A tripterine nasal nanoparticle and a preparation method thereof

Abstract

The present application relates to the technical field of biological medicine and pharmaceutical science, and particularly relates to a tripterine nanoparticle for nasal administration and a preparation method thereof. The present application uses lactoferrin as a carrier to prepare the tripterine nanoparticle through reverse solvent induced self-assembly and ultrasonic treatment. The nanoparticle has a particle size range of about 100 nm to 300 nm, a surface potential of about -20 mV to 40 mV, and uniform particle size. The nanoparticle can be used for nasal administration to improve brain drug delivery, improve drug bioavailability, and ultimately enhance the treatment effect of central nervous system diseases.

Description

Technical Field

[0001] This invention relates to the interdisciplinary field of biomedicine and pharmaceutics, and in particular to a triptolide nanoparticle administered via nasal cavity and its preparation method. Background Technology

[0002] Central nervous system diseases, including stroke, Alzheimer's disease, Parkinson's disease, brain tumors, epilepsy, and multiple sclerosis, are characterized by high rates of disability and mortality, as well as complex pathological mechanisms. While drug development for these diseases has made continuous progress, few drugs have been able to achieve effective intracerebral delivery and satisfactory clinical efficacy.

[0003] The main reason for this is that the central nervous system possesses physiological barriers such as the blood-brain barrier and the blood-cerebrospinal fluid barrier, which are highly selective for exogenous substances. These barriers maintain the homeostasis of the brain's microenvironment, but also significantly limit the entry of therapeutic drugs into brain tissue. Typically, most small-molecule drugs and almost all large-molecule drugs struggle to cross these barriers and reach effective therapeutic concentrations in the brain. Furthermore, efflux transporters such as P-glycoproteins can actively pump drugs out of the brain, further reducing drug accumulation and residence time at the lesion site. Therefore, developing therapeutic strategies that combine highly effective brain targeting, low systemic toxicity, and synergistic effects through multiple mechanisms has become an urgent need in this field.

[0004] Celastrol, a natural active product extracted from the root bark of Tripterygium wilfordii, possesses various pharmacological activities including anti-inflammatory, antioxidant, and anti-apoptotic effects. It exerts neuroprotective effects through multiple signaling pathways and shows promising application potential in the treatment of central nervous system diseases such as cerebral ischemia-reperfusion injury. However, celastrol's physicochemical properties and in vivo processing characteristics have significant drawbacks, severely restricting its clinical translation. First, its water solubility is extremely low, with a solubility of only about 0.04 mg / mL at 25°C, classifying it as a poorly soluble drug. Second, its oral bioavailability is low, and its therapeutic index is narrow, easily causing significant systemic toxicity, thus limiting its clinical application under conventional administration methods. Therefore, developing new routes of administration and formulations with better tolerability and delivery efficiency has become an important direction for improving the drug development of celastrol.

[0005] Intranasal drug delivery, as a non-invasive route, can partially bypass the blood-brain barrier through the olfactory nerve and trigeminal nerve pathways, enabling direct drug delivery to the central nervous system. It offers advantages such as being minimally invasive, painless, having a rapid onset of action, and high patient compliance, thus attracting widespread attention in the development of drugs for central nervous system diseases. For active ingredients like triptolide, which are difficult to deliver effectively into the brain using traditional methods, intranasal delivery shows great potential.

[0006] However, intranasal drug delivery still faces a number of limitations in practical applications. The limited volume of the nasal cavity and the small single-dose volume restrict drug loading and formulation design flexibility. Simultaneously, the nasal mucus layer and mucociliary clearance mechanism rapidly remove exogenous drugs, resulting in a short drug retention time and impacting absorption efficiency. Furthermore, the nasal mucosal epithelial barrier limits drug permeability, and local enzymatic degradation may reduce drug stability and bioavailability. For naso-brain delivery, further challenges remain, including insufficient local retention, limited nasal mucosal transport efficiency, and uneven distribution after entering the brain.

[0007] Therefore, when developing nasal delivery formulations of triptolide, it remains an urgent technical challenge to improve the drug's adhesion and stability in the nasal cavity, prolong its retention time, enhance transmucosal absorption and naso-brain delivery efficiency, and reduce local irritation, while also improving its poor solubility and reducing systemic toxicity. Summary of the Invention

[0008] This invention addresses the shortcomings of existing technologies by providing a triptolide nasal nanoparticle formulation and its preparation method, comprising the following steps: An organic solution containing triptolide was mixed with an aqueous antisolvent system containing lactoferrin. Triptorelide nanoparticles with lactoferrin as a carrier were formed by antisolvent-induced self-assembly and ultrasonic treatment. The organic solvent in the resulting dispersion system was then removed. The mass ratio of triptolide to lactoferrin is 2:(0.7-5), and the volume ratio of aqueous antisolvent to organic solvent is (3.5-14):1.

[0009] Tripterygium wilfordii forms a nano-dispersed structure under antisolvent-induced conditions. Lactoferrin can be used as a carrier component to encapsulate or stabilize the tripterygium wilfordii, forming a nanoparticle system with potential for brain-targeted delivery.

[0010] In some embodiments, the mass ratio of triptolide to lactoferrin can be 2:(0.8-3), preferably 2:(0.8-1.5). In some embodiments, the volume ratio of the aqueous antisolvent to the organic solvent can be (4-12):1, preferably (6-10):1. By adjusting the ratio of triptolide to lactoferrin and the volume relationship between the antisolvent system and the organic solvent system, it is beneficial to control the formation process, particle size distribution, and system stability of nanoparticles, and to form a nano-dispersion system suitable for nasal administration.

[0011] In some embodiments, the power of the ultrasonic treatment can be 120 W to 400 W, preferably 200 W to 320 W. In some embodiments, the ultrasonic treatment time can be 2 min to 25 min, preferably 12 min to 18 min. The ultrasonic treatment can facilitate the self-assembly process between triptolide and lactoferrin, and improve the uniformity and dispersion stability of the nanoparticle system.

[0012] In some embodiments, the organic solvent may include dichloromethane, ethyl acetate, acetone, ethanol, methanol, or combinations thereof. The method for removing the organic solvent may include rotary evaporation, vacuum evaporation, nitrogen blowing, or freeze-drying. In some embodiments, by removing the organic solvent, a stable triptolide nanoparticle dispersion system or its freeze-dried product can be obtained.

[0013] The present invention also provides triptolide nanoparticles prepared by the above preparation method.

[0014] The triptolide nanoparticles can form a nanoscale particle structure and exhibit good dispersibility and stability.

[0015] The lactoferrin can serve as a delivery carrier and brain-targeting component, enhancing the delivery and accumulation of nanoparticles in brain tissue by binding to relevant receptors in the brain; the triptolide, as an active pharmaceutical component, can exert anti-inflammatory, antioxidant, and neuroprotective effects.

[0016] The present invention further provides a nasal administration formulation comprising the above-mentioned triptolide nanoparticles and pharmaceutically acceptable excipients.

[0017] In some embodiments, the nasal delivery formulation may be a nasal drop, spray, in-situ gel, nanosuspension, lyophilized powder, or nasal dry powder. The nasal delivery formulation can improve the delivery efficiency of the active drug to the central nervous system via the naso-brain delivery pathway and help prolong the drug's residence time in the nasal cavity.

[0018] The present invention also provides the use of the triptolide nanoparticles in the preparation of nasal administration formulations for the treatment of nervous system diseases, inflammation or autoimmune diseases.

[0019] In some embodiments, the nasal delivery formulation may be a nasal drop, spray, in-situ gel, nanosuspension, lyophilized powder, or nasal dry powder.

[0020] The triptolide nanoparticle system provided by this invention can improve the dispersibility, stability, and delivery efficiency of triptolide, and in some embodiments, enhance the drug's accumulation capacity in brain tissue. The nanoparticle system formed by antisolvent-induced self-assembly combined with ultrasonic treatment facilitates the formation of smaller and more uniformly dispersed nanoparticles, and is suitable for nasal administration. The nanodelivery system formed by lactoferrin and triptolide can also help improve neurological deficits, reduce the degree of brain tissue damage, alleviate oxidative stress-related damage, and improve the intervention effect on related neurological diseases. Attached Figure Description

[0021] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0022] Figure 1 The image shows the morphology of the triptolide nanoparticles and their freeze-dried products in Example 1.

