Efficient brain-targeted oxytocin nasal spray for resisting oxycodone addiction

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an efficient brain-targeted oxytocin nasal spray for resisting oxycodone addiction. The nasal spray comprises a polyelectrolyte complex formed by amino acid grafted chitosan and anionic polysaccharide, wherein oxytocin is loaded in the polyelectrolyte complex. The amino acid is selected from tryptophan, leucine or tyrosine. The preparation is administrated through a nasal cavity, the brain targeting property and bioavailability of oxytocin are remarkably improved by utilizing an LAT-1 transporter mediated active transport mechanism, and the preparation is used for preventing or treating addiction of opioid drugs such as oxycodone and the like. Experiments show that the preparation can significantly enhance distribution of oxytocin in the brain, inhibit formation and relapse of addiction and promote drug withdrawal, and has a good application prospect.

Description

Technical Field

[0001] This invention pertains to the field of biomedical technology, specifically relating to a highly effective brain-targeting oxytocin nasal spray for combating oxycodone addiction. Background Technology

[0002] Oxytocin (OT) is an endogenous peptide hormone composed of a cyclic peptide of nine amino acids. It is primarily synthesized in the hypothalamus and stored in the posterior pituitary gland for release. Clinically, it is mainly used to induce labor or stop postpartum bleeding. Furthermore, oxytocin has shown positive therapeutic effects in a range of neurological and psychiatric disorders, such as drug addiction, epilepsy, schizophrenia, Alzheimer's disease, and Parkinson's disease. In drug addiction, oxytocin not only inhibits the reward effect during binge drinking but also alleviates stress responses and social dysfunction during withdrawal, thereby improving neurobehavioral changes induced by drug abuse.

[0003] The effectiveness of drug addiction treatment largely depends on the concentration of the drug in the brain. Traditional methods of oxytocin administration, such as intravenous and intramuscular injections, often have limited efficacy due to their difficulty in penetrating the blood-brain barrier. Nasal administration, as a highly efficient and non-invasive method, has emerged. Its advantages include bypassing the first-pass effect, achieving rapid absorption, reducing gastrointestinal and systemic side effects, improving bioavailability, and enhancing patient compliance, thus attracting widespread attention and research. Furthermore, the nasal-to-brain drug delivery pathway, achieved through the olfactory and trigeminal nerve pathways, allows drugs to bypass the blood-brain barrier and be delivered directly to the brain, providing a significant advantage for treating central nervous system diseases.

[0004] While nasal-to-brain drug delivery enhances brain targeting, protein and peptide drugs, due to their strong hydrophilicity and large molecular weight, often have difficulty penetrating the nasal mucosa, resulting in low bioavailability after nasal administration. To improve nasal absorption efficiency, absorption enhancers are typically used during the delivery of these drugs. Chitosan (CTS) is widely used in nasal drug delivery systems due to its good biodegradability, biocompatibility, low toxicity, and mucosal adhesion. Furthermore, CTS can promote nasal absorption by opening tight junctions between epithelial barrier cells. To date, various strategies have been employed to enhance the absorption of proteins or peptides. Among these, nanoparticles (NPs), especially nanoscale polyelectrolyte complexes (PECs), are particularly promising, as they can protect proteins / peptides from degradation and promote in vivo absorption. Because of their positive charge, biodegradability, and tunable structural modifiability, CTS is often used in conjunction with anionic polysaccharides (including hyaluronic acid, sodium alginate, chondroitin sulfate, and glucosamine sulfate) to prepare polyelectrolyte complexes. Furthermore, hydrophobic or hydrophilic modifications to CTS have been shown to effectively enhance protein or peptide delivery. Among various anionic polysaccharides, chondroitin sulfate (CS) also exhibits good biocompatibility and biodegradability, and is widely used for protein mucosal delivery.

[0005] Besides passive transport mechanisms via chitosan-mediated tight junction opening, active transporters can also effectively improve the transcellular transport efficiency of protein and peptide drugs. L-amino acid transporter 1 (LAT-1) is an amino acid transporter independent of Na+. + LAT-1 is a 12-transmembrane transporter that regulates pH and facilitates the entry of large and neutral amino acids into the brain, maintaining the normal function of the central nervous system. The main amino acids transported by LAT-1 include branched-chain amino acids (leucine, isoleucine, valine), aromatic amino acids (phenylalanine, tyrosine, tryptophan, histidine), and methionine, most of which are essential amino acids. Because LAT-1 is highly expressed on both the luminal and basal surfaces of the blood-brain barrier, it has become an important target for improving drug delivery to the brain to treat neurodegenerative diseases.

[0006] Currently, there is a lack of nasal delivery systems that can significantly increase oxytocin concentration in the brain, prolong its residence time, and enhance its anti-addiction effects. Therefore, developing a nasal delivery system for oxytocin based on an amino acid-based active targeting mechanism has significant clinical implications and application prospects. Summary of the Invention

[0007] To address the problems of existing technologies, this invention provides a highly efficient brain-targeting oxytocin nasal spray for combating oxytocin addiction. Amino acids are grafted onto chitosan molecules, enabling them to cross the blood-brain barrier and achieve brain-targeting action. The amino acid-grafted chitosan forms a polyelectrolyte complex with anionic polysaccharides and encapsulates oxytocin, which can be administered via nasal cavity to treat oxytocin addiction.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solutions.

[0009] This invention discloses a highly efficient brain-targeted oxytocin nasal spray, characterized in that it comprises a polyelectrolyte complex formed by amino acid-grafted chitosan and anionic polysaccharides, wherein the polyelectrolyte complex is loaded with oxytocin.

[0010] Furthermore, the amino acid is selected from at least one of tryptophan, leucine, or tyrosine.

[0011] Furthermore, the anionic polysaccharide includes chondroitin sulfate, sodium alginate, hyaluronic acid, and gelatin.

[0012] Furthermore, the mass ratio of the amino acid-grafted chitosan to chondroitin sulfate is 1:0.5 to 1:2.

[0013] Furthermore, the pH value of the nasal spray is 4.5 to 5.5.

[0014] Furthermore, the use of any of the above-described nasal sprays in the preparation of medicaments for the prevention or treatment of opioid addiction.

[0015] Furthermore, the opioid drug is oxycodone.

