficial effects of this application are as follows:

[0030] In this application, long-circulating liposomes serve as a drug reservoir. The hydrophilic oxytocin-like drug synthase is encapsulated within a phospholipid bilayer, effectively avoiding side effects caused by free prodrugs during blood circulation and exhibiting good biocompatibility. The long-circulating liposomes possess hydrophilic properties and modify surface potential, effectively prolonging the blood circulation time of the formulation and reducing its clearance rate by cerebrospinal fluid circulation, resulting in a long in vivo circulation time.

[0031] The long-circulating liposomes of this application have a long retention effect, which can enable the drug to accumulate in the central nervous system after nasoencephalectomy, effectively prolonging the half-life.

[0032] This application utilizes a penetrant reaction design to ensure the successful crossing of the blood-brain barrier by oxytocin. Penetrants, as short peptides that mediate the entry of biomolecules or drug carriers into cells, can be modified to effectively improve intracellular drug delivery efficiency, significantly increasing the permeability of nanocarriers in the olfactory epithelium during intranasal administration to the brain. Liposomes show promising application prospects for intranasal administration to the brain, exhibiting good blood-brain barrier penetration, and penetrant modification can effectively enhance brain distribution.

[0033] The liposomes have a particle size of approximately 190±20 nm, exhibiting uniform size and good dispersibility. The liposome nanoparticles have a potential of approximately -38.5 mV; this negative potential effectively prolongs the circulation time of the formulation in vivo, reduces its clearance rate by cerebrospinal fluid circulation, and extends its central nervous system circulation time. The particle size and zeta potential of the suspension did not show significant changes within 7 days, demonstrating good stability.

[0034] The synthesis method described in this application is simple, the materials are readily available, the cost is low, and it does not require a large amount of manpower and resources. Attached Figure Description

[0035] Figure 1 For the liposomes (pPAM@Tf-Pen-OTR) provided according to the embodiments of this application Ab A schematic diagram of the molecular structure of LCLipo;

[0036] Figure 2 The images show two types of liposome electron micrographs provided according to embodiments of this application (A: liposomes carrying free oxytocin; B: liposomes carrying pPAM plasmids).

[0037] Figure 3 The following are schematic diagrams illustrating different morphological states of liposomes according to embodiments of this application (A: freshly prepared liposomes; B: lyophilized liposomes; C: lyophilized and reconstituted liposomes).

[0038] Figure 4 This is a graph showing the changes in particle size and zeta potential stability of liposomes during 7 days of storage according to an embodiment of this application.

[0039] Figure 5 This is a graph showing the cumulative in vitro release curve of pPAM plasmid in liposomes according to the embodiments of this application;

[0040] Figure 6 This is a graph showing the biocompatibility results of hemolysis experiments at different concentrations of liposomes according to the embodiments of this application;

[0041] Figure 7This is a graph showing the comparison of oxytocin half-life in the rat brain after administration of different oxytocin preparations according to the embodiments of this application;

[0042] Figure 8 This is a schematic diagram of the Vogel drinking water electric shock stress experiment for rats according to the embodiments of this application, and a graph showing the anxiety scores of rats in each group. Detailed Implementation

[0043] Various exemplary embodiments of this application are now described in detail. This detailed description should not be considered as a limitation of this application, but rather as a more detailed description of certain aspects, features, and implementations of this application.

[0044] It should be understood that the terminology used in this application is merely for describing particular implementations and is not intended to limit the application. Furthermore, for numerical ranges in this application, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this application. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0045] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art described herein. While only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this application. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0046] Unless otherwise specified, all materials and instruments used in this application are commonly purchased by those skilled in the art, and all methods used are well-known to those skilled in the art. Unless otherwise specified, "room temperature" in this application refers to 25±2℃.

[0047] In the embodiments of this application, the structure of the obtained liposomes is as follows: Figure 1 As shown, after PEG 2000 The modified liposomes have Tf-Pen and OTR attached to their surface. Ab The fragment, a plasmid internally loaded with PAM (pPAM), can cross the blood-brain barrier and enter the central nervous system to exert its effects after nasal delivery.