[0023] Figure 2 This is a particle size distribution diagram of the triptolide nanoparticles in Example 1.

[0024] Figure 3 This is a surface charge distribution diagram of the triptolide nanoparticles in Example 1.

[0025] Figure 4 This is a morphological image of triptolide nanoparticles under a transmission electron microscope in Example 1.

[0026] Figure 5 The image shows a scanning electron microscope image of triptolide raw material and triptolide nanoparticles in Example 1.

[0027] Figure 6 The graph shows the changes in particle size and PDI of the triptolide nanoparticles in Example 1 during storage at 4°C for 2 months.

[0028] Figure 7 The following are in vitro drug release curves of triptolide raw material and triptolide nanoparticles in Experiment Example 1; (a) is the release curve of triptolide raw material and triptolide nanoparticles in artificial nasal liquid (ANF) at pH 6.8; (b) is the release curve of triptolide raw material and triptolide nanoparticles in artificial cerebrospinal fluid (ACSF) at pH 7.4; where Cel-ethanol represents the triptolide raw material group and LF-Cel NPs represents the triptolide nanoparticle group.

[0029] Figure 8 The images show in vivo fluorescence imaging and quantitative analysis of fluorescence intensity in the nasal cavity region of mice after intranasal administration of free triptolide and triptolide nanoparticles in Experiment Example 3; (a) In vivo fluorescence imaging of the nasal cavity region of mice at different time points (0, 2, 4, 6, 12, 24 h); (b) Quantitative analysis of fluorescence intensity in the corresponding nasal cavity region, with the fluorescence intensity normalized to 100% at 0 h after administration.

[0030] Figure 9 The images show in vivo fluorescence imaging and quantitative analysis of free triptolide and triptolide nanoparticles in brain tissue after intranasal administration in Experiment Example 4; (a) In vivo fluorescence imaging of rat brain tissue at 2, 4, 6 and 12 h after administration; (b) Quantitative analysis curve of average fluorescence intensity of brain tissue at different time points.

[0031] Figure 10 The bar chart shows the neurological function scores of rats in the cerebral ischemia-reperfusion injury model after intranasal administration in Experiment Example 5; where Sham represents the sham-operated group, MCAO / R represents the model group, LF represents the lactoferrin solution group, Cel represents the triptolide free drug group, and LF-Cel NPs represents the triptolide nanoparticle group.

[0032] Figure 11 The image shows TTC staining of brain tissue in rats with cerebral ischemia-reperfusion injury model after intranasal administration of the drug in Experiment Example 5. In the image, Sham represents the sham-operated group, MCAO / R represents the model group, LF represents the lactoferrin solution group, Cel represents the free triptolide group, and LF-Cel NPs represents the triptolide nanoparticle group.

[0033] Figure 12 This is a statistical chart showing the percentage of cerebral infarction volume in rats with cerebral ischemia-reperfusion injury model after intranasal administration in Experiment Example 5; where Sham represents the sham-operated group, MCAO / R represents the model group, LF represents the lactoferrin solution group, Cel represents the free triptolide group, and LF-Cel NPs represents the triptolide nanoparticle group.

[0034] Figure 13The results of nasal administration of inflammatory factors in brain tissue of rats with cerebral ischemia-reperfusion injury in Experiment Example 6 are as follows: (a) Interleukin-1β (IL-1β) level, (b) Tumor necrosis factor-α (TNF-α) level, (c) Interleukin-10 (IL-10) level, and (d) Interleukin-4 (IL-4) level. Among them, Sham represents the sham operation group, MCAO / R represents the model group, LF represents the lactoferrin solution group, Cel represents the triptolide free drug group, and LF-Cel NPs represents the triptolide nanoparticle group.

[0035] Figure 14 The results of peripheral blood routine tests in SD rats after intranasal administration in Experiment Example 7 are shown in (af); the results of platelet, erythrocyte, leukocyte, lymphocyte, granulocyte and monocyte related indicators are shown in (af); Normal represents the normal group, LF represents the lactoferrin solution group, Cel represents the triptolide free drug group, and LF-Cel NPs represents the triptolide nanoparticle group.

[0036] Figure 15 HE staining images of various organs of SD rats after intranasal administration in Experiment Example 7. In the images, Normal represents the normal group, LF represents the lactoferrin solution group, Cel represents the free triptolide group, and LF-Cel NPs represents the triptolide nanoparticle group. Detailed Implementation

[0037] The embodiments of the present invention will now be described in detail. The following examples are only used to explain the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. After reading this specification, those skilled in the art can make various modifications, substitutions or variations to the technical solutions of the present invention without departing from the concept and technical principles of the present invention, and all such modifications, substitutions or variations should fall within the scope of protection of the present invention.

[0038] Unless otherwise stated, the technical and scientific terms used herein shall have the meanings commonly understood by one of ordinary skill in the art. Unless otherwise specified, the experimental methods described herein shall be performed under standard experimental conditions, in accordance with relevant laboratory manuals, or as recommended by the manufacturers of reagents or instruments, or using other equivalent methods known in the art.

[0039] The terms "comprising," "including," and "containing" as used herein are open-ended expressions, and unless explicitly specified, they do not exclude the presence of other components, steps, or structures not explicitly listed. The terms "preferred," "optional," and "optional" as used herein are only for illustrating some embodiments and do not constitute a limitation on the scope of protection of this invention.

[0040] Unless otherwise specified, the numerical ranges mentioned in this article include any integers, fractions, decimals, and their upper and lower limits within that range.

[0041] Unless otherwise specified, the singular expressions such as "a," "a kind," and "the" used in this article also include the plural forms; "multiple" usually refers to two or more.

[0042] As used in this document, the term "and / or" refers to any one or any combination of the relevant objects.

[0043] Where there is no contradiction, the technical features of the various embodiments and examples in this specification can be combined with each other. Any conventional adjustments, substitutions, or equivalent modifications made by those skilled in the art based on the content of this specification should be considered part of the disclosure herein.

[0044] The references cited in this invention are only used to illustrate the background technology or related technical content of this invention, and their disclosures may be used for reference only if they do not conflict with the technical solutions of this invention.

[0045] In this article, "celastrol" refers to a pentacyclic triterpenoid compound and its pharmaceutically acceptable form derived from the Celastraceae plant Tripterygium wilfordii or obtained through chemical synthesis, semi-synthesis, biotransformation, or other means. Celastrol may include its amorphous form, crystalline form, solvate, hydrate, prodrug, pharmaceutically acceptable salt, or combinations thereof.

[0046] In this document, "lactoferrin" refers to an iron-binding glycoprotein capable of forming a nanodelivery system with triptolide. The lactoferrin can be derived from mammalian milk, recombinant expression systems, or other biological sources, and may include natural lactoferrin, recombinant lactoferrin, its functional fragments, derivatives, or combinations thereof. In this invention, lactoferrin should not be construed as limited to a specific source or purity level; it can be used as a carrier component, a stabilizing component, and a brain-targeting component.

[0047] In this paper, "triptocarmine nanoparticles" refers to a nanoscale particle system formed by the antisolvent-induced self-assembly of lactoferrin and triptocarmine. The nanoparticles can exhibit spherical, near-spherical, or other nanoscale dispersed structures and can exist in dispersions, lyophilized powders, or other redispersible forms. In this invention, the nanoparticles should not be construed as necessarily containing additional polymeric carriers, liposome frameworks, or polymer encapsulation structures.

[0048] In this paper, "aqueous antisolvent" refers to a liquid-phase system with water as the continuous phase that can reduce the solubility of triptolide in the system and induce its formation of a nano-dispersion structure. The aqueous antisolvent can be used to promote the antisolvent-induced self-assembly process between triptolide and lactoferrin, and is beneficial to the formation of a stable triptolide nanoparticle system.