[0016] The present invention also discloses a method for preparing the nasal spray according to any one of the above claims, comprising the following steps: S1 grafts amino acids onto chitosan to obtain an amino acid-chitosan copolymer; S2 The copolymer and anionic polysaccharide are mixed under acidic conditions, and oxytocin solution is added to form a polyelectrolyte complex; S3 was used to adjust the pH and purify the product to obtain a polyelectrolyte complex loaded with oxytocin. S4 formulated it into a nasal spray.

[0017] Furthermore, in step S2, the acidic conditions are pH 4.0 to 6.0.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0019] By grafting chitosan with amino acids (tryptophan / leucine / tyrosine) and utilizing LAT-1 active transport, the concentration of oxytocin in the olfactory bulb, forebrain, and hippocampus (key brain regions for addiction) was significantly increased. Among them, the drug concentration in key brain regions such as the hippocampus of the tryptophan-modified group was significantly higher than that of the unmodified chitosan group, thus solving the problem of blood-brain barrier penetration.

[0020] The encapsulation rate of polyelectrolyte complex (PEC) in this nasal spray is over 80%. After nasal administration, the relative bioavailability of oxytocin is increased by 1.92 to 3.12 times compared with oxytocin solution, and the time to peak concentration (Tmax) is extended to 10 minutes, indicating that the drug absorption is more sustained and is conducive to maintaining effective blood drug concentration.

[0021] The PEC carrier can protect the conformational stability of oxytocin (circular dichroism spectroscopy proves that its α-helical structure is consistent with natural oxytocin), and it can be continuously released in artificial nasal solution for 16 hours (cumulative release of 80%), avoiding the problem of traditional solutions releasing 93.4% of their contents within 1 hour.

[0022] By enhancing brain targeting, it can increase intracerebral drug concentrations while helping to reduce peripheral blood drug concentrations required to achieve the same central therapeutic effect, thereby potentially reducing the risk of peripheral oxytocin side effects (such as effects on uterine contractions) and improving medication safety.

[0023] Nasal administration allows direct access to the brain via the olfactory pathway, reaching peak levels in the brain within 0.5 hours, which is faster than oral administration. It can also significantly inhibit the acquisition of oxycodone CPP (p<0.001 in the tryptophan-modified group), block its expression, promote withdrawal expiration (2-4 days earlier than natural expiration), and eliminate relapse. Its anti-addiction effect is superior to naked oxytocin and unmodified PEC.

[0024] This invention overcomes the difficulties of low efficiency and poor bioavailability of oxytocin delivered to the brain via the nose. It can significantly reduce peripheral oxytocin concentrations and decrease systemic effects. Simultaneously, this formulation can improve the stability of oxytocin. Nasal administration, compared to traditional oral methods for treating addiction, allows the drug to enter the brain more quickly and has a faster onset of action. Attached Figure Description

[0025] Figure 1 Fourier transform infrared spectra of CTS-Tyr (A), CTS-Leu (B), and CTS-Trp (C).

[0026] Figure 2 Schematic diagram of the CTS-AA reaction and ¹H NMR spectra of CTS-Tyr (A), CTS-Leu (B) and CTS-Trp (C).

[0027] Figure 3Particle size and zeta potential of PECs prepared under different CS concentrations (A and B), different pH values ​​(C and D), and different AA-coupled CTS conditions (E and F). Data are expressed as mean ± standard deviation (n=3).

[0028] Figure 4 Transmission electron microscopy images of oxytocin-loaded chitosan-chondroitin sulfate polyelectrolyte complexes (OT-CTS-CS PECs) (A), oxytocin-loaded tryptophan-chitosan-chondroitin sulfate polyelectrolyte complexes (OT-Trp-CTS-CS PECs) (B), oxytocin-loaded leucine-chitosan-chondroitin sulfate polyelectrolyte complexes (OT-Leu-CTS-CS PECs) (C), and oxytocin-loaded tyrosine-chitosan-chondroitin sulfate polyelectrolyte complexes (OT-Tyr-CTS-CS PECs) (D); (E) Circular dichroism spectra of oxytocin solution and various oxytocin-loaded polyelectrolyte complex formulations; (F) In vitro release behavior of oxytocin solution and various oxytocin-loaded polyelectrolyte complex formulations in artificial nasal solutions; (G) In vitro permeation test results of various polyelectrolyte complex formulations through sheep nasal mucosa. Data are expressed as mean ± standard deviation (n=3).

[0029] Figure 5 The concentration-time curves of oxytocin (OT) in plasma (A) and its distribution in the brain (B) after intranasal administration to rats. Data are expressed as mean ± standard deviation (n=6).

[0030] Figure 6 Distribution of oxytocin in rat brain regions acted upon by (A) CTS-CS polyelectrolyte complexes, (B) tryptophan-chitosan-chondroitin sulfate polyelectrolyte complexes (Trp-CTS-CS PECs), (C) leucine-chitosan-chondroitin sulfate polyelectrolyte complexes (Leu-CTS-CS PECs), and (D) tyrosine-chitosan-chondroitin sulfate polyelectrolyte complexes (Tyr-CTS-CSPECs) after intranasal administration. Data are expressed as mean ± standard deviation (n=6).

[0031] Figure 7 Inhibition of oxytocin (OT) on conditioned position preference (CPP) of oxycodone (Oxy). (A) Experimental design of the conditioned position preference test. (B) Acquisition, (C) expression, (DF) decay and (G) re-ignition of conditioned position preference of oxycodone, data are expressed as mean ± standard deviation (n=6).

[0032] Figure 8Representative images of double immunofluorescence staining with c-Fos (red) and NeuN (green). Scale bar = 20 μm. ### indicates p < 0.001 compared to the control group (Con group); p < 0.05 indicates p < 0.01 indicates p < 0.001 compared to the oxycodone group (Oxy group); + indicates p < 0.05 compared to the post-reignition data group (presumably referring to the data group after reignition); ++ indicates p < 0.01; and +++ indicates p < 0.001. Detailed Implementation

[0033] The present invention will be further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the above-described subject matter of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0034] Unless otherwise specified, all reagents and materials used in this invention are commercially available.

[0035] I. Experimental Materials.

[0036] II. Experimental Methods.

[0037] 1. Laboratory animals.

[0038] Healthy male Sprague-Dawley rats weighing 180–220 g were obtained from Liaoning Changsheng Biotechnology Co., Ltd. (Benxi, China). Healthy male ICR mice aged 6–7 weeks were purchased from Beijing Huafukang Biotechnology Co., Ltd. (Beijing, China). All animal experimental protocols were approved by the Institutional Animal Care and Use Committees of Shenyang Pharmaceutical University and Shengjing Hospital of China Medical University (Approval numbers: SYPU-IACUC-2022-0302-022 and 2022PS692K), and experiments were conducted in accordance with the National Research Council's Guidelines for the Care and Use of Laboratory Animals.

