A nano-thermosensitive assembled gel for nasal administration, its preparation method and uses

Abstract

The present invention discloses a nano-thermosensitive assembled gel for nasal administration, a preparation method thereof and its use, namely, mitochondrion-targeted puerarin nanoparticles, a thermosensitive gel, a preparation method thereof and its use. The nano-thermosensitive assembled gel for nasal administration of the present invention uses chitosan modified with SS-31 peptide having mitochondrion-targeting and transmembrane properties as a carrier material, and uses a thioketal cross-linking agent sensitive to reactive oxygen species (ROS) to cross-link the modified chitosan to prepare nanoparticles loaded with puerarin. Through a poloxamer thermosensitive gel system, a thermosensitive nanogel drug reservoir encapsulating the nanoparticles is constructed, and its application in the preparation of drugs for treating nervous system diseases realizes the targeting of the drug to the mitochondria of cerebral ischemic stroke injury, responsive cleavage in an environment rich in ROS, release of the drug, increase of the drug concentration in the mitochondria, and improvement of the brain bioavailability of puerarin, providing a more suitable and effective new dosage form for the treatment of stroke.

Description

Technical Field

[0001] The present invention relates to a thermosensitive gel and a preparation method thereof, in particular to a thermosensitive gel for nasal administration and a preparation method thereof, belonging to the field of medicine and health. Background Art

[0002] Stroke, also known as apoplexy, is an acute cerebrovascular disease. Due to the rupture or blockage of intracranial blood vessels, it causes brain tissue damage, and then a series of symptoms occur, including cerebral hemorrhage and cerebral infarction. More than 70% of stroke patients will have varying degrees of functional loss, and those with severe disability account for more than 40%, seriously affecting the quality of life.

[0003] The pathological mechanism of ischemic stroke is cerebral ischemia-reperfusion injury (CIRI), which refers to that after cerebral ischemia for a period of time and then blood supply is restored, the function of the brain not only fails to recover in time, but instead aggravates nerve disorders. Modern pathological studies have shown that the pathological mechanism of CIRI is very complex, mainly involving mitochondrial damage, oxidative stress, inflammatory response, calcium ion overload, excessive release of excitatory amino acids and other links. Intravenous injection of tissue plasminogen activator (tPA) is the only Class 1A recommended treatment in the acute ischemic stroke treatment guidelines, but its application has strict time window limitations.

[0004] Puerarin is the main active ingredient of the traditional Chinese medicine Pueraria lobata. Modern pharmacological studies have found that puerarin has a variety of pharmacological activities, such as dilating blood vessels, increasing cerebral blood flow, anti-cerebral ischemia-reperfusion injury, protecting nerve cells in the brain, antioxidant, anti-inflammatory and other effects. Puerarin injection is also commonly used in clinical practice for diseases such as myocardial ischemia and cerebral ischemia. A large number of studies have shown that puerarin has a good protective effect on the nerves in the brain. In terms of protecting mitochondria, puerarin relieves mitochondrial dysfunction by inhibiting the reduction of mitochondrial mass mediated by mitophagy, maintaining mitochondrial membrane potential, preventing the release of cytochrome C, and reducing caspase activity. Puerarin can significantly reduce the water content in the brains of rats in the middle cerebral artery occlusion (MCAO) model, and improve the activity of Na + -K + -ATPase, Ca 2+ -Mg 2+ -ATPase, has strong antioxidant activity and the ability to scavenge free radicals, and can improve indicators such as superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GSH-Px) in brain tissue, and play a neuroprotective role by reducing free radical damage. Therefore, puerarin is an ideal active ingredient of traditional Chinese medicine for treating CIRI diseases.

[0005] To develop puerarin into a drug for treating CIRI, it is necessary to solve the problem of its low solubility and targetedly deliver it to the mitochondrial site. Therefore, this problem is solved through a nano-drug delivery system and targeted delivery technology. The nano-drug delivery system can improve the poor solubility of drugs, prevent drug degradation, and is expected to increase drug absorption by virtue of the transmembrane ability of the carrier. The nano-drug delivery system has the advantages of small particle size and easy phagocytosis by cells. Using biodegradable materials such as chitosan as carrier materials to construct a nano-drug delivery system has good stability, a high drug loading rate and encapsulation rate, which is more conducive to preventing drug degradation and improving drug stability. However, traditional chitosan nanoparticles have the disadvantages of poor targeting and transmembrane ability.

[0006] SS-31 peptide (D-Arg-Dmt-Lys-Phe-NH2) is an aromatic cationic short peptide with mitochondrial targeting ability and transmembrane properties. SS-31 is easily taken up by neuroblastoma cells and reaches a stable concentration after 30 minutes. The intracellular concentration of SS-31 is 6 times that outside the cell. Mitochondrial uptake of SS-31 can reach maximum absorption in less than 2 minutes, and the enrichment in the inner mitochondrial membrane is up to 5000 times the administered concentration. Studies have found that SS-31 can aggregate on the inner mitochondrial membrane and interact with cardiolipin. During ischemia-reperfusion, the sudden supply of oxygen and nutrients reactivates mitochondrial aerobic respiration, leading to a further burden of mitochondrial Ca 2+ and the opening of the mitochondrial permeability transition pore (mPTP) and a burst of ROS (reactive oxygen species) production, which in turn triggers mitochondrial dysfunction. The occurrence of these cascading reactions aggregated on mitochondria ultimately leads to cell death and tissue infarction. Therefore, mitochondria are the main target of neuroprotection in ischemic brain injury.

[0007] The existence of the blood-brain barrier (BBB), the natural physiological barrier of the brain tissue, makes many lead compounds effective in vitro unable to take effect in vivo because they cannot pass through the BBB.

[0008] The present invention selects to modify the SS-31 peptide as a targeting molecule on the surface of the nanoparticles to improve the targeted distribution of the drug in mitochondria, an important organelle in cerebral ischemia-reperfusion injury, and thus improve the therapeutic effect of the drug. Summary of the Invention

[0009] The primary objective of the present invention is to address the problems that drugs have difficulty passing through the blood-brain barrier and nano-drugs are prone to premature release. A mitochondrion-targeted puerarin nanoparticle, a thermosensitive gel, their preparation methods and uses are provided. The puerarin nanoparticles and thermosensitive gel of the present invention can be used for the treatment of ischemic nervous system diseases, especially the treatment of cerebral ischemia-reperfusion injury, and are suitable for the nasal administration route, solving the technical problem of premature release of nano-drugs and ensuring the stable release of drugs.