[0049] In this paper, "antisolvent-induced self-assembly" refers to the process by which triptolide forms a nanostructure due to changes in solubility in an antisolvent environment, and then forms a stable nanoparticle system with the participation of lactoferrin. The self-assembly process may include hydrophobic interactions, electrostatic interactions, hydrogen bonding interactions, van der Waals interactions, or combinations thereof, but this invention is not limited to a specific self-assembly mechanism.

[0050] In this article, "pharmaceuticalally acceptable excipients" refers to excipient components that can be used to prepare pharmaceutical formulations and are compatible with triptolide nanoparticles.

[0051] The first aspect of the present invention relates to a method for preparing triptolide nasal nanoparticles, comprising the following steps: An organic solution containing triptolide was mixed with an aqueous antisolvent system containing lactoferrin. Triptorelide nanoparticles with lactoferrin as a carrier were formed by antisolvent-induced self-assembly and ultrasonic treatment. The organic solvent in the resulting dispersion system was then removed. The mass ratio of triptolide to lactoferrin is 2:(0.7-5), and the volume ratio of aqueous antisolvent to organic solvent is (3.5-14):1.

[0052] Due to its strong hydrophobicity, triptolide has low solubility in aqueous systems. Therefore, upon contact between the organic phase and the aqueous antisolvent system, it can undergo desolvation during solvent exchange, forming a nanoscale dispersion structure. In this invention, lactoferrin not only participates in the formation of triptolide nanoparticles but also plays multiple functional roles at different levels. In some aspects, lactoferrin acts as a carrier component, forming a stable self-assembled structure with triptolide through hydrophobic interactions, electrostatic interactions, hydrogen bonding, van der Waals interactions, or combinations thereof, thereby achieving nanoscale delivery of triptolide. In other aspects, lactoferrin can also act as a stabilizing component, improving particle dispersion, reducing particle aggregation tendency, and enhancing system storage stability, thus facilitating the acquisition of a uniform and long-term stable nanoparticle system. Furthermore, in other aspects, lactoferrin can also act as a brain-targeting component, interacting with brain tissue-related receptors to promote the transport of nanoparticles to the central nervous system via the naso-brain delivery pathway and enhance drug accumulation in brain tissue.

[0053] The ultrasonic treatment can promote rapid mixing between the organic phase and the aqueous system, and is beneficial for reducing particle aggregation, improving particle size uniformity, and enhancing system stability. In some embodiments, the antisolvent induction process and the ultrasonic treatment process can be performed simultaneously or partially overlapped.

[0054] The resulting triptolide nanoparticles can exhibit spherical or near-spherical nanostructures and form a stable nanodispersion system. These nanoparticles can have a small particle size and low polydispersity, which is beneficial for improving system stability and subsequent nasal delivery efficiency.

[0055] In some embodiments, the resulting nanoparticle system can be further concentrated, lyophilized, reconstituted, or formulated.

[0056] In this invention, the aqueous antisolvent should not be construed as limited to pure water systems, but may also include other aqueous systems capable of maintaining system stability, adjusting osmotic pressure, adjusting pH, or improving dispersibility. In some embodiments, the water may include ultrapure water, deionized water, distilled water, purified water, or combinations thereof. In some embodiments, the aqueous antisolvent may be a pure water system, an aqueous buffer system, a saline system, an aqueous system containing a stabilizer, an alcohol-water system, or combinations thereof. The aqueous buffer system may include phosphate buffer, Tris buffer, Hepes buffer, or other pharmaceutically acceptable buffer systems; the saline system may include physiological saline, isotonic saline, or other aqueous systems containing inorganic salts; the aqueous system containing a stabilizer may include an aqueous system containing sugars, amino acids, or other stabilizing components; the alcohol-water system may include an ethanol-water system, a methanol-water system, or other mixed systems formed by lower alcohols and water. In this invention, the aqueous antisolvent may be used alone or in combination.

[0057] The mass ratio of triptolide to lactoferrin can be adjusted according to the stability of nanoparticle formation, particle size distribution, drug loading capacity, and system dispersibility. In some embodiments, the mass ratio of triptolide to lactoferrin can be 2:(0.8-3), for example, 2:0.9, 2:1, 2:1.2, 2:1.5, 2:1.8, 2:2, or 2:2.5. In some preferred embodiments, the mass ratio of triptolide to lactoferrin can be 2:(0.8-1.5), with 2:1 being the most preferred. In some embodiments, appropriately increasing the proportion of lactoferrin can improve the stability of the nanoparticle system and reduce particle aggregation, while appropriately increasing the proportion of triptolide can improve the drug loading capacity and the drug loading level of the nanoparticles.

[0058] In some embodiments, the volume ratio of the aqueous antisolvent to the organic solvent can be adjusted according to the desolvation rate of triptolide, the self-assembly kinetics, the nanoparticle size, and the system stability. The volume ratio of the aqueous antisolvent to the organic solvent can be (4–12):1, for example, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, or 12:1. In some preferred embodiments, the volume ratio of the aqueous antisolvent to the organic solvent can be (6–10):1, more preferably 8:1. In some embodiments, appropriately increasing the proportion of the aqueous antisolvent can promote rapid desolvation of triptolide and facilitate the formation of a nanoparticle system with smaller particle size and uniform dispersion.

[0059] In some embodiments, the ultrasonic treatment power can be adjusted according to the nanoparticle formation efficiency, particle size distribution, system homogeneity, and protein stability. The ultrasonic treatment power can be from 120 W to 400 W, for example, 150 W, 180 W, 200 W, 220 W, 240 W, 260 W, 280 W, 300 W, 320 W, 350 W, or 380 W. In some preferred embodiments, the ultrasonic treatment power can be from 200 W to 320 W. In some embodiments, appropriately increasing the ultrasonic treatment power can facilitate rapid mixing between the organic phase and the aqueous antisolvent system, and can help reduce particle aggregation and improve the uniformity of nanoparticle dispersion; however, excessively high ultrasonic power may lead to decreased protein structural stability or particle re-aggregation. Therefore, it can be adjusted according to the target particle size and system stability.

[0060] In some embodiments, the ultrasonic treatment time can be adjusted according to the nanoparticle formation efficiency, system stability, and particle size distribution. The ultrasonic treatment time can be 2 min to 25 min, for example, 5 min, 8 min, 10 min, 12 min, 15 min, 18 min, 20 min, or 22 min. In some preferred embodiments, the ultrasonic treatment time can be 12 min to 18 min. Appropriately extending the ultrasonic treatment time can help improve the nanoparticle formation efficiency and system uniformity, but excessively long ultrasonic time may lead to increased system temperature rise, decreased protein structure stability, or particle aggregation. Therefore, it can be adjusted according to the nanoparticle size, PDI (Polydispersity Index), and system stability.

[0061] The organic solvent used for triptolide can be selected based on the solubility of triptolide, the volatility of the system, the antisolvent induction efficiency, and the subsequent organic solvent removal requirements. In some embodiments, the organic solvent may include dichloromethane, ethyl acetate, acetone, ethanol, methanol, or combinations thereof. For example, the organic solvent may be a dichloromethane / ethyl acetate mixture, a dichloromethane / acetone mixture, an ethyl acetate / ethanol mixture, an acetone / ethanol mixture, an ethanol / methanol mixture, or combinations thereof. In some embodiments, the organic solvent may also include other pharmaceutically acceptable organic solvent systems capable of dissolving triptolide and forming a solvent exchange process with the aqueous antisolvent system. In some embodiments, the organic solvent can form a homogeneous and stable drug organic phase and induce desolvation and nano-precipitation of triptolide after contact with the aqueous antisolvent system. Different organic solvent systems can affect the precipitation rate, self-assembly kinetics, nanoparticle size distribution, and system stability of triptolide, and therefore can be adjusted according to the target particle size, PDI, and drug loading requirements.

[0062] In some embodiments, the organic solvent may further contain small amounts of co-solvents, stabilizers, or dispersing aids. For example, the organic solvent system may further contain lower alcohols, esters, or ketones as co-solvents to adjust the solubility, evaporation rate, and nanoparticle formation process of triptolide.