[0048] The two types of liposomes from examples (1) and (4) that have been prepared were collected, and their morphology was observed and recorded by electron microscopy. The results are as follows: Figure 2 As shown, Figure 2A is a complex liposome encapsulating oxytocin (OT@OT@Tf-CPP-OTR). Ab -LCLipo). Figure 2 B is a complex liposome (pPAM@Tf-CPP-OTR) carrying a peptidylglycine-α-amidyl monooxygenase plasmid. Ab -LCLipo).

[0049] Figure 3 It is in liposome form. Figure 3 A represents freshly prepared liposomes. Figure 3 B represents lyophilized liposomes. Figure 3 Liposomes reconstructed after C-site freeze-drying.

[0050] Figure 4 The results of stability tests on the particle size and zeta potential of the nanoparticles show that the particle size and zeta potential of the liposomes are stable within seven days.

[0051] Figure 5 This experiment measured the cumulative release of liposomes in PBS, with a molecular weight cutoff of 100 kDa. The liposomes were incubated at 37 °C with continuous shaking at 50 rpm. At predetermined time intervals, 300 μL of the suspension was collected and centrifuged at 5,000 rpm for 30 minutes at 4 °C. DNA quantification was used to determine the release rate, and the cumulative release of pPAM from the liposomes was calculated.

[0052] Figure 6 The results of the hemolysis experiment showed that the hemolytic effect of liposomes at different concentrations was tested. The results indicated that the liposomes exhibited low toxicity and good tissue compatibility at phospholipid concentrations below 800 nM.

[0053] Figure 7 The test of the brain half-life of oxytocin after nasal administration revealed that administration using its synthase plasmid resulted in a longer half-life than administration using its peptide, while the use of transmembrane peptide long-circulating liposomes significantly prolonged the half-life of oxytocin in the central nervous system.

[0054] Figure 8 The results of the Vogel drinking water electric shock test on rats after oxytocin liposomes were applied showed that the long circulating liposomes carrying the transmembrane peptide pPAM greatly improved the anxiety symptoms of rats and reduced their mental stress scores.

[0055] In a first aspect, this application provides a pPAM long-circulating liposome, wherein the long-circulating liposome uses a blank liposome as a carrier, internally encapsulates the plasmid pPAM, and is surface-modified with a transferrin-penetrin conjugate complex (Tf-Pen) and an oxytocin receptor antibody (OTR). Ab ;

[0056] The plasmid pPAM carries the lentiviral cDNA sequence of peptidylglycine α-amidyl monooxygenase PAM, and the species origin includes, but is not limited to, humans, mice, monkeys, rats, etc.

[0057] The blank liposomes are composed of DSPC and PEG. 2000 Cholesterol and lecithin.

[0058] As a preferred embodiment of this application, the penetrant includes the unmodified subtype Pen1 and the capped analog subtype Pen2;

[0059] The amino acid sequence of the unmodified subtype Pen1 is SEQ ID: No. 1;

[0060] The amino acid sequence of the capped analog subtype Pen2 is SEQ ID: No. 2.

[0061] As a preferred embodiment of this application, the oxytocin receptor antibody (OTR) Ab This includes both natural and synthetic polyclonal and monoclonal antibodies.

[0062] As a preferred embodiment of this application, the long-circulating liposomes contain DSPC-PEG. 2000 The molar ratio of cholesterol to total cholesterol is 0.5:1 to 2:1;

[0063] The DSPC-PEG 2000 The molar percentage in liposomes is 0.5–3 mol%;

[0064] Using maleimide (Mal) as the coupling medium, the DSPC-PEG 2000 -Mal and transferrin-penetrin conjugate complex Tf-Pen, oxytocin receptor antibody OTR Ab The coupling molar ratio is 1:1 to 1:10.

[0065] Secondly, this application provides a method for preparing pPAM long-circulating liposomes, comprising:

[0066] Example 1: Preparation of pPAM@Tf-Pen-OTRAb-LCLipo (Formula 1)

[0067] (1) Lentiviral vector plasmids containing PAM (pPAM) cDNA are amplified and isolated in Escherichia coli. Then, according to the instructions, the corresponding plasmids are prepared using the GenCatch™ Plasmid DNA Kit (Epoch Life Science). The concentration of pPAM plasmids is determined and then frozen for later use. The plasmid concentration is generally 200-500 ng / μL.