[0010] To achieve the objective of the present invention, on the one hand, the present invention provides a preparation method of a nanoparticle for nasal administration (i.e., a mitochondrion-targeted puerarin nanoparticle), wherein the puerarin nanoparticle has ROS responsiveness and comprises the following steps:

[0011] (1) Add 2,2'-[propane-2,2-diylbis(thio)]diacetic acid, EDC, and NHS to DMSO, stir under light-shielded conditions for an esterification reaction to obtain a ROS-responsive crosslinking agent TKDA (i.e., a DMSO solution of TKDA);

[0012] (2) Add sodium 4-(N-maleimidomethyl)cyclohexane-1-carboxylate sulfosuccinimide ester to an aqueous chitosan solution and stir for a first crosslinking reaction to obtain a first crosslinked solution; then add SS-31 peptide to the first crosslinked solution and stir for a second crosslinking reaction to obtain SS-31 peptide-chitosan;

[0013] (3) Mix liquid paraffin and an emulsifier to obtain a paraffin-emulsifier mixture;

[0014] (4) Under stirring, add the SS-31-chitosan aqueous solution to the paraffin-emulsifier mixture, stir for emulsification treatment to form an emulsification system, and obtain a paraffin-emulsifier-SS-31-chitosan mixture;

[0015] (5) Add puerarin to the ROS-responsive crosslinking agent TKDA prepared in step (1), mix evenly and then add the paraffin-emulsifier-SS-31-chitosan mixture prepared in step (4), stir for a third crosslinking reaction to obtain (i.e., prepare puerarin-SS-31 peptide-ROS-responsive nanoparticles).

[0016] The solution of puerarin-SS-31 peptide ROS-responsive nanoparticles obtained by the third crosslinking reaction is simply referred to as the puerarin-peptide-ROS nanoparticle solution, that is, the mitochondrion-targeted puerarin nanoparticle described above.

[0017] ROS responsiveness means that the nanoparticles can rapidly release drugs by cleavage in a high-level ROS environment.

[0018] Among them, the molar ratio of 2,2'-[propane-2,2-diylbis(thio)]diacetic acid to EDC in step 1) is 1:(1.5 - 2), preferably 1:2; the molar ratio of 2,2'-[propane-2,2-diylbis(thio)]diacetic acid to NHS is 1:(1.5 - 3), preferably 1:2.

[0019] In particular, the mass-to-volume ratio of 2,2'-[propane-2,2-diylbis(thio)]diacetic acid to DMSO is (0.4 - 0.5):1, that is, the mass of 2,2'-[propane-2,2-diylbis(thio)]diacetic acid and DMSO dissolved in every 1 mL of DMSO is 400 - 500 mg, preferably 0.448:1.

[0020] In particular, the esterification reaction temperature is room temperature, preferably 10 - 30 °C; the esterification reaction time is 5 - 6 h.

[0021] In particular, the ROS-responsive crosslinker TKDA in step (1) is a DMSO solution of TKDA.

[0022] Among them, in the process of the first crosslinking reaction in step (2), the mass / volume concentration of the chitosan aqueous solution is 0.5 - 1.5% (w / v), preferably 1.0% (w / v); the pH is 5 - 6; the molar ratio of sodium 4-(N-maleimidomethyl)cyclohexane-1-carboxylate sulfosuccinimide ester to the amino group on chitosan in the chitosan aqueous solution is 1:(10 - 20), preferably 1:10.

[0023] In particular, in the process of the second crosslinking reaction, the molar ratio of the sulfhydryl group in SS-31 peptide to the amino group on chitosan in the chitosan aqueous solution is 1:(15 - 25), preferably 1:20.

[0024] In particular, the temperatures of the first and second crosslinking reactions are room temperature, usually 10 - 30 °C; the times of the first and second crosslinking reactions are 20 - 45 min, preferably 30 min.

[0025] In particular, it further includes performing a first dialysis treatment on the mixture after the first crosslinking reaction in pure water to remove unreacted Sulfo-SMCC, obtaining the first crosslinked solution, and then performing the second crosslinking reaction.

[0026] In particular, the first dialysis treatment uses a dialysis bag with a molecular weight cut-off of 3500 and is dialyzed for 20 - 24 h.

[0027] Specifically, it further includes subjecting the mixture after the second cross-linking reaction to a second dialysis treatment in pure water to remove the unreacted SS-31 peptide, thereby obtaining an SS-31-chitosan aqueous solution.

[0028] Specifically, the time of the second dialysis treatment is 18 - 24 h, preferably 24 h; the cut-off molecular weight of the dialysis bag during the dialysis treatment is 3500 to remove the unreacted SS-31 peptide.

[0029] Specifically, it further includes subjecting the SS-31-chitosan aqueous solution after the second dialysis treatment to freeze-drying to obtain the SS-31-chitosan as described above.

[0030] That is, subject the mixture after the second cross-linking reaction to dialysis treatment, and dry the product retained in the dialysis bag to obtain a dry SS-31-chitosan solid, which is the SS-31-chitosan as described above.

[0031] Wherein, the volume ratio of the liquid paraffin to the emulsifier in step (3) is 100:(3.5 - 5.2), preferably 100:3.71 (i.e., 35:1.3).

[0032] Specifically, the emulsifier selected in step (3) is Span85 and Tween80.

[0033] Specifically, the volume ratio of Span-85 to Tween-80 in the emulsifier is 1:(0.3 - 0.6), preferably 1:0.3.

[0034] Specifically, the volume ratio of Span-85 to the liquid paraffin is 1:(30 - 35), preferably 1:35; the volume ratio of Tween-80 to the liquid paraffin is 1:(50 - 150), preferably 1:116.7.

[0035] Wherein, the SS-31-chitosan aqueous solution in step 4) is prepared according to the following method: add the SS-31-chitosan prepared in step 2) to water with a water temperature of 45 ± 2 °C, stir, and dissolve evenly to obtain it; wherein the mass / volume concentration of the SS-31-chitosan solution is (0.5 - 1.5) % (w / v), preferably 1 % (w / v).

[0036] Specifically, the mass / volume concentration of the SS-31-chitosan aqueous solution in step (4) is 0.5 % - 1.5 % (w / v), preferably 1 % (w / v).

[0037] Specifically, the volume ratio of the SS-31-chitosan aqueous solution to the paraffin-emulsifier mixture is 1:8 - 10, preferably 1:9.075.