[0039] 2. Synthesis of CTS-AA.

[0040] 0.5 g of chitosan (CTS, purity >98%, degree of deacetylation ≥95%, molecular weight: 50 kDa) was dissolved in 50 mL of acetic acid solution (0.5%, v / v), and the pH was adjusted to 6.0 with 4M sodium hydroxide. Tyrosine (Tyr), leucine (Leu), and tryptophan (Try) were dissolved in 50 mL of 2-(N-morpholino)ethanesulfonic acid buffer (25 mM, pH = 6), respectively. Subsequently, NHS and EDC were added to each solution according to the molar ratio of amino acid (AA): N-hydroxysuccinimide (NHS): 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) = 1:1:3, and the mixture was activated at room temperature in the dark for 2 hours. Under continuous stirring, the activated amino acid solution was slowly added dropwise to the above CTS solution, and the mixture was stirred at 30°C in the dark for 48 hours. After the reaction was completed, three volumes of anhydrous ethanol were added to terminate the reaction. The reaction product was dialyzed with distilled water (molecular weight cutoff: 8000~14000 Da) for 72 hours, and then freeze-dried at -40°C for 24 hours to obtain the CTS-AA copolymer material.

[0041] 3. Characterization of CTS-AA polymer.

[0042] 3.1 Fourier transform infrared spectroscopy (FTIR).

[0043] To assess the potential interaction between CTS and AAs, Fourier transform infrared spectroscopy (FTIR) was used to detect the synthesized CTS-AA copolymer, the corresponding individual components, and their physical mixtures. The specific procedure was as follows: Before FTIR detection, the detection platform was wiped with anhydrous ethanol. A quantitative sample powder was placed on a diamond surface for experimental determination. The sample was fixed and pressed into a transparent sheet by rotating the fixing knob of the SMART ITR accessory. The sample was scanned in the wavenumber range of 4000–400 cm⁻¹, with 32 scans and a resolution of 4 cm⁻¹.

[0044] 3.2 Hydrogen nuclear magnetic resonance spectrum (¹H-NMR).

[0045] To further verify the synthesis of the CTS-AA copolymer, a 600 MHz hydrogen nuclear magnetic resonance spectrometer from Bruker (Switzerland) was used for detection with deuterated dimethyl sulfoxide as the solvent. ¹H-NMR spectra were acquired at 25 °C using the Bruker standard pulse sequence “noesyigld1d”. The parameters were set as follows: 2 blank scans, 32 scans, 64 K data points, acquisition time 3.28 seconds, receiver gain 114, and relaxation delay 30 seconds.

[0046] 4. Preparation and characterization of polyelectrolyte complex (PEC).

[0047] A chitosan-chondroitin sulfate (purity >98%, molecular weight: 50 kDa) polyelectrolyte complex (OT-CTS-CS) loaded with oxytocin (OT) was prepared and optimized via a self-assembly process. First, CTS and CS were dissolved in glacial acetic acid (0.1%, v / v) and distilled water of different concentrations, respectively. Then, the CTS and CS solutions were sonicated at 300 W for 10 minutes. While stirring, an appropriate amount of OT powder was slowly added to the CS solution until a clear solution was formed. Furthermore, the CTS solution was added dropwise to the CS solution while stirring at 400 rpm to obtain the OT-CTS-CS polyelectrolyte complex. Simultaneously, based on the above optimization method, an OT-loaded chitosan-amino acid-chondroitin sulfate polyelectrolyte complex (OT-CTS-AA-CS) was also prepared.

[0048] The particle size and zeta potential of PECs were evaluated using a laser particle size analyzer (Malvin Instruments GmbH, Germany, Zetasizer Nano ZS 90) at 25 °C and a scattering angle of 90 °C. The morphology of OT-CTS-AA-CS and OT-CTS-CS PECs was observed using transmission electron microscopy at an accelerating voltage of 200 kV. To determine the encapsulation efficiency (EE) and drug loading (DL) of OT in PECs, freshly prepared PECs were centrifuged at 4500 rpm for 30 min using an ultrafiltration tube (molecular weight cutoff: 3.5 kDa). The supernatant was further diluted and filtered through a 0.22 μm filter membrane. OT content was determined using a high-performance liquid chromatograph (Agilent 1260, Shanghai, China) at 220 nm with a C18 column (4.6 mm × 250 mm, 5 μm) and the following gradient elution program: 0–30 min: mobile phase A 70–40%, mobile phase B 30–60%; 30–30.1 min: mobile phase A 40–70%, mobile phase B 60–30%. Mobile phase A was 0.1 mol / L sodium dihydrogen phosphate solution, and mobile phase B was acetonitrile:distilled water = 50:50 (volume ratio). The drug loading and encapsulation efficiency were calculated according to Formulas 1 and 2 as follows: Encapsulation efficiency = (Total amount of OT added - Amount of OT in centrifugation supernatant) / Total amount of OT added × 100% Formula 1 Drug loading = (Total amount of OT added - Amount of OT in centrifugation supernatant) / Amount of electrolyte complex × 100% Formula 2 5. Circular dichroism (CD).

[0049] To verify whether the conformational stability of OT changed after PEC preparation, the secondary structure of OT was recorded in the far-ultraviolet wavelength range of 180 nm to 330 nm using a circular dichroism spectrometer (Japan Spectrophotometer, J-1500), with a slit width and step size of 5 nm and 2 nm, respectively. All fresh samples with a concentration of approximately 0.5 mg / mL OT were transferred to a quartz cell with an optical path length of 1 mm, and the molecular ellipticity of OT was measured at a speed of 100 nm / min.

[0050] 6. In vitro release of PEC.

[0051] The in vitro release behavior of the selected formulation was studied using the dialysis tube method. One mL of OT-loaded PEC (OT concentration 1 mg / mL) was placed in a dialysis tube (molecular weight cutoff 12000 Da). The dialysis tube was immersed in a receiving cell containing 10 mL of artificial nasal solution, and the mixture was stirred at 100 rpm while maintaining the temperature at 37 ± 1 °C. Samples were collected periodically and replaced with an equal volume of fresh artificial nasal solution. The collected samples were filtered through a 0.45 μm filter membrane, and the filtrate was injected into a high-performance liquid chromatography (HPLC) system. The cumulative percentage of drug release was plotted against time to visually represent the results of the in vitro drug release study.