[0063] In some embodiments, the organic solvent removal method may include rotary evaporation, reduced pressure evaporation, nitrogen purging, or freeze-drying. For example, the organic solvent can be removed by rotary evaporation under reduced pressure, or by nitrogen purging for volatilization. In some embodiments, the organic solvent removal process may also be combined with processes such as heating, reduced pressure, centrifugation, filtration, concentration, or freeze-drying. In some embodiments, after removing the organic solvent, a stable triptolide nanoparticle dispersion system can be obtained.

[0064] In some embodiments, the obtained triptolide nanoparticle dispersion system can be further freeze-dried to form a lyophilized powder nanoparticle system. During the freeze-drying process, freeze-drying protectants, stabilizers, or dispersion protectants can be added. For example, the freeze-drying protectant may include mannitol, trehalose, sucrose, glucose, lactose, glycine, arginine, or combinations thereof. In some embodiments, the freeze-dried nanoparticle system can maintain good particle size distribution, PDI, and dispersion stability after reconstitution, and can be further used to prepare nasal drops, sprays, nanosuspensions, in-situ gels, or nasal dry powders, etc., for nasal administration.

[0065] The second aspect of the present invention relates to triptolide nanoparticles prepared by the preparation method described above.

[0066] The nanoparticles can exist in the form of nanodispersions, nanosuspensions, lyophilized powders, resolvable nanosystems, or other nanodelivery systems. In some embodiments, the nanoparticles can maintain good particle size distribution and system stability after resolvation.

[0067] The nanoparticles prepared by the method of the present invention can have a particle size range of about 100 nm to 300 nm. For example, the average particle size of the nanoparticles can be 120 nm, 150 nm, 160 nm, 180 nm, 190 nm, 200 nm, 220 nm, 240 nm, 250 nm, or 280 nm. In some preferred embodiments, the particle size is 150 nm to 250 nm, and more preferably 180 nm to 220 nm.

[0068] In some embodiments, the surface potential of the triptolide nanoparticles can be -20 mV to 40 mV, -10 mV to 0 mV, or any combination thereof. For example, the surface potential can be -15 mV, -10 mV, -9 mV, -8 mV, -7 mV, -6 mV, -5 mV, -4 mV, -3 mV, -2 mV, -1 mV, 0 mV, 10 mV, 20 mV, or 30 mV. In some preferred embodiments, it is -8 mV to -2 mV, more preferably -6 mV to -4 mV. In some embodiments, an appropriate surface potential can help improve the stability of the nanoparticle system and reduce particle aggregation.

[0069] In some embodiments, the polydispersity index (PDI) of the triptolide nanoparticles can be less than 0.3, for example, the PDI can be 0.25, 0.2, 0.18, 0.15, or 0.1. A lower PDI can improve the homogeneity and long-term storage stability of the nanoparticle system. In some embodiments, the triptolide nanoparticles can maintain good dispersion stability under artificial nasal solutions, artificial cerebrospinal fluid, physiological conditions, or storage conditions. In some embodiments, after the nanoparticles are stored at 4°C for several days, weeks, or months, the changes in particle size and PDI remain within acceptable ranges. In some embodiments, the nanoparticles can also exhibit sustained-release properties, thereby improving the drug retention capacity in the nasal cavity and the efficiency of brain tissue delivery.

[0070] A third aspect of the invention relates to a nasal delivery formulation comprising triptolide nanoparticles as described above, and pharmaceutically acceptable excipients.

[0071] The nasal delivery formulation can be used to improve the dispersion stability, mucosal retention capacity, and naso-brain delivery efficiency of triptolide nanoparticles in the local nasal environment. In some embodiments, the triptolide nanoparticles in the nasal delivery formulation may exist in the form of nanodispersions, nanosuspensions, lyophilized powders, resolvable nanosystems, or other nanodelivery systems.

[0072] In some embodiments, the pharmaceutically acceptable excipients may include osmotic pressure regulators, buffers, stabilizers, preservatives, suspending agents, solubilizers, lyophilization protectants, mucosal adhesives, wetting agents, dispersants, pH adjusters, chelating agents, or combinations thereof. For example, the osmotic pressure regulator may include sodium chloride, glucose, mannitol, sorbitol, or combinations thereof; the buffer may include phosphate buffer systems, Tris buffer systems, Hepes buffer systems, citrate buffer systems, or combinations thereof; the stabilizer may include polyethylene glycol, polyvinyl alcohol, poloxamer, Tween surfactants, polysorbate surfactants, or combinations thereof; the preservative may include benzalkonium chloride, phenethyl alcohol, parabens, potassium sorbate, or combinations thereof; the suspending agent or dispersant may include sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, xanthan gum, carbomer, or combinations thereof; and the lyophilization protectant may include mannitol, trehalose, sucrose, lactose, glucose, glycine, arginine, or combinations thereof.

[0073] In some embodiments, the nasal delivery formulation may further include a mucosal adhesion component to improve the retention time of the nanoparticles in the local nasal environment. For example, the mucosal adhesion component may include chitosan, hyaluronic acid, carbomer, alginate, cellulose derivatives, or combinations thereof. In some embodiments, the nasal delivery formulation may further include an in-situ gel-related component to improve retention and sustained-release properties after local nasal administration. For example, the in-situ gel-related component may include poloxamer, carbomer, gellan gum, sodium alginate, chitosan, or combinations thereof.

[0074] In some embodiments, the nasal delivery formulation can be a nasal drop, a spray, an in-situ gel, a nanosuspension, a lyophilized powder, or a nasal dry powder. For example, the nasal drop can be in the form of a nano-dispersion; the spray can be in the form of a liquid spray or atomization; the in-situ gel can form a gel structure in the local environment of the nasal cavity; the nanosuspension can be a stable nanoparticle suspension system; the lyophilized powder can be restored to a nanoparticle dispersion state after reconstitution; and the nasal dry powder can be delivered to the local nasal cavity by spraying or inhalation.

[0075] In some embodiments, the nasal delivery formulation may have a pH, osmotic pressure, and viscosity range suitable for nasal delivery. The pH may be 4.0–8.0, 4.5–7.5, 5.0–7.0, or any combination thereof. For example, the pH may be 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0. In some embodiments, the osmotic pressure may be close to physiological osmotic pressure, thereby improving nasal tolerance and reducing mucosal irritation.

[0076] The triptolide nanoparticles in the nasal delivery formulation can enter the central nervous system via the naso-brain delivery route and, in some embodiments, can enhance drug accumulation in brain tissue. The nasal delivery formulation can also improve the dispersion stability, bioavailability, and intracerebral delivery efficiency of triptolide, and is beneficial in improving pathological states related to neurological diseases.

[0077] A fourth aspect of the invention relates to the use of triptolide nanoparticles as described above in the preparation of nasal administration formulations for neurological diseases, inflammation, or autoimmune diseases.

[0078] In some embodiments, the neurological disease is a central nervous system disease. The central nervous system disease may include cerebral ischemia-reperfusion injury, ischemic stroke, cerebral infarction, cerebral edema, neuroinflammatory injury, neurodegenerative diseases, or oxidative stress-related neurological damage. In some preferred embodiments, the neurological disease is cerebral ischemia-reperfusion injury or ischemic stroke. The results of the embodiments of the present invention show that intranasal administration of the triptolide nanoparticles can improve the neurological function scores of animals with cerebral ischemia-reperfusion injury, reduce cerebral infarction volume, and alleviate brain tissue damage, thereby demonstrating a good neuroprotective effect.

[0079] In some embodiments, the inflammatory disease may include a disease or pathological state associated with abnormal activation of the inflammatory response, such as acute inflammation, chronic inflammation, tissue injury-related inflammation, neuroinflammation, ischemia-reperfusion-related inflammation, inflammation-mediated tissue damage, or a combination thereof. Since triptolide has known anti-inflammatory activity, and the nanodelivery system provided by this invention can improve its delivery efficiency and lesion tissue exposure level, it is expected to have potential application value for inflammation-related diseases. In particular, significant inflammatory factor release and inflammatory cell activation accompany cerebral ischemia-reperfusion injury; therefore, the neuroprotective effect observed in the embodiments of this invention is at least partially related to the improvement of the inflammatory response.