[0038] In particular, the emulsification treatment temperature in step (4) is 50 - 60 °C; the emulsification treatment time is 2 - 3 h; the stirring rate during the emulsification treatment is 1000 - 1500 rpm, preferably 1000 rpm.

[0039] Among them, the SS-31-chitosan aqueous solution in step 4) is prepared according to the following method: After heating pure water to 45 ± 2 °C, add the SS-31-chitosan prepared in step 2) and stir to dissolve it.

[0040] Among them, the reaction temperature of the third cross-linking reaction in step 5) is 50 - 60 °C; the third cross-linking reaction time is 2 - 3 h.

[0041] In particular, the mass-to-volume ratio of puerarin to the ROS-responsive cross-linking agent TKDA in step (5) is 1:(4 - 6), preferably 1:5, that is, for every 1 mg of puerarin, add it to 4 - 6 μL of the ROS-responsive cross-linking agent TKDA solution, preferably for every 1 mg of puerarin, add it to 5 μL of the ROS-responsive cross-linking agent TKDA solution.

[0042] In particular, the ROS-responsive cross-linking agent TKDA solution is a DMSO solution of the ROS-responsive cross-linking agent TKDA.

[0043] In particular, the mass-to-volume ratio of puerarin to the paraffin-emulsifier-SS-31-chitosan mixture is 10:(30 - 50), preferably 10:(40 - 40.5), that is, for every 10 mg of puerarin, add it to 30 - 50 mL of the paraffin-emulsifier-SS-31-chitosan mixture, preferably for every 10 mg of puerarin, add it to 40 - 40.5 mL of the paraffin-emulsifier-SS-31-chitosan mixture.

[0044] Among them, the mass ratio of puerarin to SS-31-chitosan in the paraffin-emulsifier-SS-31-chitosan mixture in step 5) is (20 - 25):100, preferably 25:100;

[0045] In step (5), the molar ratio of the N-hydroxysuccinimide group in the ROS-responsive cross-linking agent TKDA to the amino group in SS-31-chitosan in the paraffin-emulsifier-SS-31-chitosan mixture is 1:(9 - 10), preferably 1:10.

[0046] In particular, it also includes centrifuging the reaction solution after the third cross-linking reaction. The centrifuged precipitate is the puerarin-peptide-ROS nanoparticles.

[0047] On the other hand, the present invention provides a nasal administration nanoparticle (i.e., a mitochondrion-targeted puerarin nanoparticle) prepared according to the above method.

[0048] Among them, the use of the mitochondrion-targeted puerarin nanoparticles in the preparation of a medicament for treating nervous system diseases.

[0049] In particular, the nervous system diseases are Parkinson's disease, depression, stroke or ischemic stroke, preferably ischemic stroke.

[0050] On the other hand, the present invention provides a preparation method of a nasal administration nano-thermosensitive assembled gel (i.e., mitochondrion-targeted puerarin thermosensitive gel), comprising the following steps:

[0051] Adding the high molecular materials poloxamer P407 and poloxamer P188 into the above-mentioned nasal administration nanoparticles (i.e., mitochondrion-targeted puerarin nanoparticles), stirring and mixing evenly, and standing for at least 18 h after the high molecular materials are dissolved to make the molecular segments of the high molecular materials stretch and form a stable colloidal solution, thus obtaining the mitochondrion-targeted puerarin thermosensitive gel.

[0052] Among them, the mass ratio of SS-31 peptide-chitosan in the mitochondrion-targeted puerarin nanoparticles (i.e., Ge-peptide-ROS nanoparticles, namely the puerarin-SS-31 peptide ROS-responsive nanoparticle solution prepared in step (5)) to the mass of poloxamer P407 is 1:(16-22), preferably 1:18; the mass ratio of SS-31 peptide-chitosan in the mitochondrion-targeted puerarin nanoparticles (i.e., Ge-peptide-ROS nanoparticles, namely the puerarin-SS-31 peptide ROS-responsive nanoparticle solution prepared in step (5)) to the mass of poloxamer P188 is 1:(2-5), preferably 1:2.

[0053] In particular, the standing treatment time is 18-24 h, preferably 24 h.

[0054] The prepared mitochondrion-targeted puerarin thermosensitive gel will solidify into a gel state below 34 °C, namely the puerarin-SS-31 peptide-ROS-responsive nanoparticle-thermosensitive assembled gel system (abbreviated as SS-31NP / PU Gel);

[0055] On the other hand, the present invention provides a mitochondrion-targeted puerarin thermosensitive gel prepared according to the above method.

[0056] Among them, the use of the mitochondrion-targeted puerarin thermosensitive gel in the preparation of a medicament for treating nervous system diseases.

[0057] In particular, the nervous system diseases are Parkinson's disease, depression, stroke or ischemic stroke, preferably ischemic stroke.

[0058] The present invention provides, on the one hand, an application of puerarin and SS-31 peptide in the preparation of a drug or health product for preventing, alleviating and / or treating ischemic nervous system diseases.

[0059] Among them, the ischemic nervous system diseases mainly include ischemic stroke.

[0060] The present invention provides, on the other hand, an application of puerarin in the preparation of a drug for reperfusion therapy of ischemic stroke.

[0061] In particular, the drug is composed of puerarin, SS-31 peptide and a pharmaceutically acceptable carrier.

[0062] When the SS-31 peptide described in the present invention is used for preventing, alleviating or treating ischemic stroke reperfusion, it can mediate the penetration of nanoparticles through the cell membrane and actively target to the mitochondrial site.

[0063] When the puerarin described in the present invention is used for preventing, alleviating or treating ischemic stroke reperfusion, it can be responsive released in mitochondria rich in ROS.

[0064] The nanoparticles in the present invention can target puerarin to mitochondria and then release it in a responsive manner.

[0065] The drug of the present invention is administered through the nasal route, and is a pharmaceutical preparation and corresponding pharmaceutical dosage form with SS-31 peptide and puerarin as active ingredients for preventing, alleviating or treating ischemic stroke. The pharmaceutical preparation takes the SS-31 peptide and puerarin as effective active ingredients and includes other carrier components acceptable in pharmacy.

[0066] Among them, the selected puerarin has a purity > 98%.

[0067] The puerarin-SS-31 peptide-ROS-responsive nanoparticles-thermosensitive gel prepared by the present invention through nasal administration is used for preventing, treating and / or alleviating ischemic nervous system diseases, including diseases such as cerebral ischemia reperfusion.