[0052] 7. In vitro permeation studies.

[0053] To investigate the permeability of different formulations through mucosa, in vitro permeation studies were conducted using sheep nasal mucosa. Fresh inferior nasal turbinate mucosa from slaughtered goats was obtained, rinsed with physiological saline, and stored at 4°C. An in vitro permeation study was performed using a Franz diffusion cell. The nasal mucosa was sandwiched between the donor and recipient cells, stabilized in both cells with physiological saline for 30–60 minutes, and then the receiving medium was replaced. The sample was placed in the donor cell, and the Franz diffusion cell was placed in a transdermal diffusion apparatus, operated at 200 rpm and 34 ± 1°C in a constant-temperature water bath. The donor cell was kept sealed throughout the experiment. Samples were collected from the recipient cell at predetermined time points and replaced with fresh diffusion medium. The collected samples were filtered through a 0.45 μm filter membrane, and the filtrate was injected into a high-performance liquid chromatography (HPLC) system for OT quantitative analysis.

[0054] 8. In vivo analysis of OT.

[0055] The concentrations of OT in brain and plasma were analyzed using liquid chromatography-tandem mass spectrometry (Agilent 1260-6420 LC-MS / MS). A ZORBAX SB C18 column (2.1 mm × 50 mm, 1.8 μm) was used, with a mobile phase consisting of 80% methanol and 20% formic acid (0.1%, v / v). The injection volume was 5 μL, and the flow rate was 0.3 mL / min. OT was detected using electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode. 100 μL of plasma was added to 400 μL of diethyl ether, mixed for 1 min, and then centrifuged at 10,000 rpm for 10 min. The supernatant was collected and dried at 40 °C in the presence of nitrogen. The residue was then redissolved with 400 μL of mobile phase. For brain tissue, 100 μL of tissue homogenate was mixed with 500 μL of methanol / acetonitrile solution (1:4, v / v) containing 0.3% trifluoroacetic acid for 30 seconds and centrifuged at 12,000 rpm for 10 minutes. The supernatant was filtered through a 0.22 μm filter and analyzed by LC-MS / MS. No endogenous chemical interference was found. A linear correlation was found between OT concentration and OT peak area ratio (plasma: A = 0.030C + 18.71, R² = 0.994; brain: A = 0.059C + 20.49, R² = 0.994). The corresponding mean extraction recoveries for brain and plasma samples were 88.86% and 82.29%, respectively. The mean method recoveries were 98.34% and 97.62%, respectively. The intra-day and inter-day relative standard deviations (RSDs) of precision were both less than 15.0%, and the matrix effects were within the range of 80–120%.

[0056] 9. Pharmacokinetic studies in rats.

[0057] Pharmacokinetic studies were conducted using male SD rats weighing 200–220 g. Animals were randomly assigned to five groups (n=6 per group), with the OT nasal solution group used as the standard for relative bioavailability calculation. The PEC-based nasal formulation was administered intranasally using a microsyringe (Shanghai Gaoge Industry & Trade Co., Ltd., China) with a needle attached. The needle was connected to a PE-10 tube approximately 6 mm in diameter placed inside the nostril. The dose for unilateral nostril administration was 50 μL, and the dose for bilateral nostril administration was 100 μL (OT concentration 1 mg / mL). Approximately 0.2 mL blood samples were collected at 5, 10, 15, 30, 60, 90, and 180 minutes post-administration and centrifuged at 3500 rpm for 10 minutes. Pharmacokinetic parameters, including the area under the concentration-time curve (AUC0₋₋₀), were determined using DAS 2.0 software based on the plasma concentration-time curve. t ), the time required to reach the concentration (T) max ) and maximum drug concentration (C maxBy comparing the AUC0₋ after intranasal administration of PEC. t AUC0₋ of the nasal solution group t The relative bioavailability (Fr) of PEC was determined.

[0058] 10. Study on the distribution of rat brain.

[0059] Rats were randomly assigned to five groups (n=18 per group), as in the pharmacokinetic studies. For brain distribution studies, rats were sacrificed at 0.25, 0.5, 1, 2, and 4 hours post-administration, and brain tissue was obtained after cardiac perfusion with saline. For brain region distribution studies, rats were sacrificed at 0.25, 0.5, 1, 2, 4, and 6 hours post-administration, and olfactory bulb, hippocampus, and forebrain tissue were collected after perfusion with saline. The concentration of OT in brain tissue homogenates was determined using the technique described in Method 9. Furthermore, Cy5-labeled OT was loaded into various PECs, and fluorescence images of OT distribution were captured using a small animal in vivo imaging system (PerkinElmer IVISLumina III, Shanghai, China).

[0060] 11. Conditional position preference (CPP).

[0061] The CPP device consisted of two rectangular chambers (42 × 40 × 50 cm) separated by a door (10 × 14 cm). The two chambers were distinguished by unique visual and tactile cues. One chamber had alternating black and white horizontal stripes on the walls and hollow squares on the floor; the other chamber had vertical stripes on the walls and hollow dots on the floor. The time spent in each chamber was recorded and analyzed using an Ethovision XT8.0 computerized video tracking system. CPP testing was conducted in a soundproof room to eliminate any potential interference.

[0062] CPP experiments were conducted using 6-7 week old adult ICR mice. 168 mice were used to detect four different phases of CPP: formation, expression, regression, and recurrence. In each phase, mice were randomly assigned to seven groups: control group (saline), oxycodone group (Oxy), oxycodone + OT group (Oxy + OT), oxycodone + OT-CTS-CS group (Oxy + OT-CTS-CS PEC), oxycodone + OT-Leu-CTS-CS group (Oxy + OT-Leu-CTS-CS PEC), oxycodone + OT-Tyr-CTS-CS group (Oxy + OT-Tyr-CTS-CS PEC), and oxycodone + OT-Trp-CTS-CS group (Oxy + OT-Trp-CTS-CSPEC). Oxycodone was administered once daily via intraperitoneal injection at a dose of 6 mg / kg. OT was administered nasally 30 minutes before oxycodone administration, with a volume of 25 μL per nostril.