[0080] In some embodiments, the autoimmune disease may include multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, autoimmune encephalitis, or other immune imbalance-related diseases. Tripterygium wilfordii has been reported to have immunomodulatory and inhibitory effects on abnormal immune activation, and the nanodelivery system provided by this invention can improve its pharmacokinetic characteristics and delivery efficiency; therefore, it is expected to be further used for the intervention or treatment of autoimmune diseases. It should be noted that the embodiments of this invention mainly verified the neuroprotective effect in a cerebral ischemia-reperfusion injury model. Its application to inflammatory diseases and autoimmune diseases can be reasonably deduced based on the known pharmacological activities of triptolide and the delivery advantages brought by the delivery system of this invention.

[0081] The triptolide nanoparticles can enter the central nervous system via the naso-brain delivery route and, in some embodiments, can enhance the drug's accumulation capacity in brain tissue. Lactoferrin can participate in the nanodelivery process as a brain-targeting component, while triptolide can exert anti-inflammatory, antioxidant, neuroprotective, and tissue damage-improving effects as an active pharmaceutical ingredient.

[0082] In some embodiments, the triptolide nanoparticles can be delivered to the central nervous system via nasal administration, which facilitates bypassing the blood-brain barrier and thus improves drug delivery efficiency in brain tissue. In some embodiments, the triptolide nanoparticles can enter brain tissue via the olfactory nerve pathway, the trigeminal nerve pathway, or a combination thereof, and in some embodiments, can enhance the local accumulation of the drug in brain tissue.

[0083] In some embodiments, the triptolide nanoparticles can also help improve neurological deficits, reduce cerebral infarction area, alleviate cerebral edema, reduce inflammatory factor levels, reduce oxidative stress damage, reduce apoptosis levels, or improve the pathological state of neurological tissue. For example, the inflammatory factors may include TNF-α, IL-1β, IL-6, or combinations thereof; the oxidative stress-related indicators may include ROS levels, MDA levels, SOD activity, GSH levels, or combinations thereof.

[0084] The description of the nasal delivery formulation in the third aspect of the present invention also applies to the fourth aspect of the present invention.

[0085] The embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that these embodiments are only used to illustrate the technical content of the present invention and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the specific experimental conditions in the following embodiments are given priority reference to the guidelines provided in this specification, or may be carried out according to generally accepted experimental manuals or conventional experimental conditions, or other experimental methods known in the art, or according to the conditions recommended by the relevant reagent or instrument manufacturers. In specific embodiments, unless otherwise specified, minor deviations within the weighing accuracy range are allowed for the measurement parameters involving raw material components; reasonable deviations due to instrument detection accuracy or operational accuracy are also allowed for parameters such as temperature and time.

[0086] Example 1 This embodiment provides triptolide nanoparticles, which include: the active drug triptolide and lactoferrin.

[0087] The triptolide is a hydrophobic drug, a pentacyclic triterpenoid natural active product extracted from the root bark of *Tripterygium wilfordii*, a plant in the Celastraceae family. It was purchased from Chengdu Pufeed Biotechnology Co., Ltd., with a purity ≥99.9% (HPLC). The lactoferrin is derived from bovine milk and purchased from Nanjing Kangmanlin Chemical Industry Co., Ltd. (production batch number: 20250624, Jiangsu, China). It has a protein content of 98.3% and an iron saturation of 15%. The preparation method of the triptolide nanoparticles is as follows: Step 1: Completely dissolve triptolide in an organic solvent; completely dissolve lactoferrin in water: Weigh 10 mg of triptolide and dissolve it thoroughly in 0.5 mL of dichloromethane; separately weigh 5 mg of lactoferrin and dissolve it thoroughly in 4 mL of ultrapure water. Step 2: Slowly add the lactoferrin solution obtained in Step 1 to the tripterygium sorbitol solution, vortex to mix, and then sonicate the mixture at a power of 260 W for 15 min. Step 3: Remove the organic solvent from the mixture after sonication in Step 2 by rotary evaporation. The process is carried out in the dark and at room temperature to obtain tripterygium nanoparticles.

[0088] Observation of traits: Figure 1 Part (a) shows the appearance of the triptolide nanoparticles, which are a clear orange-yellow solution without layering or precipitation. Figure 1 Part (b) shows the appearance of the freeze-dried triptolide nanoparticles. After freeze-drying, they exhibit a smooth and loose morphology without collapse or shrinkage. The particle size after reconstitution with water did not show any significant change compared to before freeze-drying, indicating that freeze-drying has no effect on triptolide nanoparticle formulations and can be prepared into freeze-dried products for storage.

[0089] Particle size and surface potential determination: The particle size, particle size distribution, and surface potential of the nanoparticles were determined using a Zetasizer Nano ZS90 laser particle size analyzer (Malvin, UK). 20 µL of triptolide nanoparticles were diluted to 3 mL with ultrapure water. The sample was added to the sample cell, and after removing air bubbles, the analysis was performed at 25 °C. Figure 2 The image shows the particle size distribution of triptolide nanoparticles. The average particle size of the triptolide nanoparticles is 187.87 ± 5.25 nm; the polydispersity index (PDI) is 0.133, indicating a uniform particle size distribution. Figure 3 The potential distribution diagram of triptolide nanoparticles is shown, with a surface potential of -5.11±1.39 mV.

[0090] Morphological observation: Approximately 20 μL of triptolide nanoparticles were dropped onto a sample plate. A copper mesh was placed face down on the surface of the droplet, and after 3 minutes, it was removed and allowed to air dry at room temperature. The plate was then placed on a 2% phosphotungstic acid staining solution for 1-2 minutes. The copper mesh was removed, and excess staining was blotted off with filter paper. The sample was then observed and photographed under a transmission electron microscope at 200 kV. Figure 4 As shown, the triptolide nanoparticles exhibit a relatively regular spherical morphology and are uniformly dispersed. The particle size is around 190 nm, consistent with the results measured by laser particle size analyzer.

[0091] Tripterygium wilfordii raw material and triptolide nanoparticles were uniformly dispersed on a sample stage coated with a layer of conductive adhesive. Any loose samples were blown away with a syringe. The sample stage was then placed in an ion sputtering apparatus for gold sputtering treatment, and finally observed and imaged using a scanning electron microscope. Figure 5 As shown, the microstructure of triptolide raw material is characterized by irregular particles and blocky structures, with particle sizes mostly ranging from 2 to 10 μm. The surface exhibits varying degrees of roughness and defects, and some areas show crystalline features, displaying a multiphase state and complex structure. In contrast, triptolide nanoparticles exhibit a spherical appearance, with a particle size ranging from 180 to 200 nm. They are relatively well dispersed, with a smooth surface, and no obvious protrusions, pores, or other complex surface structures were observed.

[0092] Stability study: Tripterygium wilfordii nanoparticles were stored in the dark at 4°C and removed at 1, 2, 3, 7, 14, 21, 30, and 60 days. The particle size and PDI distribution of the nanoparticles were measured using a Zetasizer Nano ZS90 laser particle size analyzer to assess the stability of the triptolide nanoparticles. Figure 6 As shown, it can be observed that the particle size and PDI of triptolide nanoparticles did not change significantly within 60 days, and the PDI was less than 0.3, indicating that triptolide nanoparticles are relatively stable under light-proof and cold storage.

[0093] Example 2 This embodiment investigated the effect of different feed ratios of triptolide / lactoferrin on the particle size and PDI of the obtained triptolide nanoparticles.

[0094] Step 1: Completely dissolve triptolide in an organic solvent; completely dissolve lactoferrin in water: Weigh out triptolide and lactoferrin respectively according to the triptolide / lactoferrin feeding ratio of 4:1, 2:1, 1:1, and 1:2; dissolve triptolide in 0.5 mL of dichloromethane; dissolve lactoferrin in 4 mL of ultrapure water. Step 2: Slowly add the lactoferrin solution obtained in Step 1 to the tripterygium sorbitol solution, vortex to mix, and then sonicate the mixture at a power of 260 W for 5 min. Step 3: Remove the organic solvent from the mixture after sonication in Step 2 by rotary evaporation. The process is carried out in the dark and at room temperature to obtain tripterygium nanoparticles.