[0068] Through the interaction between mitochondrion-targeted and ROS-responsive puerarin nanoparticles and the molecular segments of poloxamer, a thermosensitive assembled nanogel is constructed. On the one hand, the chitosan nanoparticles serve as a drug reservoir and can actively target damaged mitochondria under the mediation of SS-31 peptide. On the other hand, in the mitochondria rich in ROS, the thioketal bond in the crosslinker TKDA of the nanoparticles will break responsively, causing the nanoparticles to disintegrate and release the drug. In addition, the chitosan drug-loaded nanoparticles and poloxamer are used to construct a thermosensitive assembled hydrogel, which is beneficial to prolong the residence time of the drug in the nasal cavity. Traditional administration methods such as intravenous injection and oral administration are difficult for drugs to cross the blood-brain barrier and enter the brain tissue to exert their efficacy. By using the nasal administration method, the gel changes from a flowing liquid to an immobile hydrogel state at body temperature and adheres to the nasal mucosa surface to continuously release the drug. Importantly, this drug delivery system can directly enter the brain through the olfactory region, actively target the mitochondrial site, and respondently lyse in an environment rich in ROS to release puerarin, thereby improving the efficacy of the drug.

[0069] Compared with the prior art, the present invention has the following obvious advantages:

[0070] 1. First, the drug puerarin is loaded into chitosan nanoparticles modified with a polypeptide (SS-31), which overcomes the problem of poor water solubility of puerarin and endows the delivery and release of puerarin with mitochondrion-targeted and ROS-responsive characteristics.

[0071] A mitochondrion-targeted polypeptide SS-31 peptide is modified on chitosan using a coupling agent to prepare SS-31 peptide-chitosan. Then, an active oxygen (ROS)-sensitive crosslinker TKDA is used to carry out a crosslinking reaction with the modified SS-31 peptide-chitosan to prepare ROS-responsive sensitive chitosan nanoparticles, enabling the modified chitosan nanoparticles to actively target the mitochondria of cerebral ischemia-reperfusion injury and respondently release puerarin in the mitochondria rich in ROS, which is beneficial to the efficient accumulation of the drug puerarin in the organelles of cerebral ischemia-reperfusion injury and rapidly treat cerebral ischemia-reperfusion injury.

[0072] 2. Poloxamer is added to the ROS-sensitive chitosan nanoparticles in the present invention to make the nanoparticles into a thermosensitive gel preparation with temperature sensitivity. At body temperature, it can prolong the residence time of the drug in the nasal cavity. By using the nasal administration method, through the nasal-brain pathway, the drug can directly enter the brain without passing through the blood-brain barrier.

[0073] In addition, the present invention can be prepared into a drug or health food for preventing, conditioning and / or treating ischemic nervous system diseases, thus developing a new direction for the clinical application of puerarin and protein drugs.

[0074] 3. The puerarin of the present invention has strong pharmacological effects, and has remarkable efficacy in preventing, conditioning and treating cerebral ischemia-reperfusion injury, with quick effect, small toxic and side effects, good safety, and its mechanism of action in treating cerebral ischemia-reperfusion injury can be analyzed by modern pharmacology, having good medicinal prospects.

[0075] 4. The product of the present invention has rich raw material sources, is safe for clinical use, has a simple preparation process, can be made into various dosage forms, and has a small dosage and convenient use, so it is easy to promote.

[0076] 5. The drug for preventing and treating cerebral ischemia-reperfusion injury prepared by the present invention using puerarin and SS-31 peptide realizes the targeted delivery and responsive release of effective components of traditional Chinese medicine, and can be used to prepare drugs for targeted treatment of ischemia-reperfusion injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 In vitro responsive drug release curve of puerarin-SS-31 peptide-ROS responsive nanoparticles-thermosensitive gel.

[0078] Figure 2 Temperature scanning curve of rheological study of puerarin-SS-31 peptide-ROS responsive nanoparticles-thermosensitive gel.

[0079] Figure 3 Effect diagram of puerarin-SS-31 peptide-ROS responsive nanoparticles on the survival rate of H2O2-induced SH-SY5Y cells.

[0080] Figure 4 Microscopic observation diagram of mitochondrial targeting of puerarin-SS-31 peptide-ROS responsive nanoparticles to SH-SY5Y cells.

[0081] Figure 5 Research diagram of the effect of puerarin-SS-31 peptide-ROS responsive nanoparticles on intracellular ROS in H2O2-induced SH-SY5Y cells.

[0082] Figure 6 Observation and analysis diagram of the cerebral infarction volume of puerarin-SS-31 peptide-ROS responsive nanoparticles-thermosensitive gel in cerebral ischemia-reperfusion injury.

[0083] Figure 7 Research and analysis diagram of the expression of mitochondrial-related apoptotic proteins (Bax, Bcl-2) in the brain tissue of rats with cerebral ischemia-reperfusion injury by puerarin-SS-31 peptide-ROS responsive nanoparticles-thermosensitive gel. DETAILED DESCRIPTION OF THE INVENTION

[0084] The present invention will be further described below in conjunction with specific embodiments. However, these embodiments are only for illustrating the present invention and not for limiting the scope of the present invention. The experimental methods without specific experimental conditions noted in the following embodiments are generally carried out under conventional conditions or according to the conditions recommended by the manufacturers.

[0085] The therapeutic effect of the nasal administration of puerarin thermosensitive gel, namely the puerarin-SS-31 peptide-ROS-responsive nano-thermosensitive gel, on cerebral ischemia-reperfusion injury of the present invention will be further described through experiments below. These test examples include the in vitro characterization and drug release test of the puerarin-SS-31 peptide-ROS-responsive nanoparticles-thermosensitive gel of the present invention and the in vitro and in vivo pharmacodynamic tests of the puerarin-SS-31 peptide-ROS-responsive nanoparticles-thermosensitive gel of the present invention.

[0086] Example 1 Preparation of SS-31 peptide-chitosan (abbreviated as peptide-chitosan)

[0087] 1A. Accurately measure pure water into a beaker, heat it, and add accurately weighed chitosan while maintaining the water temperature at 45°C. Stir, and slowly add concentrated hydrochloric acid to the mixture until the chitosan is dissolved to obtain a chitosan aqueous solution, where the mass / volume concentration of the chitosan aqueous solution is 1% (w / v) (usually 0.5%-1.5% (w / v)); the pH is 5.5 (usually 5-6).