[0063] Phase 1 - Oxycodone CPP Formation: On days 1-2, mice were acclimatized to the CPP device for 15 minutes. On day 3, each mouse was allowed free exploration of the CPP chambers for 15 minutes, and the time spent in each chamber was recorded. On days 4, 6, 8, and 10, mice were intraperitoneally injected with either oxycodone (oxycodone group) or saline (control group) 30 minutes after OT pretreatment. Afterward, all mice were confined to the drug-paired chambers for 1 hour. Conversely, on days 5, 7, 9, and 11, all mice were intraperitoneally injected with saline 30 minutes after nasal administration and confined to the non-drug-paired chambers for 1 hour. On day 12, mice were allowed free access to both chambers for 15 minutes, and the time spent in the drug-paired chambers was recorded.

[0064] Phase 2 - Oxycodone CPP Expression Period: On days 1-2, mice were acclimatized to the CPP device for 15 minutes. On day 3, each mouse was allowed to freely explore the CPP chambers for 15 minutes, and the time spent in each chamber was recorded. On days 4, 6, 8, and 10, mice were intraperitoneally injected with either oxycodone (oxycodone group) or saline (control group) and confined to the drug-paired chamber for 1 hour; on days 5, 7, 9, and 11, mice were intraperitoneally injected with saline and confined to the non-drug-paired chamber for 1 hour. On day 12, 30 minutes before the behavioral test, mice were intranasally administered either oxytocin (oxycodone group) or saline (control group).

[0065] Phase 3 - Oxycodone CPP Extinction Phase: After establishing CPP formation in mice, all mice were allowed to freely explore the CPP chambers for 30 minutes. For six consecutive days (days 13-18), mice were administered either oxytocin (oxycodone group) or saline (control group). The time spent in the drug-pairing chamber was recorded daily. The criterion for determining preference extinction was that there was no statistically significant difference in the time spent in the drug-pairing chamber compared to the CPP expression phase on the test day.

[0066] Phase 4 - Recurrence period of oxycodone CPP induced by restraint stress: After a two-week regression period, mice were subjected to 30 minutes of restraint stress. For restraint, mice were allowed to spontaneously enter a fine acrylic mesh restraint device, 20 cm long, 7 cm wide, and 6 cm high. On day 27, 30 minutes before the start of restraint stress, mice were administered oxytocin (oxycodone group) or saline (control group) intranasally. Five minutes after the start of restraint, the time spent in the drug-paired chamber was recorded.

[0067] 12. Immunofluorescence staining (IF) One hour after the oxycodone CPP recurrence period ended, mice were perfused with phosphate-buffered saline (PBS), followed by perfusion with 4% paraformaldehyde. Whole brains were removed, post-fixed with 4% paraformaldehyde for 48 hours, dehydrated with 20 / 30% sucrose for 72 hours, and then frozen into 15 μm thick sections. These sections were blocked with 5% goat serum and incubated overnight at 4°C with primary antibodies against c-Fos and NeuN (dilution: 1 / 1000). The next day, the sections were warmed to room temperature for 1 hour, washed with PBS, and incubated with secondary antibodies at room temperature for 2 hours. The sections were then stained with DAPI for 15 minutes. Images were captured blindly using a Nikon Ci confocal microscope (excitation wavelengths of 405, 488, and 561 nm). All raw images were analyzed using ImageJ software (National Institutes of Health).

[0068] 13. Statistical Analysis Experimental results are expressed as mean ± standard deviation (SD) of at least three measurements. One-way ANOVA was used to assess the significance of differences at a significance level of less than 0.05.

[0069] III. Experimental Results.

[0070] 1. Synthesis and characterization of chitosan-amino acid (CTS-AA) copolymer.

[0071] To verify whether AA was successfully synthesized onto the CTS molecule, Fourier transform infrared spectroscopy analysis was performed on the prepared CTS-AA graft copolymer and its components. Figure 1As shown in Figure A, taking the CTS-tyrosine (Tyr) copolymer as an example, CTS exhibits a stretching vibration peak of the -NH2 group at 3422 cm⁻¹, and characteristic absorption peaks of the amide at 1645 cm⁻¹ and 1606 cm⁻¹, respectively. Tyr exhibits a stretching vibration peak of the -NH2 group at 3423 cm⁻¹, and C=O stretching vibration peak and NH bending vibration peak at 1621 cm⁻¹ and 1592 cm⁻¹, respectively. Simultaneously, a stretching vibration peak of the carboxylic acid OH in Tyr can also be observed at 3207 cm⁻¹. For the CTS-Tyr graft copolymer, the characteristic peak of the -NH2 group in CTS shifts from 3422 cm⁻¹ to 3433 cm⁻¹. The C=O stretching vibration peak and NH bending vibration peak of Tyr at 1621 cm⁻¹ and 1592 cm⁻¹ shifted to 1608 cm⁻¹ and 1588 cm⁻¹, respectively, indicating that a covalent bond was formed between the -NH₂ group of CTS and the -COOH group of Tyr. Furthermore, the amide peak of CTS shifted from 1645 cm⁻¹ and 1606 cm⁻¹ to 1608 cm⁻¹ and 1588 cm⁻¹, which can also be attributed to the formation of secondary amides. For CTS-leucine (Leu) graft copolymers (… Figure 1 (B) Compared to pure CTS and Leu, the newly formed 3420 cm⁻¹ characteristic peak in the CTS-Leu copolymer replaces the -NH₂ group peaks of CTS and Leu. Correspondingly, the C=O stretching vibration peak and NH bending vibration peak of Leu at 1613 cm⁻¹ and 1569 cm⁻¹ also change to 1645 cm⁻¹ and 1588 cm⁻¹, respectively. In the CTS-tryptophan (Trp) copolymer (… Figure 1 A similar trend was observed in C).

[0072] Furthermore, the structure of the synthesized CTS-AA graft copolymer was characterized by ¹H-NMR. Figure 2 As shown in Figure A, taking CTS-Tyr as an example, compared with pure CTS and Tyr, a new doublet (d) appears at 6.0 ppm in the spectrum of CTS-Tyr. This can be attributed to the cis-trans isomerism of the secondary amide compound, and its ¹H-NMR spectrum usually shows signals corresponding to both isomers simultaneously, indicating that the -COOH group of Tyr has been successfully attached to the NH2 group of CTS. In the CTS-Leu and CTS-Trp graft copolymers ( Figure 2 Similar results were observed in B and C, which further confirms the successful linkage between CTS and amino acids.

[0073] 2. Preparation and optimization of polyelectrolyte complexes (PECs).