[0068] (2) DSPE-PEG 2000 Synthesis of -Pen: DSPE-PEG at a molar concentration of 300 μM was used. 2000 -NHS and 100 μM Pen were dissolved in anhydrous dimethylformamide (DMF), and the pH was adjusted to 8.5 ± 0.1 with triethylamine (TEA). The reactants were mixed and stirred at room temperature for 3 days. The resulting product was then dialyzed against a MWCO dialysis membrane with a molecular weight cutoff of 3.5 kDa for 48 hours to remove free, uncoupled Pen. The dialysate was lyophilized and stored at -20°C for later use.

[0069] (3) DSPE-PEG 2000 Synthesis of Tf: Tf and DSPE-PEG 2000 -NHS was dissolved in anhydrous DMF at a ratio of 125 μg / μM. The pH was adjusted to 8.5 ± 0.1 with triethylamine. The mixture was stirred with a magnetic stirrer at room temperature for 24 hours. The resulting product was separated from uncoupled transferrin by G-100 Sephadex column chromatography, and the coupling efficiency was evaluated using the micro-BCA method.

[0070] (4) Plasmid Encapsulation Preparation: To improve the stability of the plasmid before encapsulation, we prepared a chitosan-pPAM complex. Specifically, 1% chitosan (w / v, molecular weight 30 kDa, degree of deacetylation 85-90%) was dissolved in 0.2 M acetate buffer (pH 4.5), and then pPAM was added at a 5:1 N / P ratio. Subsequently, we used a "post-insertion" method to encapsulate the chitosan-pPAM complex into liposomes. This method has high encapsulation efficiency and ensures the stability of the liposomes.

[0071] (5) Thiolized antibody OTR Ab-SDSP Preparation: The thiolized antibody was prepared by conjugating an anti-OTR monoclonal antibody (25 µg) with SPDP (6.25 mg / mL; SPDP / mAb molar ratio = 10:1). Excess SPDP was removed using a PD-10 column equilibrated with distilled water, and the pyridyl dithiopropionic acid-containing antibody (PDP-Ab) conjugate (assessed by absorbance at 280 nm) was lyophilized and stored at 4°C under nitrogen protection. PDP-Ab was reduced with 5 mM TCEP for 5 min to generate the thiolized antibody (Ab-SH), and absorbance was checked at 280 nm (protein concentration) and 343 nm (SPDP modification) to ensure compound stability.

[0072] (6) Connect to OTR Ab Preparation of long-circulating immunoliposomes with Pen-Tf and encapsulation with pPAM: Prepared DSPE-PEG2000-Pen (2 mol%), DOPE (45 mol%), DOTAP (45 mol%), and OTR were loaded onto the liposomes. Ab SDSP (1 mol%) and cholesterol (2 mol%) were dissolved in a chloroform / methanol (2:1, v / v) mixture. The solvent was evaporated to form a thin lipid membrane, which was then rotary evaporated to form a dried liposome membrane, followed by hydration with HEPES buffer. The buffer was then sonicated to form liposomes, which were then subjected to Pen-OTR. Ab -LCLipo and DSPE-PEG2000-Tf (2 mol%) were mixed with micelles and stirred overnight to obtain Tf-OTR. Ab -LCLipo. Liposomes were subjected to Sephadex G-100 column chromatography to remove unbound drug, and further purified using a 0.2 μm polycarbonate membrane.

[0073] (7) Liposome collection and storage: Resuspend the liposomes in PBS (pH 7.4), store at 4°C under nitrogen and in the dark, and use within 2 weeks. Immediately mix the thiolized antibody with the liposomes and stir at room temperature in the dark for 1 hour. Remove unbound antibody and unencapsulated drug using TLX ultracentrifugation (Optima™) (100,000 × g; 45 min). After lyophilization, reconstitute the liposomes as needed.

[0074] Example 2: Preparation of pPAM@Tf-Pen-OTRAb-LCLipo (Formula 2)

[0075] (1) Same as Example 1.

[0076] (2) DSPE-PEG 2000Synthesis of -Pen: 250 μM DSPE-PEG was used. 2000 -NHS and 70 μM Pen were dissolved in anhydrous dimethylformamide (DMF), and the pH was adjusted to 8.2 ± 0.1 with triethylamine (TEA). The reactants were mixed and stirred at room temperature for 3 days. The resulting product was then dialyzed against a MWCO dialysis membrane with a molecular weight cutoff of 3.5 kDa for 48 hours to remove free, uncoupled Pen. The dialysate was lyophilized and stored at -20°C for later use.