[0088] For example, measure 100 mL of pure water and add it to a beaker, preheat it at 45°C, weigh 1 g of chitosan, add it to the preheated pure water and stir, and then slowly add concentrated hydrochloric acid to the mixture until the chitosan is dissolved to obtain a chitosan aqueous solution, where the mass / volume concentration of the chitosan aqueous solution is 1% (w / v).

[0089] The mass / volume concentration of the chitosan aqueous solution in the specific implementation manner of the present invention is described by taking 1% (w / v) as an example, and other concentrations such as 0.5%-1.5% (w / v) are also applicable to the present invention.

[0090] 1B. Add sodium 4-(N-maleimidomethyl)cyclohexane-1-carboxylate sulfosuccinimide ester (Sulfo-SMCC, 271 mg) to an aqueous chitosan solution (100 mL, containing 1 g of chitosan). Here, the molar ratio of Sulfo-SMCC to the amino groups on chitosan in the aqueous chitosan solution is 1:10 (usually 1:10 - 20). Stir at room temperature to carry out the first cross-linking reaction. The amino groups on Sulfo-SMCC and chitosan are coupled. After 30 min (usually 20 - 45 min) of the first cross-linking reaction, place the first cross-linking reaction mixture in a dialysis bag with a molecular weight cut-off of 3500 and conduct the first dialysis treatment in pure water. Dialyze in pure water for 24 h (usually 20 - 24 h) to remove the unreacted Sulfo-SMCC and obtain the first cross-linked solution.

[0091] 1C. Add SS-31 peptide (230 mg) to the first cross-linked solution and stir at room temperature to carry out the second cross-linking reaction. Here, the molar ratio of the sulfhydryl groups in SS-31 peptide to the amino groups in chitosan in the aqueous chitosan solution is 1:20 (usually 1:15 - 25). The sulfhydryl groups on Sulfo-SMCC and SS-31 peptide are coupled. After 30 min (usually 20 - 45 min) of the second cross-linking reaction, conduct the second dialysis treatment, that is, dialyze in pure water for 24 h (usually 18 - 24 h), where the molecular weight cut-off of the dialysis bag is 3500, to remove the unreacted SS-31 peptide.

[0092] Sodium 4-(N-maleimidomethyl)cyclohexane-1-carboxylate sulfosuccinimide ester (Sulfo-SMCC) is a sulfhydryl-amino coupling agent. It first couples with the amino groups in chitosan and then with the sulfhydryl groups in SS-31 peptide.

[0093] 1D. Freeze-dry the SS-31 peptide-chitosan retained in the dialysis bag after the second dialysis treatment to obtain SS-31 peptide-chitosan (abbreviated as peptide-chitosan, 1.2 g) for standby.

[0094] Example 2. Synthesis of ROS-responsive cross-linking agent (TKDA)

[0095] Weigh 2,2'-[propane-2,2-diylbis(thio)]diacetic acid (112.15 mg) and add it to DMSO (0.5 mL), stir to dissolve. Then add EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 115.09 mg) and NHS (N-hydroxysuccinimide, 191.70 mg). Under room temperature, avoid light and stir for 5 - 6 h to obtain the crosslinker TKDA (0.95 mL, that is, 0.95 mL of the DMSO solution of TKDA. The crosslinker TKDA has ROS responsiveness) by esterification reaction. Store the crosslinker TKDA in the dark at 4 °C for later use. It is directly used for the preparation of mitochondria-targeted puerarin nanoparticles, where the molar ratio of 2,2'-[propane-2,2-diylbis(thio)]diacetic acid to EDC is 1:2 (usually 1:1.5 - 2); the molar ratio of 2,2'-[propane-2,2-diylbis(thio)]diacetic acid to NHS is 1:2 (usually 1:1.5 - 3); the mass ratio of 2,2'-[propane-2,2-diylbis(thio)]diacetic acid to the volume of the solvent DMSO is 0.448:1 (usually 0.4 - 0.5:1).

[0096] TKDA: 2,2'-[propane-2,2-diylbis(thio)]diacetic acid NHS ester. TKDA is the ester intermediate formed by activating the carboxyl group of 2,2'-[propane-2,2-diylbis(thio)]diacetic acid with EDC and NHS.

[0097] Example 3. Preparation of puerarin-ROS-responsive nanoparticles (SS-31NP / PU)

[0098] 3A. Preparation of paraffin-emulsifier mixture

[0099] Connect and fix the multifunctional vacuum stirrer to the overhead stirrer. First, add liquid paraffin (35 mL) to a three-necked flask (250 mL), then add the emulsifiers Span-85 (1 mL) and Tween-80 (0.3 mL). Under water bath conditions, stir and mix evenly to prepare the paraffin-emulsifier mixture (36.3 mL, as the oil phase), where the volume ratio of Span-85 to Tween-80 is 1:0.3 (usually 1:0.3 - 0.6); the volume ratio of Span-85 to liquid paraffin is 1:35 (usually 1:30 - 35); the volume ratio of Tween-80 to liquid paraffin is 1:116.7 (usually 1:100 - 150); the water bath temperature is 50 °C (usually 50 - 60 °C); the stirring rate is 500 rpm (usually 500 - 800 rpm), and the stirring time is 30 min (usually 30 - 60 min).

[0100] 3B. Weigh the SS-31 peptide-chitosan (0.1 g) prepared in Example 1, add it to pure water (10 mL), stir to dissolve, and prepare an SS-31 peptide-chitosan aqueous solution (abbreviated as peptide-chitosan aqueous solution, 10 mL, aqueous phase). The mass / volume concentration of the prepared SS-31 peptide-chitosan aqueous solution is 1% (w / v, usually 0.5 - 1.5%).

[0101] 3C. Under the condition of maintaining the water bath temperature at 50 °C (usually 50 - 60 °C), slowly drip the SS-31 peptide-chitosan aqueous solution (aqueous phase, 4 mL) into the paraffin-emulsifier mixture (oil phase, 36.3 mL) prepared in step 3A) using a peristaltic pump while stirring. Control the dripping flow rate at 0.75 mL / min (usually 0.6 - 1 mL / min). After the dripping is completed, continue to stir for 2 h to fully mix the aqueous phase and the oil phase evenly for emulsification treatment, and obtain a paraffin-emulsifier-SS-31-chitosan mixture (40.3 mL, oil phase-aqueous phase emulsion system). The volume ratio of the aqueous phase to the oil phase is 1:9.075 (usually 1:8 - 10), and the rotation speed of the stirrer is 1000 rpm (usually 1000 - 1500 rpm).