[0074] CTS and chondroitin sulfate (CS) are commonly used carriers for preparing polyelectrolyte complexes via electrostatic interactions to deliver biomolecules. In this study, a CTS-CS polyelectrolyte complex loaded with oxytocin (OT) was prepared and optimized. First, the concentrations of CTS and OT were kept constant at 0.5 mg / mL, and the effect of CS concentration on the properties of the polyelectrolyte complex was investigated. Figure 3 As shown in A and B, the CS concentration significantly affects the particle size of the prepared polyelectrolyte complexes. This is attributed to the different charge densities provided by different CS concentrations, with the CTS-CS polyelectrolyte complex exhibiting the smallest particle size at a CS concentration of 0.25 mg / mL. Notably, when the CS concentration is below 1 mg / mL, all polyelectrolyte complexes exhibit a positive potential, indicating that the negatively charged OT and CS are encapsulated within the polyelectrolyte complex, which is also reflected by the high encapsulation efficiency (EE, %). Upon further increasing the CS concentration, a negative potential was observed in the polyelectrolyte complexes, indicating that the CTS within the polyelectrolyte complexes has reversed.

[0075] Furthermore, the effect of medium pH on the prepared polyelectrolyte composite was investigated. As the pH increased from 4 to 5, the particle size of the polyelectrolyte composite gradually decreased from approximately 268 nm to 223 nm. Figure 3 C). When the pH value was increased to 5.5, a significant increase in particle size was observed to be approximately 297 nm (p<0.05). However, pH value had no significant effect on zeta potential and encapsulation efficiency (%).

[0076] Based on the above results, various CTS-amino acids (CTS-AA) and CS polyelectrolyte complexes loaded with OT were prepared at concentrations of 0.25 mg / mL and 0.5 mg / mL, respectively, under pH 5 conditions. Figure 3 As shown in E and F, the polyelectrolyte complexes based on the three amino acid-modified CTS have similar particle sizes, ranging from 216 to 254 nm. Notably, all three polyelectrolyte complexes exhibit strong positive charges and high encapsulation efficiencies (%), approximately 80%, indicating the successful preparation of polyelectrolyte complexes based on amino acid-coupled CTS.

[0077] Table 1. Encapsulation efficiency (EE, %) and drug loading (DL, %) of PECs prepared under different conditions.

[0078] 3. Characterization of polyelectrolyte complexes (PECs).

[0079] The morphology of oxytocin (OT)-loaded chitosan-chondroitin sulfate (CTS-CS) or amino acid-chitosan-chondroitin sulfate (AA-CTS-CS) polyelectrolyte complexes was observed using transmission electron microscopy (TEM). Figure 4 As shown in AD, all polyelectrolyte complex formulations exhibited spherical to elliptical particle shapes and good dispersibility. The CTS-CS polyelectrolyte complex and polyelectrolyte complexes based on various amino acid-coupled CTSs had comparable particle sizes, ranging from 180 to 250 nm, consistent with dynamic light scattering (DLS) measurements. Furthermore, regardless of amino acid modifications, the CTS-CS polyelectrolyte complexes displayed similar morphologies, indicating that different amino acid modifications did not alter the structure of the polyelectrolyte complexes.

[0080] To further investigate whether the conformation and structure of the polyelectrolyte complex changed after preparation, circular dichroism spectroscopy (CD) was used for detection. This technique is widely used to determine structural changes in proteins. Figure 4 As shown in Figure E, the circular dichroism spectra of OT immediately recovered from various polyelectrolyte complex formulations exhibit typical double negative peaks at 208 nm and 216 nm, indicating that OT possesses a large number of α-helical structures and that the spectra are highly consistent with those of natural OT. This suggests that the random coil structure of OT remains unchanged, indicating that OT exhibits good physicochemical stability in polyelectrolyte complexes.

[0081] 4. In vitro release study of oxytocin-loaded polyelectrolyte complexes (PECs) This study also investigated and compared the in vitro release of oxytocin-loaded chitosan-chondroitin sulfate polyelectrolyte complexes (OT-CTS-CS PECs) and various amino acid-coupled chitosan-chondroitin sulfate polyelectrolyte complexes (AA-CTS-CSPECs) with oxytocin solution. Figure 4 As shown in Figure F, oxytocin solution was rapidly released within 1 hour, with approximately 93.4% of the oxytocin being released, and the release process conformed to a first-order kinetic model (R² = 0.9936). However, all polyelectrolyte complex formulations exhibited sustained release behavior. Over 16 hours, the release curves of oxytocin in various polyelectrolyte complexes showed a relatively rapid release in the initial stage (approximately 2 hours), exhibiting a burst release effect (approximately 55%), followed by sustained release conforming to the Riger-Pappas model (R² > 0.9), with approximately 80% of the oxytocin being released within 16 hours. Meanwhile, there was no significant difference between the CTS-CS polyelectrolyte complex and various amino acid-grafted CTS-CS polyelectrolyte complexes. f 2 >50), indicating that the modification of different amino acids on chitosan did not affect the in vitro release behavior of the encapsulated oxytocin.

[0082] 5. In vitro permeation studies.

[0083] In vitro permeation is a key parameter for evaluating the delivery effect of nasal mucosa. This study used a Franz diffusion cell to investigate the in vitro permeation behavior of naked OT-CTS-CS polyelectrolyte complex and CTS-CS polyelectrolyte complex grafted with three amino acids on freshly excised goat nasal mucosa, and compared it with oxytocin solution. Figure 4 As shown in G, the nasal mucosal permeability of the oxytocin solution was low, with less than 10% of oxytocin able to pass through the nasal mucosa. In contrast, the cumulative permeation of oxytocin from the CTS-CS polyelectrolyte complex increased significantly over 6 hours, reaching approximately 353.57 ± 8.9 μg / cm². This may be attributed to the nanoparticle size characteristics of the polyelectrolyte complex and the absorption-enhancing effect of chitosan, which can reversibly open tight junctions between cells. Interestingly, the leucine-chitosan-chondroitin sulfate polyelectrolyte complexes (Leu-CTS-CS PECs) and tyrosine-chitosan-chondroitin sulfate polyelectrolyte complexes (Tyr-CTS-CS PECs) exhibited even higher oxytocin permeability, with cumulative permeations of 360.51 ± 9.6 μg / cm² and 374.74 ± 8.75 μg / cm², respectively. Meanwhile, compared to the CTS-CS polyelectrolyte complex, the steady-state flow rate (J) and permeability coefficient (Kp) of the leucine or tyrosine-grafted CTS-CS polyelectrolyte complex were also higher, indicating that amino acid coupling can promote faster oxytocin permeation through the mucosa. This may be attributed to the presence of L-amino acid transporter 1 (LAT1) distributed in the mucosa, which can promote the transport of amino acid-grafted polyelectrolyte complexes. For the tryptophan-chitosan-chondroitin sulfate polyelectrolyte complex (Trp-CTS-CS PECs), although the observed cumulative oxytocin permeation was lower, approximately 331.70 ± 11.3 μg / cm², its J and Kp values ​​were similar to those of the leucine or tyrosine-modified CTS-CS polyelectrolyte complex. Based on these results, compared to naked CTS-CS particles, the polyelectrolyte complexes prepared by grafting selected amino acids onto chitosan and chondroitin sulfate can further improve oxytocin transport and exhibit excellent mucosal permeability.