[0077] (3) DSPE-PEG 2000 Synthesis of Tf: Tf and DSPE-PEG 2000 -NHS was dissolved in anhydrous DMF at a ratio of 100 μg / μM. The pH was adjusted to 8.2 ± 0.1 with triethylamine. The mixture was stirred with a magnetic stirrer at room temperature for 24 hours. The resulting product was separated from uncoupled transferrin by G-100 Sephadex column chromatography, and the coupling efficiency was evaluated using the micro-BCA method.

[0078] (4) Plasmid Encapsulation Preparation: To improve the stability of the plasmid before encapsulation, we prepared a chitosan-pPAM complex. Specifically, 1% chitosan (w / v, molecular weight 30 kDa, degree of deacetylation 85-90%) was dissolved in 0.3 M acetate buffer (pH 4.5), and then pPAM was added at a 6:1 N / P ratio. Subsequently, we used a "post-insertion" method to encapsulate the chitosan-pPAM complex into liposomes. This method has high encapsulation efficiency and ensures the stability of the liposomes.

[0079] (5) Same as Example 1.

[0080] (6) Connect to OTR Ab Preparation of long-circulating immunoliposomes with Pen-Tf and encapsulation with pPAM: Prepared DSPE-PEG2000-Pen (1.8 mol%), DOPE (42 mol%), DOTAP (42 mol%), and OTR were loaded onto the liposomes. Ab SDSP (1.2 mol%) and cholesterol (2.4 mol%) were dissolved in a chloroform / methanol (2:1, v / v) mixture. The solvent was evaporated to form a thin lipid membrane, which was then rotary evaporated to form a dried liposome membrane, followed by hydration with HEPES buffer. The buffer was then sonicated to form liposomes, which were then subjected to Pen-OTR. Ab -LCLipo and DSPE-PEG2000-Tf (2.4 mol%) micelles were mixed and stirred overnight to obtain Tf-OTR. Ab-LCLipo. Liposomes were subjected to Sephadex G-100 column chromatography to remove unbound drug, and further purified using a 0.2 μm polycarbonate membrane.

[0081] (7) Same as Example 1.

[0082] Example 3: Preparation of pPAM@Tf-Pen-OTRAb-LCLipo (Formula 3)

[0083] (1) Same as Example 1

[0084] (2) DSPE-PEG 2000 Synthesis of -Pen: DSPE-PEG at a molar concentration of 150 μM was used. 2000 -NHS and 50 μM Pen were dissolved in anhydrous dimethylformamide (DMF), and the pH was adjusted to 8.5 ± 0.1 with triethylamine (TEA). The reactants were mixed and stirred at room temperature for 3 days. The resulting product was then dialyzed against a MWCO dialysis membrane with a molecular weight cutoff of 3.5 kDa for 48 hours to remove free, uncoupled Pen. The dialysate was lyophilized and stored at -20°C for later use.

[0085] (3) DSPE-PEG 2000 Synthesis of Tf: Tf and DSPE-PEG 2000 -NHS was dissolved in anhydrous DMF at a concentration of 75 μg / μM. The pH was adjusted to 8.3 ± 0.1 with triethylamine. The mixture was stirred with a magnetic stirrer at room temperature for 24 hours. The resulting product was separated from uncoupled transferrin by G-100 Sephadex column chromatography, and the coupling efficiency was evaluated using the micro-BCA method.

[0086] (4) Plasmid Encapsulation Preparation: To improve the stability of the plasmid before encapsulation, we prepared a chitosan-pPAM complex. Specifically, 1% chitosan (w / v, molecular weight 30 kDa, degree of deacetylation 85-90%) was dissolved in 0.10M acetate buffer (pH 4.5), and then pPAM was added at a 6:1 N / P ratio. Subsequently, we used a "post-insertion" method to encapsulate the chitosan-pPAM complex into liposomes. This method has high encapsulation efficiency and ensures the stability of the liposomes.

[0087] (5) Same as Example 1.