[0102] 3D. Weigh puerarin (10 mg), dissolve it in the ROS-responsive crosslinker TKDA (50 μL) prepared in Example 2 to obtain a puerarin-TKDA solution (50 μL). The mass ratio of puerarin to the volume of the crosslinker TKDA is 1:5 (usually 1:4 - 6), that is, 10 mg of puerarin is dissolved in every 50 μL of the DMSO solution of the crosslinker TKDA.

[0103] 3E. Under the condition of maintaining the water bath temperature at 50 °C (usually 50 - 60 °C), after the oil phase and the aqueous phase are fully mixed, slowly drip the puerarin-TKDA solution (50 μL) into the paraffin-emulsifier-SS-31-chitosan mixture (i.e., the oil phase-aqueous phase mixture system, 40.3 mL) using a peristaltic pump while stirring. Among them:

[0104] The molar ratio of the amino group contained in chitosan in SS-31-chitosan in the aqueous phase to the N-hydroxysuccinimide group in TKDA is 10:1 (usually (9 - 10):1);

[0105] The mass ratio of SS-31 peptide-chitosan in the aqueous phase to the mass of puerarin is 100:25 (usually 100:(20 - 25)).

[0106] Control the flow rate of the ROS-responsive crosslinking agent TKDA added to be 0.15 mL / min (usually 0.1 - 0.2 mL / min); the stirring speed is 1000 rpm (usually 1000 - 1500 rpm). After the dropping is completed, continue stirring for 3 h (usually 2 - 4 h) to carry out the third crosslinking reaction, so that SS-31-chitosan and the TKDA crosslinking agent react fully to form ROS-responsive nanoparticles, and puerarin is uniformly dispersed in the ROS-responsive nanoparticles and will enter the interior of the nanoparticles.

[0107] 3F. Transfer the mixed liquid of the third crosslinking reaction in the three-necked flask to a 50 mL centrifuge tube and carry out centrifugation treatment, where centrifuge at 3800 rpm for 10 min to separate the aqueous phase and the oil phase, discard the oil phase, and collect the lower aqueous phase to obtain a puerarin-SS-31 peptide-ROS-responsive nanoparticle solution (i.e., puerarin-peptide-ROS nanoparticle solution, abbreviated as SS-31NP / PU, 4 mL).

[0108] Example 4. Preparation of puerarin-peptide-ROS nanoparticle-thermosensitive assembled gel system (SS-31NP / PU Gel)

[0109] Take 1 mL of the puerarin-peptide-ROS nanoparticle solution prepared in Example 3 (where the mass of SS-31 peptide-chitosan in the puerarin-peptide-ROS nanoparticle solution is 10 mg) and place it in a vial (2.7 cm × 4.7 cm), then add poloxamer P407 (P407, 180 mg) and poloxamer P188 (P188, 20 mg), stir. After the poloxamer is dissolved, let the mixed solution stand for 24 h (usually 18 - 24 h) so that the molecular chain segments of the polymer material fully stretch to form a stable colloidal solution, and this colloidal solution is the thermosensitive gel stock solution, that is, puerarin-SS-peptide-ROS nanoparticle-thermosensitive gel stock solution (abbreviated as SS-31NP / PU Gel stock solution), where the mass ratio of P407 to P188 is 9:1 (usually 8 - 9:1); the mass ratio of SS-31 peptide-chitosan in the puerarin-peptide-ROS nanoparticles to poloxamer P407 is 1:18 (usually 1:16 - 22); the mass ratio of SS-31 peptide-chitosan in the puerarin-peptide-ROS nanoparticles to poloxamer P188 is 1:2 (usually 1:2 - 5).

[0110] The SS-31NP / PU Gel stock solution will solidify into a gel state at a temperature of 34 °C to form a puerarin-SS-31 peptide-ROS-responsive nanoparticle-thermosensitive assembled gel system (abbreviated as SS-31NP / PU Gel).

[0111] Test Example 1 Particle size and Zeta potential measurement of puerarin-SS-31 peptide-ROS-responsive nanoparticles

[0112] Dilute the nanoparticle solution prepared in Example 3 by 10 times, and use a nanoparticle size analyzer to measure the particle size, particle size dispersion index and surface Zeta potential of the nanoparticles. Repeat the measurement 3 times at room temperature, and take the average value of the results. The test results are shown in Table 1.

[0113] Table 1 Particle size and Zeta-potential of puerarin-SS-31 peptide-ROS responsive nanoparticles (Means±SD)

[0114] Particle size (nm) Polydispersity index Zeta potential (mV) Puerarin-SS-31 peptide-ROS-responsive nanoparticles 36.50±2.2 0.418±0.11 16.0±3.1

[0115] It can be seen from the test results in Table 1 that the puerarin-SS-31 peptide-ROS responsive nanoparticles of the present invention have a small particle size and good uniformity; the dispersion index of the nanoparticles is small, and the particle size distribution of the nanoparticles is uniform; the Zeta-potential of the nanoparticles is high, and the stability of the nanoparticles is high.

[0116] Test Example 2 Determination of drug loading rate of puerarin-SS-31 peptide-ROS responsive nanoparticles

[0117] Measure 1 mL of the puerarin-SS-31 peptide-ROS responsive nanoparticle solution in Example 3, freeze-dry it and record the weight m1 of the nanoparticle solid. Then add HCl solution (200 μL, 0.1 M) and H2O2 solution (200 μL) to the nanoparticle solid. HCl is used to dissolve the chitosan of the nanoparticles, and the H2O2 solution is used to cleave TKDA. Finally, add 1 mL of methanol solution and perform ultrasonic treatment 3 times. Methanol extracts puerarin. The ultrasonic extraction treatment time is 30 min each time, and 1 mL of fresh methanol solution is replaced each time. Transfer the three methanol extraction solutions to a 10 mL volumetric flask and make up to the mark. Filter through a microporous membrane with a pore size of 0.45 μm to obtain the sample solution for measuring the drug loading amount.