[0095] Take 20 μL of each of the triptolide nanoparticles prepared in this embodiment, dilute them to 3 mL with ultrapure water, and place the samples into the sample cell for detection. The measurement temperature is set to 25℃. The effective particle size and PDI data of each formulation are shown in Table 1 below: Table 1. Effect of the ratio of leucocarbamate to lactoferrin on particle size and PDI ( ±s, n=3)

[0096] As shown in Table 1, the ratio of triptolide to lactoferrin significantly affects the effective particle size of the obtained nanoparticles. A higher proportion of triptolide results in a larger particle size; increasing the proportion of lactoferrin decreases the particle size, but beyond a certain ratio, the particle size increases again. When the ratio of triptolide to lactoferrin is 2:1, the particle size is relatively the smallest, and the PDI < 0.3, meeting the requirements. Therefore, the ratio of triptolide to lactoferrin in the preparation method provided by this invention is 2:1.

[0097] Example 3 This embodiment investigates the effects of different ultrasonic powers on the particle size and PDI of the obtained triptolide nanoparticles.

[0098] Step 1: Completely dissolve triptolide in an organic solvent; completely dissolve lactoferrin in water: Weigh 10 mg of triptolide and dissolve it thoroughly in 0.5 mL of dichloromethane; separately weigh 5 mg of lactoferrin and dissolve it thoroughly in 4 mL of ultrapure water. Step 2: Slowly add the lactoferrin solution obtained in Step 1 to the tripterygium solution, vortex to mix, and then sonicate the mixture for 5 min at ultrasonic power of 130 W, 260 W and 390 W respectively. Step 3: Remove the organic solvent from the mixture after sonication in Step 2 by rotary evaporation. The process is carried out in the dark and at room temperature to obtain tripterygium nanoparticles.

[0099] Take 10 μL of each of the triptolide nanoparticles prepared in this example, dilute with ultrapure water to 3 mL, and place the samples into the sample cell for detection. The measurement temperature is set to 25℃. The effective particle size and PDI data of each formulation are shown in Table 2 below: Table 2. Effect of ultrasonic power on particle size and PDI ( ±s, n=3)

[0100] As can be seen from the results in Table 2, when the ultrasonic power is 260 W, the particle size is relatively small and the PDI is less than 0.3. However, increasing or decreasing the ultrasonic power increases the particle size to varying degrees. Therefore, the ultrasonic power in the preparation method provided by this invention is 260 W.

[0101] Example 4 This embodiment verifies the effect of different ultrasonic treatment times on the particle size and PDI of the obtained triptolide nanoparticles.

[0102] Step 1: Completely dissolve triptolide in an organic solvent; completely dissolve lactoferrin in water: Weigh 10 mg of triptolide and dissolve it thoroughly in 0.5 mL of dichloromethane; separately weigh 5 mg of lactoferrin and dissolve it thoroughly in 4 mL of ultrapure water. Step 2: Slowly add the lactoferrin solution obtained in Step 1 to the tripterygium sorbitol solution, vortex to mix, and then sonicate the mixture at a power of 260 W for 3 min, 5 min, 15 min, and 20 min respectively. Step 3: Remove the organic solvent from the mixture after sonication in Step 2 by rotary evaporation. The process is carried out in the dark and at room temperature to obtain tripterygium nanoparticles.

[0103] Take 10 μL of each of the triptolide nanoparticles prepared in this example, dilute with ultrapure water to 3 mL, and place the samples into the sample cell for detection. The measurement temperature is set to 25℃. The effective particle size and PDI data of each formulation are shown in Table 3 below: Table 3. Effects of ultrasonic time on particle size and PDI ( ±s, n=3)

[0104] As can be seen from the results in Table 3, when the ultrasonic time is 15 min, the particle size is relatively small and the PDI is less than 0.3. However, increasing or decreasing the ultrasonic time increases the particle size to varying degrees. Therefore, the ultrasonic treatment time in the preparation method provided by this invention is 15 min.

[0105] Example 5 This embodiment verifies the effect of the antisolvent / solvent volume ratio on the particle size and PDI of the obtained triptolide nanoparticles.

[0106] Step 1: Dissolve triptolide completely in dichloromethane (solvent); dissolve lactoferrin completely in water (antisolvent): Weigh 10 mg of triptolide and 5 mg of lactoferrin, and prepare systems with antisolvent / solvent volume ratios of 4:1, 8:1, and 12:1, respectively. Step 2: Slowly add the lactoferrin solution obtained in Step 1 to the tripterygium sorbitol solution, vortex to mix, and then sonicate the mixture at a power of 260 W for 15 min. Step 3: Remove the organic solvent from the mixture after sonication in Step 2 by rotary evaporation. The process is carried out in the dark and at room temperature to obtain tripterygium nanoparticles.

[0107] Take 20 μL of each of the triptolide nanoparticles prepared in this example, dilute them to 3 mL with ultrapure water, and place the samples into the sample cell for detection. The measurement temperature is set to 25℃. The effective particle size and PDI data of each formulation are shown in Table 4 below: Table 4. Effect of antisolvent / solvent volume ratio on particle size and PDI ( ±s, n=3)

[0108] The results are shown in Table 4. When the antisolvent / solvent volume ratio is less than 4:1, the system contains a large proportion of dichloromethane. After rotary evaporation to remove the organic solvent, drug precipitation occurs. When the antisolvent / solvent ratio exceeds 8:1, the low organic solvent content in the system cannot provide sufficient desolvation energy, resulting in increased particle size. The experimental results indicate that when the antisolvent / solvent volume ratio is 8:1, the nanoparticles have the smallest particle size, and the PDI < 0.3, exhibiting uniform and stable particle size. Therefore, the optimal antisolvent / solvent volume ratio is selected as 8:1.

[0109] Experimental Example 1 This experimental example evaluates the drug release performance of the triptolide nanoparticles provided by the present invention.

[0110] Release behavior study: Tripterygium wilfordii nanoparticle solution and triptolide ethanol solution were placed in dialysis bags (Mw: 8000-14000), fixed and sealed, and then placed in artificial nasal fluid (ANF, pH 6.8) and artificial cerebrospinal fluid (ACSF, pH 7.4) containing 1% Tween 80, respectively. The dialysis apparatus was placed in a 37℃ constant temperature shaker with a rotation speed of 120 r / min. 1 mL samples were taken at 1, 2, 4, 6, 8, 10, 12, and 24 hours, and 1 mL of fresh medium was added accordingly. The samples were filtered through a 0.22 μm aqueous microporous membrane, and the filtrate was collected for high-performance liquid chromatography-ultraviolet chromatography (HPLC-UV) analysis to determine the drug concentration in the dialysis medium at different time points. The monitoring conditions were as follows: the chromatographic column was Ultimate® LP-C18 (5 μm, 4.6 × 150 mm); the mobile phase was methanol and 0.5% formic acid aqueous solution in a ratio of 88:12; the detection wavelength was 426 nm; the column temperature was 30℃; the injection volume was 10 μL; and the flow rate was 1 mL / min.

[0111] Figure 7 The figures show the drug release profiles of triptolide raw material and triptolide nanoparticles in artificial nasal solution (ANF, pH 6.8) and artificial cerebrospinal fluid (ACSF, pH 7.4). The results are shown in the figure. Figure 7 In sample a), the release of both the active pharmaceutical ingredient (API) and nanoparticles was significantly inhibited. The cumulative release rate of the API over 24 hours was less than 25%, while the cumulative release rate of the nanoparticles over 24 hours was only 12.37%. This indicates that the nanoparticles are not easily released in nasal fluid and, after nasal absorption, can enter the brain with a relatively intact structure. In artificial cerebrospinal fluid (... Figure 7 In (b) of the study, the release rate of triptolide API was approximately 75% after 24 hours, while that of triptolide nanoparticles was approximately 65%. Initially, triptolide API was released faster, but the release trends of both products became more similar later on, indicating that nanoparticle formulations can delay release to some extent in artificial cerebrospinal fluid. In all media, the release rate of triptolide API was generally faster than that of triptolide nanoparticles, demonstrating that nanoparticle encapsulation technology can effectively achieve sustained drug release.