[0088] (6) Connect to OTR AbPreparation of long-circulating immunoliposomes with Pen-Tf and encapsulation with pPAM: Prepared DSPE-PEG2000-Pen (3 mol%), DOPE (40 mol%), DOTAP (40 mol%), and OTR were loaded onto the liposomes. Ab SDSP (2 mol%) and cholesterol (4 mol%) were dissolved in a chloroform / methanol (2:1, v / v) mixture. The solvent was evaporated to form a thin lipid membrane, which was then rotary evaporated to form a dried liposome membrane, followed by hydration with HEPES buffer. The buffer was then sonicated to form liposomes, which were then used for Pen-OTR. Ab -LCLipo and DSPE-PEG2000-Tf (2 mol%) were mixed with micelles and stirred overnight to obtain Tf-OTR. Ab -LCLipo. Liposomes were subjected to Sephadex G-100 column chromatography to remove unbound drug, and further purified using a 0.2 μm polycarbonate membrane.

[0089] (7) Same as Example 1.

[0090] Preferably, the drug-loaded liposome pPAM@Tf-Pen-OTR Ab -LCLipo's components include Tf-Pen-DSPC-PEG in a molar ratio of 0.5:1 to 2:1. 2000 -OTR Ab And cholesterol, containing 0.5~3 mol% Tf-Pen-DSPC-PEG. 2000 -OTR Ab DSPC-PEG with maleimide (Mal) as the coupling medium 2000 -Mal was obtained by coupling reaction at a molar ratio of 1:1 to 1:10, and each liposome encapsulated 1500 to 2500 PAM plasmids; preferably, the drug-loaded liposome pPAM@Tf-Pen-OTR Ab -LCLipo components include Tf-Pen-DSPC-PEG in a molar ratio of 1.5:1. 2000 -OTR Ab And cholesterol, containing 1.5 mol% DSPC-PEG. 2000 The liposomes were obtained by coupling with the coupling medium DSPC-PEG2000-Mal at a molar ratio of 1:5, and each liposome encapsulated approximately 2000 PAM plasmids.

[0091] Example 4: Preparation of OT@Tf-Pen-OTRAb-LCLipo

[0092] (1) DSPE-PEG 2000Synthesis of -Pen: DSPE-PEG at a molar concentration of 300 μM was used. 2000 -NHS and 100 μM Pen were dissolved in anhydrous dimethylformamide (DMF), and the pH was adjusted to 8.5 ± 0.1 with triethylamine (TEA). The reactants were mixed and stirred at room temperature for 3 days. The resulting product was then dialyzed against a MWCO dialysis membrane with a molecular weight cutoff of 3.5 kDa for 48 hours to remove free, uncoupled Pen. The dialysate was lyophilized and stored at -20°C for later use.

[0093] (2) DSPE-PEG 2000 Synthesis of Tf: Tf and DSPE-PEG 2000 -NHS was dissolved in anhydrous DMF at a ratio of 125 μg / μM. The pH was adjusted to 8.5 ± 0.1 with triethylamine. The mixture was stirred with a magnetic stirrer at room temperature for 24 hours. The resulting product was separated from uncoupled transferrin by G-100 Sephadex column chromatography, and the coupling efficiency was evaluated using the micro-BCA method.

[0094] (3) Thiolized antibody OTR Ab -SDSP Preparation: The thiolized antibody was prepared by conjugating an anti-OTR monoclonal antibody (25 µg) with SPDP (6.25 mg / mL; SPDP / mAb molar ratio = 10:1). Excess SPDP was removed using a PD-10 column equilibrated with distilled water, and the pyridyl dithiopropionic acid-containing antibody (PDP-Ab) conjugate (assessed by absorbance at 280 nm) was lyophilized and stored at 4°C under nitrogen protection. PDP-Ab was reduced with 5 mM TCEP for 5 min to generate the thiolized antibody (Ab-SH), and absorbance was checked at 280 nm (protein concentration) and 343 nm (SPDP modification) to ensure compound stability.

[0095] (4) Connect to OTR Ab Preparation of long-circulating immunoliposomes with Pen-Tf and encapsulation of OT: The prepared DSPE-PEG2000-Pen (4 mol%), DOPE (43.5 mol%), DOTAP (43.5 mol%), and OTR were encapsulated. Ab SDSP (1 mol%), free OT (1 mol%), and cholesterol (2 mol%) were dissolved in a chloroform / methanol (2:1, v / v) mixture. The solvent was evaporated to form a thin lipid membrane, which was then dried by rotary evaporation to form a dried liposome membrane. The membrane was then hydrated with HEPES buffer. Liposomes were formed by sonication of the buffer. OT@Pen-OTR was then applied. Ab -LCLipo and DSPE-PEG2000 -Tf (2 mol%) and micelles were mixed and stirred overnight to obtain Tf-OTR. Ab -LCLipo. Liposomes were subjected to Sephadex G-100 column chromatography to remove unbound drug, and further purified using a 0.2 μm polycarbonate membrane.