[0118] The content of puerarin in the sample solution is determined by high performance liquid chromatography. The determination conditions are as follows: chromatographic column: Kromasil 100-5 C18 column (250×4.6 mm, 5 μm), phase A is acetonitrile (0.1% trifluoroacetic acid), phase B is ultrapure water (0.1% trifluoroacetic acid), flow rate: 1 mL / min; column temperature: 30 °C, detection wavelength: 306 nm; injection volume: 10 μL;

[0119] Gradient elution conditions: 15% acetonitrile (0.1% trifluoroacetic acid) at 0 min, 40% acetonitrile (0.1% trifluoroacetic acid) at 25 min, 100% acetonitrile (0.1% trifluoroacetic acid) at 30 min

[0120] Under the above chromatographic conditions, the components in the sample are baseline separated. There is interference in the determination of the chitosan component in the sample at 254 nm. Therefore, 306 nm is selected as the detection wavelength for puerarin. The method is feasible under these chromatographic conditions.

[0121] The mass (m2) of puerarin in puerarin-SS-31 peptide-ROS-responsive nanoparticles was determined by HPLC, and the drug loading rate was calculated according to the following formula (1):

[0122] Drug loading rate (%) = mass of the drug encapsulated in the nanoparticles (m2) / mass of the drug-loaded nanoparticles (m1) × 100% (1)

[0123] The results of three parallel experiments were as follows: the mass m2 of the drug encapsulated in the nanoparticles was 0.27, 0.27, 0.27 mg; the mass m1 of the drug-loaded nanoparticles was 10.01, 10.12, 9.98 mg; the puerarin drug loading rate was approximately 2.69%, and the ROS-responsive nanoparticles of the present invention had a high drug loading capacity.

[0124] Experimental Example 3 Study on in vitro responsive release of puerarin from puerarin-SS-31 peptide-ROS-responsive nanoparticles-thermosensitive gel

[0125] The release behavior of the gel was explored by the membrane-free release method.

[0126] (1) SS-31NP / PU Gel was prepared according to the method prepared in Example 4, and a release experiment was carried out (the sample was in parallel 3 times).

[0127] (2) Phosphate buffer solution (PBS) with pH = 6.4 simulating the nasal cavity environment was selected as the release medium, and in vitro nasal cavity simulation release was carried out at a temperature of 34 °C and a rotation speed of 55 rpm.

[0128] (3) The gel stock solution (1 mL, SS-31NP / PU Gel stock solution) was placed in a glass bottle of known weight and placed in a shaker preheated to and maintained at a temperature of 34 °C. After the gel stock solution formed a gel, it was weighed, and the initial weight of the gel and the glass bottle was recorded. Then, PBS buffer solution (2 mL) preheated to a temperature of 34 °C was added to the glass bottle. Subsequently, the shaker was started, and at 0.25, 0.5, 0.75, 1, 2, 3, 4, 6, 8, 12, 24 h, a total of 11 times of liquid was taken. Each time, all the release medium was taken out, the weight of the remaining gel and the glass bottle was weighed, and 2 mL of fresh release medium (PBS buffer solution) was added to continue the release until all the gels were eroded. The release medium taken out each time was filtered through a microporous filter with a pore size of 0.45 μm, and the filtrate was placed in a 1.5 mL centrifuge tube and stored in a 4 °C refrigerator to determine the concentration of puerarin in the release solution.

[0129] The filtrate (200 μL) of the solution taken at each of the above time points was respectively taken as a sample for measuring the release of puerarin from the thermosensitive gel, and the puerarin concentration was determined by HPLC. The in vitro drug release performance was expressed by the cumulative drug release percentage, and the cumulative drug release rate (%) was calculated according to formulas (2) and (2-1):

[0130] Q n = C1×V1 + C2×V2 + C3×V3 + … C n ×Vn (2)

[0131]

[0132] Where: Q n is the cumulative drug release amount at the nth sampling; C n is the drug concentration in the release medium at the nth sampling; Vn is the volume of the release solution taken at the nth sampling; n is an integer, n = 1, 2, 3, ..., 11; Q 总 is the content of puerarin in the gel.

[0133] The drug release results are as Figure 1 shown: Where:

[0134] The cumulative release amounts of puerarin at 0.5, 1, 2, 3, 4, 5, 6, 8, 12, and 24 h are 10.21%, 12.31%, 23.23%, 23.85%, 32.32%, 38.26%, 45.37%, 53.43%, 65.24%, and 82.43% respectively.

[0135] At 0 - 2 h, the drug puerarin is released rapidly, but the release is slow and stable in the later stage. This release mode is more in line with the requirements of drug release through the nasal route to the brain. In the 2 - 12 h stage, the release curve of puerarin is approximately a straight line, indicating that the release of puerarin is slow and stable. In the 33 °C environment of the nasal cavity, the thermosensitive gel solidifies into a solid form, slowing down the speed of gel destruction, blocking erosion by the release medium, and enabling stable drug release.

[0136] The burst release problem of the nano - preparation causes the drug to be released too fast in the early stage, resulting in a drastic change in the drug content entering the brain. The function of the thermosensitive gel enables the drug to be released in a slow and stable manner. The thermosensitive gel for nasal administration enables the drug puerarin to enter the brain in a slow and stable manner, which is more conducive to maintaining a stable drug concentration in the brain and avoiding acute adverse reactions caused by drug burst release. Experimental Example 4 Rheological Study of Puerarin - SS - 31 Peptide - ROS Responsive Nanoparticle - Thermosensitive Gel

[0137] According to the formulation in Table 2, take P407 and P188 with 9 different dosing ratios, and add 1 mL of the puerarin-SS-31 peptide-ROS-responsive nanoparticle solution prepared in Example 3 respectively. Thermosensitive gels with different formulations were prepared according to the method of Example 4; the rheological parameters (elastic modulus G', viscous modulus G") of different formulations were measured in the range of 15 - 40 °C. The rheological parameters of the "puerarin-peptide-ROS-nano thermosensitive gel" were measured using a rheometer. The frequency of the rheometer was set to 1.00 Hz, the shear stress was 0.01, and the detection time was 30 min.