[0112] Experiment Example 2 This example is a comparative study of the ability of lactoferrin to form nanoparticles with different hydrophobic drugs.

[0113] To investigate the universality of forming nanoparticle systems between lactoferrin and different hydrophobic drugs, nanoparticles of various hydrophobic drugs and lactoferrin were prepared using the same or similar methods as in Example 1.

[0114] 1 Experimental Methods 1.1 LF-Quercetin Weigh 10 mg of quercetin into a 10 mL EP tube and dissolve it in 0.5 mL of dichloromethane. Separately, weigh 5 mg of lactoferrin (LF) and dissolve it in 4 mL of ultrapure water. Slowly add the quercetin solution to the LF solution and vortex to mix. Then, sonicate the mixture using an ultrasonic cell disruptor under ice bath conditions (260 W for 15 min). Finally, place the mixture in a rotary evaporator and remove the dichloromethane by rotary evaporation under light-protected, room temperature conditions.

[0115] 1.2 LF-Phlorisin Weigh 10 mg of phlorizin into a 10 mL EP tube and dissolve it in 0.5 mL of tetrahydrofuran. Separately, weigh 5 mg of lactoferrin (LF) and dissolve it in 4 mL of ultrapure water. Slowly add the phlorizin solution to the LF solution and vortex to mix. Then, sonicate the mixture using an ultrasonic cell disruptor under ice bath conditions (260 W for 5 min). Finally, place the mixture in a rotary evaporator and remove the tetrahydrofuran by rotary evaporation under light-protected, room temperature conditions.

[0116] 1.3 LF-gallic acid Weigh 4 mg of gallic acid into a 10 mL EP tube and dissolve it in 0.3 mL of tetrahydrofuran. Separately, weigh 2 mg of lactoferrin (LF) and dissolve it in 4 mL of ultrapure water. Slowly add the gallic acid solution to the LF solution and vortex to mix. Then, sonicate the mixture using an ultrasonic cell disruptor under ice bath conditions (260 W for 5 min). Finally, place the mixture in a rotary evaporator and remove the tetrahydrofuran under light-protected, room temperature conditions.

[0117] 1.4 LF-caffeic acid, ferulic acid Weigh 2 mg of caffeic acid or ferulic acid into a 10 mL EP tube and dissolve it in 0.3 mL of tetrahydrofuran. Weigh 4 mg of lactoferrin (LF) into two other tubes and dissolve them thoroughly in 4 mL of ultrapure water. Slowly add the caffeic acid or ferulic acid solution to the LF solution and vortex to mix. Then, sonicate the mixture using an ultrasonic cell disruptor under ice bath conditions (260 W for 5 min). Finally, place the mixture in a rotary evaporator and remove the tetrahydrofuran under light-protected, room-temperature conditions.

[0118] 1.5 LF-Trifolin Weigh 10 mg of trifolin into a 10 mL EP tube and dissolve it in 0.5 mL of tetrahydrofuran. Separately, weigh 5 mg of lactoferrin (LF) and dissolve it in 4 mL of ultrapure water. Slowly add the trifolin solution to the LF solution and vortex to mix. Then, sonicate the mixture using an ultrasonic cell disruptor under ice bath conditions (260 W for 15 min). Finally, place the mixture in a rotary evaporator and remove the tetrahydrofuran under light-protected, room temperature conditions.

[0119] 1.6 LF-Azalea Weigh 2.5 mg of rhododendron extract into a 10 mL EP tube and dissolve it in 0.5 mL of tetrahydrofuran. Separately, weigh 5 mg of lactoferrin (LF) and dissolve it in 4 mL of ultrapure water. Slowly add the rhododendron extract solution to the LF solution and vortex to mix. Then, sonicate the mixture using an ultrasonic cell disruptor under ice bath conditions (260 W for 15 min). Finally, place the mixture in a rotary evaporator and remove the tetrahydrofuran under light-protected, room temperature conditions.

[0120] 1.7 LF-Rhein Weigh 5 mg of rhein into a 10 mL EP tube and dissolve it in 1 mL of DMSO. Separately, weigh 10 mg of lactoferrin (LF) and dissolve it in 8 mL of ultrapure water. Slowly add the rhein solution to the LF solution and vortex to mix. Then, use an ultrasonic cell disruptor to sonicate the cells in an ice bath (260 W for 10 min). Finally, remove the DMSO to obtain the final product.

[0121] 2. Experimental Results The appearance of each formulation prepared in the experiment was evaluated, and the particle size, PDI, and stability were evaluated using a particle size analyzer. The results are shown in the table below. LF, along with hydrophobic drugs such as quercetin, phloretin, gallic acid, caffeic acid, ferulic acid, trifolin, rhododendronin, and emodin, could not form stable nanoparticles when prepared using the same or similar processes as tripterygium wilfordii nanoparticles. Furthermore, the resulting nanoparticles exhibited large particle size, instability, drug precipitation, and stratification. The structures are shown in the table below.

[0122]

[0123] The results showed that not all hydrophobic drugs could form stable nanoparticle systems with lactoferrin. Although the drugs mentioned above all had a certain degree of hydrophobicity, most systems exhibited phenomena such as drug precipitation, excessively large particle size, unstable particle size distribution, or stratification upon standing.

[0124] In contrast, triptolide can form a nanoparticle system with lactoferrin that has a smaller particle size, uniform dispersion, and better stability.

[0125] Experimental Example 3 This experimental example verifies the retention time of triptolide nanoparticles in the nasal cavity after intranasal administration and evaluates their retention characteristics in the nasal cavity.

[0126] Cy5-labeled triptolide was used to replace triptolide in the preparation of the formulation. C57 mice (n = 3 / group) were administered free triptolide or triptolide nanoparticles intranasally. At different time points of 0, 2, 4, 6, 12, and 24 h, images were taken using a small animal in vivo optical imaging system (excitation wavelength: 640 nm, emission wavelength: 664 nm), and the average fluorescence intensity was quantitatively analyzed.

[0127] The results are as follows Figure 8 As shown, these results indicate that lactoferrin-modified triptolide nanoparticles can significantly improve nasal retention behavior after intranasal drug administration. Compared with free triptolide, the nanoparticles can significantly prolong the drug retention time in the nasal cavity and slow down the drug clearance rate, providing more time for subsequent drug absorption via the naso-brain pathway.

[0128] Experiment Example 4 This experimental example verifies the distribution of triptolide nanoparticles in brain tissue after intranasal administration and evaluates their brain targeting ability.

[0129] Brain targeting analysis was performed using Cy5-labeled triptolide. Free triptolide and triptolide nanoparticles were administered intranasally to rats. Rats were sacrificed at 2, 4, 6, and 12 h post-administration, and brain tissue was collected. After rinsing with physiological saline and drying with filter paper, the tissue was imaged using a small animal in vivo optical imaging system (excitation wavelength: 640 nm, emission wavelength: 664 nm), and the average fluorescence intensity was quantitatively analyzed.

[0130] Imaging results as follows Figure 9 As shown, the fluorescence signals of triptolide nanoparticles in the brain were significantly higher than those of free triptolide at all time points, and the fluorescence signal of triptolide nanoparticles gradually decreased after reaching a peak at 4 hours. Preparing triptolide into nanoparticles enhances its delivery and retention time in the brain. This experimental example demonstrates that triptolide nanoparticles are more easily aggregated in brain tissue, which is beneficial for improving cerebral ischemia-reperfusion injury.

[0131] Experimental Example 5 This experimental example verifies the ameliorative effect of triptolide nanoparticles on cerebral ischemia-reperfusion injury after intranasal administration.

[0132] SPF-grade male SD rats, weighing 250-280 g, were randomly divided into 5 groups using a simple randomization method: sham-operated group, model group, lactoferrin group, free triptolide group, and triptolide nanoparticle group.