[0084] Table 2. In vitro permeation data of the nasal mucosa model (related to model equations).

[0085] 6. Rat pharmacokinetic studies.

[0086] To further investigate whether amino acid-grafted chitosan (CTS) can improve oxytocin (OT) absorption in vivo, this study investigated the pharmacokinetic behavior of various polyelectrolyte complex (PEC) formulations and compared them with oxytocin solutions. Figure 5 As shown in Figure A, after nasal administration of oxytocin solution, the plasma drug concentration rose slightly at approximately 5 minutes, then rapidly decreased. However, after nasal administration of various polyelectrolyte complex formulations, the time to peak plasma concentration (Tmax) was approximately 10 minutes, indicating that polyelectrolyte complexes allow for a more sustained absorption of oxytocin into the systemic circulation. According to Table 3, after nasal administration of the CTS-CS polyelectrolyte complex, the average relative bioavailability of oxytocin reached 192.11%, indicating that non-invasive nasal administration using polyelectrolyte complexes can increase oxytocin absorption by approximately 1.9 times compared to nasal administration of oxytocin solution. Furthermore, this study also investigated the effects of chitosan grafted with different amino acids on nasal absorption of oxytocin. Figure 5 Compared with the naked CTS-CS polyelectrolyte complex, the time to peak concentration (Tmax) of chitosans coupled with various amino acids did not change. However, the area under the curve (AUC0-∞) of chitosans modified with leucine (Leu), tyrosine (Tyr), and tryptophan (Trp) increased by 2.63 times, 2.87 times, and 3.12 times, respectively (Table 3). This indicates that amino acid coupling does not affect the absorption rate, but it can significantly improve the absorption of oxytocin in the nasal cavity. This suggests that introducing amino acids into chitosan can further enhance the ability of oxytocin to pass through the nasal mucosa, thereby improving its absorption rate. Meanwhile, among these three amino acids, the tryptophan-chitosan-chondroitin sulfate polyelectrolyte complex (Trp-CTS-CS PECs) had the highest Cmax (peak plasma concentration) and AUC0-∞, followed by the tyrosine-chitosan-chondroitin sulfate polyelectrolyte complex (Tyr-CTS-CS PECs) and the leucine-chitosan-chondroitin sulfate polyelectrolyte complex (Leu-CTS-CS PECs). This may be due to the different effects of the conformation and structure of different amino acids on mucosal transport, which is consistent with previous research results.

[0087] Table 3. Pharmacokinetic parameters of oxytocin in plasma after intranasal administration of various formulations (n=6).

[0088] 7. Distribution of the rat brain.

[0089] The above results indicate that intranasal administration of three amino acid-modified chitosan-chondroitin sulfate polyelectrolyte complexes (CTS-CSPECs) can enhance the systemic absorption and brain accumulation of oxytocin (OT). In this part of the study, Cy5-labeled oxytocin was loaded into various polyelectrolyte complexes. After intranasal administration, the intensity of intrabrain fluorescence distribution was detected at different time points and compared with Cy5-labeled oxytocin solution. Figure 5 B shows that the intranasal fluorescence intensity of Cy5-labeled oxytocin solution reached its peak 1 hour after administration, and then gradually decreased. A similar trend was observed for the CTS-CS polyelectrolyte complex, but its intrabrain distribution was faster, reaching its strongest fluorescence intensity at 0.5 hours. In contrast, the tyrosine-chitosan-chondroitin sulfate polyelectrolyte complexes (Tyr-CTS-CS), tryptophan-chitosan-chondroitin sulfate polyelectrolyte complexes (Trp-CTS-CS), and leucine-chitosan-chondroitin sulfate polyelectrolyte complexes (Leu-CTS-CS) exhibited stronger fluorescence intensity and longer duration of intrabrain fluorescence, up to 4 hours, indicating that amino acid-grafted chitosan can further enhance the absorption and distribution of oxytocin in the brain. Furthermore, among the three selected amino acids, tryptophan-grafted chitosan showed the best performance in terms of intrabrain distribution and drug duration, consistent with its pharmacokinetic behavior.

[0090] Given the crucial roles of brain regions such as the hippocampus and prefrontal cortex in the pathological characteristics of addiction, and the main mechanism by which nasal administration achieves targeted delivery to brain regions via the olfactory pathway, this study further investigated the effect of nasal administration of a tyrosine-, tryptophan-, and leucine-grafted CTS-CS polyelectrolyte complex on the brain region targeting of oxytocin. First, the oxytocin-loaded CTS-CS polyelectrolyte complex was administered nasally, and the distribution of oxytocin in various brain regions was examined. For example... Figure 6 As shown, during the experiment, only small amounts of oxytocin were detected in the olfactory bulb, hippocampus, and forebrain tissue, indicating that only a small amount of oxytocin was able to translocate to brain tissue. Interestingly, according to... Figure 6In the study of BD, the time to peak concentration (Tmax) of oxytocin in the olfactory bulb, forebrain, and hippocampus of these amino acid-grafted CTS-CS polyelectrolyte complexes was 0.5 h, 1 h, and 2 h, respectively. This indicates that the oxytocin encapsulated in these polyelectrolyte complexes is absorbed into the brain via the olfactory pathway, subsequently entering the forebrain and hippocampus. Furthermore, the Cmax (peak plasma concentration) of the amino acid-grafted CTS-CS polyelectrolyte complexes in the olfactory bulb, forebrain, and hippocampus was significantly higher than that of the naked CTS-CS polyelectrolyte complex. This demonstrates that the amino acid-grafted CTS-CS polyelectrolyte complex can further enhance the bioavailability of oxytocin and increase its accumulation in the brain through L-amino acid transporter 1 (LAT1). Furthermore, among the three amino acid-grafted CTS-CS polyelectrolyte complexes, the tryptophan-chitosan-chondroitin sulfate polyelectrolyte complex (Trp-CTS-CS) had the highest Cmax in the hippocampus, forebrain, and olfactory bulb, followed by the tyrosine-chitosan-chondroitin sulfate polyelectrolyte complex (Tyr-CTS-CS) and the leucine-chitosan-chondroitin sulfate polyelectrolyte complex (Leu-CTS-CS). Moreover, for all amino acid-grafted CTS-CS polyelectrolyte complexes, the enrichment of oxytocin consistently showed the order of forebrain > olfactory bulb > hippocampus.