[0096] (5) Liposome collection and storage: Resuspend the liposomes in PBS (pH 7.4), store them at 4°C under nitrogen and in the dark, and use them within 2 weeks. Immediately mix the thiolized antibody with the liposomes and stir at room temperature in the dark for 1 hour. Remove unbound antibody and unencapsulated drug using TLX ultracentrifugation (Optima™) (100,000 × g; 45 min). After lyophilization, reconstitute the liposomes as needed.

[0097] Thirdly, this application provides an application of pPAM long-circulating liposomes, using oxytocin synthase and its analogues as the active ingredient in liposome-based drugs for treating and improving mental stress. The application has explored and verified that pPAM liposomes can alleviate symptoms such as depression to a certain extent. Experiments show that using long-circulating PAM liposomes effectively reversed the increased number of escape-from-electric-shock errors in rats after stress and reduced anxiety scores. This provides a new pharmaceutical use for the development and application of novel oxytocin formulations and also offers a new pre-treatment approach for depression.

[0098] As a preferred embodiment of this application, the PAM enzyme generated after pPAM expression can catalyze the synthesis of oxytocin and its derivatives, including: natural hormone oxytocin, carbetocin, desmooxytocin, or acetate oxytocin, etc.

[0099] As a preferred embodiment of this application, the preparation for treating or improving mental stress is in the form of a nasal spray, a sublingual spray, or a transdermal patch.

[0100] As a preferred embodiment of this application, the final concentration of pPAM and its derivatives in the formulation is 0.5-5 μg / mL.

[0101] As a preferred embodiment of this application, the dosage form of the composition is a long-circulating liposome nasal spray, aerosol, sublingual spray, or transdermal patch.

[0102] The nasal spray of this application is to be sprayed into both nostrils several times a day, and the frequency of use can be increased when the subject needs to relieve mental stress.

[0103] The binding mechanism of this application is as follows: pPAM is encapsulated in long-circulating liposomes and delivered via PEG. 2000 Modifications prolong its intracerebral circulation time. The introduction of penetrin and oxytocin receptor antibodies further promotes cellular uptake and targeted delivery of oxytocin after synthesis.

[0104] 1. Extended drug half-life: Through long-circulating liposome technology, the half-life of PAM in vivo is significantly extended, reducing the frequency of administration.

[0105] 2. Improved bioavailability: The introduction of penetrant improves the cellular uptake efficiency and targeted delivery capability of oxytocin.

[0106] 3. Reduce side effects: By improving the accuracy of drug delivery, non-specific effects and related side effects of drugs can be reduced.

[0107] This application utilizes plasmids capable of producing oxytocin encapsulated in long-circulating liposomes to effectively prolong its retention time in the bloodstream, improve drug bioavailability, and reduce dosing frequency and dosage. PAM (Plasmin-Induced Acid) long-circulating liposomes are a novel drug delivery system combining penetrant and long-circulating liposome technologies. This system aims to improve the stability and bioavailability of oxytocin synthase PAM in vivo, increase the retention time of oxytocin in the brain, prolong its half-life (t1 / 2), and simultaneously promote drug penetration through cell membranes, thereby enhancing therapeutic efficacy.

[0108] The pPAM drug-loaded liposomes prepared in this application have a stable particle size of 190±20 nm, with uniform particles and good dispersibility, and a stable zeta potential of around -38.5 mV. Within 7 days of storage at room temperature, the particle size and potential of the formulation do not fluctuate significantly, and there is no aggregation or sedimentation. After lyophilization and reconstitution, the liposome structure is intact, the in vitro plasmid release is stable, there is no burst release effect, and the formulation has excellent stability.

[0109] The hemolysis test results showed that the liposomes of this application did not exhibit significant hemolysis within the effective dosing concentration range of phospholipid concentration ≤800 nM, had extremely low cytotoxicity, good biocompatibility, and met the safety requirements for in vivo administration.