[0133] A rat model of focal cerebral ischemia-reperfusion injury was established using the suture occlusion method. Intraoperatively, cerebral blood flow in SD rats was monitored using a laser Doppler flow monitor to confirm successful model establishment in the sham-operated group. At 4 h, 28 h, and 52 h post-modeling, each group received intranasal administration of free triptolide and triptolide nanoparticles, respectively. The sham-operated group and the model group received an equal volume of PBS.

[0134] Four days after feeding, neurological function was assessed using the Longa 5-point scoring system (scoring criteria table shown in Table 5). Following the scoring, SD rats were anesthetized via intraperitoneal injection of 2% sodium pentobarbital solution. After anesthesia, the brain tissue of the SD rats was rapidly dissected on ice, fixed at -20℃ for 20 min, sectioned, and stained with 2,3,5-triphenyltetrazolium chloride (TTC) solution. The staining was incubated at 37℃ for 60 min. After ensuring sufficient staining, the brain tissue sections were placed in 4% paraformaldehyde solution and fixed at 4℃ for 48 h. After photographing, the infarcted portion of the brain tissue was removed, and the weight of the entire brain tissue and the infarcted portion were weighed separately to calculate the percentage of infarct volume. The calculation formula is as follows: Percentage of cerebral infarction volume = (W 脑梗死 / W 脑组织 ) × 100%.

[0135] W 脑梗死 W represents the weight of brain tissue from cerebral ischemia-infarction. 脑组织 This refers to the total weight of the brain tissue.

[0136] Table 5 Longa 5-point scoring criteria

[0137] The results of the neurological deficit score are shown below. Figure 10 Compared to the sham-operated group, the neurological deficit scores of rats in the model group were significantly increased. There was no statistically significant difference in neurological deficit scores between the lactoferrin group and the model group. The scores of the free triptolide group were lower than those of the model group, and the neurological deficit scores of the triptolide nanoparticle group were significantly lower than those of the model group, with a better improvement effect than both the lactoferrin and free triptolide groups. These results suggest that intranasal administration of triptolide nanoparticles can effectively alleviate neurological deficit symptoms in rats with cerebral ischemia-reperfusion injury (CIRI).

[0138] Figure 11 and Figure 12 These are, respectively, a representative TTC staining map of cerebral infarction in rats after therapeutic administration and a statistical chart of the percentage of cerebral infarction volume. Figure 12 As shown, compared with other experimental groups, the triptolide nanoparticle treatment group significantly reduced the cerebral infarction volume in CIRI model rats, and the therapeutic effect was better than that of the free triptolide group.

[0139] Experimental Example 6 To investigate inflammatory factor levels, tissue samples from the ischemic penumbra were weighed and then mixed with PBS at a ratio of 10 mg to 90 μL. The mixture was thoroughly ground, incubated at 4°C for 30 min, and then centrifuged at 3000 rpm for 10 min. The supernatant was collected and stored at -80°C for later use. The levels of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), interleukin-4 (IL-4), and interleukin-10 (IL-10) in the brain tissue were detected using ELISA. Specific procedures: Set up standard wells, sample wells, and blank control wells in a 96-well plate. Add 50 μL of each concentration of standard to the standard wells. Add 40 μL of sample diluent to the sample wells, followed by 10 μL of the sample to be tested (diluted 5 times). Do not add sample or enzyme-labeled reagent to the blank wells. Add 100 μL of enzyme-labeled reagent to each well (except for the blank wells). Seal the plate and incubate at 37°C for 60 min. Dilute the 20-fold concentrated washing buffer with distilled water for later use. Uncover the membrane, pour out the liquid, shake dry, fill with washing buffer, shake, and pat dry. Repeat 5 times. Add 50 μL each of colorimetric reagent A and B to each well, mix well, and develop color at 37°C in the dark for 15 min. Add 50 μL of stop solution to stop the reaction and measure the absorbance value at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0140] Test results as follows Figure 13 As shown, triptolide nanoparticles can regulate the levels of inflammatory factors in rats with cerebral ischemia-reperfusion injury (CIRI): significantly inhibiting the release of pro-inflammatory factors such as IL-1β and TNF-α, while upregulating the expression of protective anti-inflammatory factors such as IL-4 and IL-10. Compared with free lactoferrin and free triptolide raw materials, it has outstanding in vivo anti-inflammatory neuroprotective advantages.

[0141] Experimental Example 7 This experimental example verifies the short-term safety of triptolide nanoparticles.

[0142] After one week of acclimatization, healthy SD rats were randomly divided into four groups: the triptolide group received triptolide suspension (0.25 mg / kg) intranasally, the lactoferrin group received lactoferrin solution (0.125 mg / kg) intranasally, the triptolide nanoparticle group received triptolide nanoparticle solution (triptolide 0.25 mg / kg, lactoferrin 0.125 mg / kg) intranasally, and the normal group received an equal volume of PBS. The administration was once a day for three consecutive days.

[0143] Rats were euthanized on the fourth day, and blood was collected from the abdominal aorta. Blood cell counts were analyzed using a fully automated veterinary blood cell analyzer, primarily detecting platelets, red blood cells, white blood cells, lymphocytes, neutrophils, and monocytes. The results are as follows: Figure 14As shown, the blood cell test results of rats after intranasal administration of triptolide nanoparticles were all within the normal range, indicating the safety of the blood test.

[0144] Heart, liver, spleen, lung, kidney, and nasal mucosa of rats in each group were subjected to HE staining (Hematoxylin and Eosin staining) and analyzed under an inverted fluorescence microscope. Figure 15 As shown in the HE staining results, no obvious lesions were observed in any organ of rats after intranasal administration of triptolide nanoparticles, indicating that it has good biocompatibility.

[0145] In summary, the triptolide nanoparticles provided by this invention have significant effects on improving neurological function loss, reducing cerebral infarction area, alleviating cerebral edema, and reducing cerebral ischemia-reperfusion injury; they also exhibit good biocompatibility. Furthermore, the preparation method of the triptolide nanoparticles provided by this invention is simple, mild, safe, and stable, and the resulting product has good stability.

[0146] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. The scope of protection of the present invention should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for preparing triptolide nasal nanoparticles, characterized in that, Includes the following steps: An organic solution containing triptolide was mixed with an aqueous antisolvent system containing lactoferrin. Triptorelide nanoparticles with lactoferrin as a carrier were formed by antisolvent-induced self-assembly and ultrasonic treatment. The organic solvent in the resulting dispersion system was then removed. The mass ratio of triptolide to the carrier is 2:(0.7~5), and the volume ratio of the aqueous antisolvent to the organic solvent is (3.5~14):

1.

2. The preparation method according to claim 1, characterized in that, The mass ratio of triptolide to lactoferrin is 2:(0.8~3); preferably 2:(0.8~1.5).

3. The preparation method according to claim 1, characterized in that, The volume ratio of the aqueous antisolvent to the organic solvent is (4~12):1; preferably (6~10):

1.

4. The preparation method according to claim 1, characterized in that, The power of the ultrasonic treatment is 120 W to 400 W; preferably 200 W to 320 W.

5. The preparation method according to claim 4, characterized in that, The ultrasonic treatment time is 2 min to 25 min; preferably 12 min to 18 min.

6. The preparation method according to any one of claims 1 to 5, characterized in that, The organic solvent includes dichloromethane, ethyl acetate, acetone, ethanol, methanol, or combinations thereof; Optionally, the method for removing organic solvents includes rotary evaporation, vacuum evaporation, nitrogen blowing, or freeze drying.

7. Tripterygium oleoresin nanoparticles prepared by the preparation method according to any one of claims 1 to 6.

8. A nasal delivery formulation, characterized in that, It comprises the triptolide nanoparticles of claim 7, and pharmaceutically acceptable excipients.

9. The nasal delivery formulation according to claim 8, characterized in that, It can be nasal drops, sprays, in-situ gels, nano-suspensions, lyophilized powders, or nasal dry powders.

10. The use of the triptolide nanoparticles according to claim 7 in the preparation of nasal delivery formulations for treating nervous system diseases, inflammation or autoimmune diseases; Optionally, the nasal delivery formulation is a nasal drop, spray, in-situ gel, nano-suspension, lyophilized powder, or nasal dry powder.

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