[0091] 8. Inhibition of oxytocin (OT) on conditioned position preference (CPP) of oxycodone (Oxy).

[0092] Figure 7 The experimental procedure for establishing the conditioned position preference test with oxytocin and the specific administration protocol for oxytocin were demonstrated. The post-conditioned position preference test (day 12) showed that, compared with the control group (Con group), oxytocin significantly increased the time mice spent on the drug-pairing side. Figure 7 (AF), indicating that oxycodone successfully induced drug addiction in mice. During the conditional positional preference acquisition phase, oxytocin pretreatment significantly reduced the time mice spent on the drug-pairing side compared to the oxycodone group (Oxy group) (Oxy+OT group, Oxy+OT-CTS-CS group: p<0.05; Oxy+OT-Leu-CTS-CS group, Oxy+OT-Tyr-CTS-CS group: p<0.01; Oxy+OT-Trp-CTS-CS group: p<0.001; Figure 7 A). During the conditional position preference expression phase, oxytocin pretreatment also significantly reduced the time mice spent on the drug-paired side (Oxy+OT group, Oxy+OT-CTS-CS group, Oxy+OT-Leu-CTS-CS group: p<0.05; Oxy+OT-Tyr-CTS-CS group: p<0.01; Oxy+OT-Trp-CTS-CS group: p<0.001; Figure 7B). During the extinction phase of conditioned position preference, mice naturally achieved complete extinction on day 6 (E6) after drug withdrawal. Figure 7 C). In contrast, mice in the oxytocin-loaded formulation showed complete regression on day 2 (E2, OT-Trp-CTS-CS group), day 3 (E3, OT-Tyr-CTS-CS group, OT-Leu-CTS-CS group), or day 4 (E4, OT group, OT-CTS-CS group) after drug withdrawal. Figure 7 DE). In the conditional position preference re-ignition phase, compared with the oxytocin group, oxytocin pretreatment significantly reduced the time mice spent on the drug-paired side (Oxy+OT group, Oxy+OT-CTS-CS group: p<0.05; Oxy+OT-Leu-CTS-CS group, Oxy+OT-Tyr-CTS-CS group: p<0.01; Oxy+OT-Trp-CTS-CS group: p<0.001; Figure 7 These results indicate that oxytocin formulations inhibit the acquisition of conditioned positional preference for oxycodone, block its expression, promote extinction, and eliminate restraint stress-induced relapse. Among various oxytocin-related formulations, this study found that OT-tryptophan-chitosan-chondroitin sulfate polyelectrolyte complexes (OT-Trp-CTS-CS PECs) had the strongest inhibitory effect on oxycodone addiction. This superior efficacy was attributed to the formulation significantly reducing the time spent on the drug-paired side in oxycodone-treated mice during the acquisition, expression, and relapse phases in the study. Furthermore, during the extinction phase, OT-Trp-CTS-CS PECs facilitated the earliest successful withdrawal in addicted mice. In contrast, the inhibitory effects of OT-tyrosine-chitosan-chondroitin sulfate polyelectrolyte complexes (OT-Tyr-CTS-CS PECs), OT-leucine-chitosan-chondroitin sulfate polyelectrolyte complexes (OT-Leu-CTS-CS PECs), and OT-CTS-CS polyelectrolyte complexes and oxytocin solution on oxycodone addiction gradually weakened. This finding is consistent with previously reported results that OT-Trp-CTS-CSPECs exhibit excellent brain distribution and brain targeting.

[0093] c-Fos, as an immediate early gene, plays a crucial role in molecular processes such as learning, memory formation, and synaptic plasticity. Its dynamic expression pattern closely reflects neuronal activity during key stages of memory encoding and consolidation, serving as a key molecular link between transient neural activation and persistent changes in synaptic connections. One hour after the reignition phase, we assessed the inhibitory effect of oxytocin on oxycodone-induced conditional place preference by detecting c-Fos expression in mouse hippocampal neurons. The results showed that oxytocin significantly reduced neuronal c-Fos expression, indicating that oxytocin can inhibit addiction by suppressing neuronal c-Fos expression. Figure 8 Furthermore, the inhibitory effect of oxytocin solution and various oxytocin-loaded polyelectrolyte complexes gradually increased, which is consistent with the results of conditional position preference tests.

[0094] The above description is merely a preferred embodiment of the present invention and is not intended to limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A highly effective brain-targeting oxytocin nasal spray, characterized in that, It comprises a polyelectrolyte complex formed by amino acid-grafted chitosan and anionic polysaccharides, wherein the polyelectrolyte complex is loaded with oxytocin.

2. The nasal spray as described in claim 1, characterized in that, The amino acid is selected from at least one of tryptophan, leucine, or tyrosine.

3. The nasal spray as described in claim 1, characterized in that, The anionic polysaccharide includes chondroitin sulfate, sodium alginate, hyaluronic acid, and gelatin.

4. The nasal spray as described in claim 1, characterized in that, The mass ratio of the amino acid-grafted chitosan to chondroitin sulfate is 1:0.5 to 1:

2.

5. The nasal spray as described in claim 1, characterized in that, The nasal spray has a pH value of 4.5–5.

5.

6. Use of the nasal spray according to any one of claims 1–5 in the preparation of a medicament for the prevention or treatment of opioid addiction.

7. The application as described in claim 6, characterized in that, The opioid drug in question is oxycodone.

8. A method for preparing a nasal spray as described in any one of claims 1-5, comprising the following steps: S1 grafts amino acids onto chitosan to obtain an amino acid-chitosan copolymer; S2 The copolymer and anionic polysaccharide are mixed under acidic conditions, and oxytocin solution is added to form a polyelectrolyte complex; S3 was used to adjust the pH and purify the product to obtain a polyelectrolyte complex loaded with oxytocin. S4 formulated it into a nasal spray.

9. The method according to claim 8, characterized in that, In step S2, the acidic conditions are pH 4.0-6.0.

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