[0110] Compared to free oxytocin nasal drops and ordinary liposomal oxytocin preparations, the pPAM long-circulation targeted liposomes of this application can continuously synthesize active oxytocin in the central nervous system, greatly prolonging the retention time and half-life of oxytocin in the rat brain, thus achieving long-lasting efficacy.

[0111] The results of the Vogel drinking water shock stress behavior experiment in rats confirmed that the formulation of this application can significantly improve the abnormal anxiety behavior of stressed rats, reduce mental stress-related scores, effectively alleviate the mental stress and anxiety state of animals, and the mood regulation effect is significantly better than that of traditional short-acting oxytocin formulations.

[0112] The above description is only a preferred embodiment of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of this application, and these improvements and modifications should also be considered within the scope of protection of this application.

Claims

1. A pPAM long-circulating liposome, characterized in that, The long-circulating liposomes use blank liposomes as carriers, internally encapsulating plasmid pPAM, and surface-modified with transferrin-penetrin conjugate complex Tf-Pen and oxytocin receptor antibody OTR. Ab ; The plasmid pPAM carries the cDNA sequence of peptidylglycine α-amidyl monooxygenase PAM. The blank liposomes were composed of distearate phosphatidylcholine (DSPC) and polyethylene glycol 2000 (PEG). 2000 Cholesterol and lecithin.

2. The pPAM long-circulating liposome according to claim 1, characterized in that, The penetrant includes the unmodified subtype Pen1 and the capped analog subtype Pen2; The amino acid sequence of the unmodified subtype Pen1 is SEQ ID: No. 1; The amino acid sequence of the capped analog subtype Pen2 is SEQ ID: No.

2.

3. The pPAM long-circulating liposome according to claim 1, characterized in that, The oxytocin receptor antibody OTR Ab This includes natural polyclonal antibodies, artificially synthesized polyclonal antibodies, and monoclonal antibodies.

4. The pPAM long-circulating liposome according to claim 1, characterized in that, DSPC-PEG in long-circulating liposomes 2000 The molar ratio of cholesterol to total cholesterol is 0.5:1 to 2:1; The DSPC-PEG 2000 The molar percentage in liposomes is 0.5–3 mol%; Using maleimide (Mal) as the coupling medium, the DSPC-PEG 2000 -Mal and transferrin-penetrin conjugate complex Tf-Pen, oxytocin receptor antibody OTR Ab The coupling molar ratio is 1:1 to 1:

10.

5. A method for preparing pPAM long-circulating liposomes, characterized in that, The method includes: The liposome suspension was subjected to ultrasonic treatment under a set temperature condition to obtain an activated liposome suspension. At room temperature, activated liposome suspension was reacted with thiol-modified oxytocin receptor antibody OTR. Ab Mix thoroughly and react; Transferrin-penetin conjugate complex Tf-Pen, oxytocin receptor antibody OTR Ab Mixed with N-succinimide-3-(2-pyridyldithio)propionate SPDP; Control of N-succinimide-3-(2-pyridyldithio)propionate SPDP and oxytocin receptor antibody OTR Ab The molar ratio is used to complete the coupling modification reaction; The reaction system was separated and purified to remove free unreacted impurities, and pPAM long-circulating liposomes were prepared by plasmid encapsulation process, followed by freeze-drying and vacuum storage.

6. The method for preparing pPAM long-circulating liposomes according to claim 5, characterized in that, The liposome suspension was placed at 50–70°C and ultrasonicated at 60 times power for 5 min to obtain an activated liposome suspension.

7. The method for preparing pPAM long-circulating liposomes according to claim 5, characterized in that, Add 1-10 μg of transferrin-penetrin conjugate complex (Tf-Pen) and 5-50 μg of oxytocin receptor antibody (OTR). Ab Mix with 5-15 mg / mL of N-succinimide-3-(2-pyridyldithio)propionate.

8. The method for preparing pPAM long-circulating liposomes according to claim 5, characterized in that, Control of N-succinimide-3-(2-pyridyldithio)propionate SPDP and oxytocin receptor antibody OTR Ab The molar ratio is 5:1 to 50:

1.

9. The method for preparing pPAM long-circulating liposomes according to claim 5, characterized in that, The reaction system was separated and purified using a PD-10 column equilibrated with distilled water.

10. The use of the pPAM long-circulating liposomes according to any one of claims 1-4 in the preparation of nanomedicines for relieving mental stress in animals.

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