[0138] Table 2 Rheological study of thermosensitive gel Poloxamer dosage ratio

[0139]

[0140]

[0141] The storage modulus, also known as the elastic modulus, refers to the amount of energy stored due to elastic (reversible) deformation when the material undergoes deformation, reflecting the elasticity of the material; the loss modulus, also known as the viscous modulus, refers to the amount of energy dissipated due to viscous deformation (irreversible) when the material undergoes deformation, reflecting the viscosity of the material; when the storage modulus is much greater than the loss modulus, the material mainly undergoes elastic deformation, so the material is in a solid state; when the loss modulus is much greater than the storage modulus, the material mainly undergoes viscous deformation, so the material is in a liquid state; when the storage modulus and the loss modulus are comparable, the material is in a semi-solid state, and the gel is a typical semi-solid substance. When the storage modulus and the loss modulus cross, the gelation temperature of the thermosensitive gel is reflected, and we can quantitatively calculate the gelation temperature of the thermosensitive gel through this method. The measurement results are as Figure 2 shown, where: keeping the P407 content in the gel system unchanged, as the P188 content increases, the gelation temperature continuously increases; keeping the P188 content in the gel system unchanged, as the P407 content increases, the gelation temperature continuously decreases. By adjusting the different ratios of P407 and P188, a thermosensitive gel formulation that meets the requirements of nasal drug delivery is obtained, and the ratio of P407 to P188 is 18:2.

[0142] Figure 2 The ratios of P407 / P188 (w / w) in A, B, C, D, E, F, G, H, I are 16:0, 16:2, 16:4, 18:0, 18:2, 18:4, 20:0, 20:2, 20:4 respectively.

[0143] Experimental Example 5 Effect of puerarin-SS-31 peptide-ROS-responsive nanoparticles on the viability of H2O2-induced SH-SY5Y cells

[0144] (1) Preparation of puerarin nanoparticles solution (NP / PU): Except that in step 3C), the aqueous phase is a chitosan solution with a mass / volume concentration of 1% (w / v); in step 3E), the third cross-linking reaction allows chitosan to fully react with the TKDA cross-linking agent to form nanoparticles; in step 3F), the lower aqueous phase is collected to obtain the puerarin-ROS-responsive nanoparticles solution (puerarin-ROS nanoparticles, abbreviated as NP / PU nanoparticles solution, abbreviated as NP / PU solution 4 mL), the rest is the same as in Example 3. Among them, the chitosan solution is prepared as follows: Weigh 0.1 g of chitosan, add it to 10 mL of pure water, add concentrated hydrochloric acid and stir to dissolve it to prepare a 1% chitosan aqueous solution, and let it stand overnight at 4 °C for use.

[0145] (2) Preparation of SS-31 peptide nanoparticles solution (SS-31NP): Except that puerarin is not added in step 3D), the rest is the same as in Example 3, and the SS-31 peptide nanoparticles solution (abbreviated as SS-31NP solution) is prepared.

[0146] (3) Use the CCK-8 reagent (Cell Counting Kit-8 cell counting reagent) to detect the effects of the puerarin nanoparticles solution (NP / PU) prepared in step (1), the SS-31 peptide nanoparticles (SS-31NP) prepared in step (2), the puerarin-peptide-ROS nanoparticles solution (SS-31NP / PU) prepared in Example 3, and free puerarin (PU) on the viability of H2O2-induced SH-SY5Y cells. The concentration of puerarin in each group is 100 μM.

[0147] Take SH-SY5Y cells in the logarithmic growth phase and inoculate them into a 96-well plate with a cell density of 2×10 4 cells / well, and place them in a constant-temperature cell incubator for 24 h. The groups are the Control group, the SS-31NP / PU group, the NP / PU group, the SS-31NP group, and the PU group. After the cells adhere to the wall, the Control group is not treated, and the other 5 groups are incubated with a medium containing 50 μM H2O2 for 12 h, and then added to the cell medium containing different drugs. After incubating for 12 h, add the medium containing the CCK-8 reagent and incubate in the dark for 3 - 4 hours. Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance of the 96-well plate at 450 nm. There are 6 replicate wells in each group. Calculate the cell viability according to formula (3).

[0148] Cell viability = [(As - Ab) / (Ac - Ab)] × 100% (3)

[0149] In formula (3): As: absorbance of the experimental well (containing cells, culture medium, CCK-8 solution and drug solution); Ac: absorbance of the control well (containing cells, culture medium and CCK-8 solution, without drug); Ab: absorbance of the blank well (containing culture medium and CCK-8 solution, without cells and drug).

[0150] The experimental results are as Figure 3 , SS-31NP / PU (prepared in Example 3): the administration group of puerarin-loaded nanoparticles prepared from chitosan modified with SS-31 peptide; NP / PU: the administration group of puerarin-loaded nanoparticles prepared from unmodified chitosan prepared in step (1); SS-31NP: the administration group of drug-free nanoparticles prepared from chitosan modified with SS-31 peptide prepared in step (2); PU: the administration group of puerarin monomer; Control: the blank control group without treatment under normal culture conditions.

[0151] SS-31NP / PU, NP / PU, SS-31NP and PU can all improve H2O2-induced cell damage; it is worth noting that compared with NP / PU, SS-31NP and free PU, SS-31NP / PU can significantly reduce the cell damage caused by H2O2 (the relative cell viability reaches (73.16±4.92)%). This may be related to the transmembrane property and mitochondrial targeting of SS-31 peptide. The modification of SS-31 peptide enhances the ability of nanoparticles to enter cells, delivers more drugs to cell mitochondria, enhances the efficacy of puerarin, and improves cell survival rate.

[0152] Figure 3 Compared with the H2O2 group, #P < 0.05, ##P < 0.01; compared with the Control group, &&P < 0.01; compared with the SS-31NP group at the same concentration, **P < 0.01; n = 6, Mean±SD).

[0153] Experimental Example 6 Study on the mitochondrial targeting of coumarin-SS-31 peptide-ROS-responsive nanoparticles to SH-SY5Y cells

[0154] Since puerarin itself has no fluorescence and coumarin-6 is a lipophilic and hydrophobic fluorescent dye, because its solubility is similar to that of puerarin, coumarin-6 was used to prepare fluorescently labeled nanoparticles, and then a laser confocal microscope was used to observe the distribution of coumarin-6-labeled nanoparticles in the mitochondria of SH-SY5Y cells to analyze whether the modification of SS-31 peptide can increase the mitochondrial targeting of nanoparticles.

[0155] (1) Preparation of coumarin nanoparticles (Cou6 NP):

[0156] 1A. Prepare a paraffin-emulsifier mixture (36.3 mL, oil phase), and the preparation method is the same as that in step 3A) of Example 3.

[0157] 1B. Weigh 0.1 g of chitosan, add it to 10 mL of pure water, add concentrated hydrochloric acid and stir to dissolve it to prepare a 1% chitosan aqueous solution (aqueous phase), and let it stand overnight at 4 °C for later use.

[0158] 1C. Except for slowly dripping the chitosan aqueous solution (aqueous phase, 4 mL) into the oil phase (36 mL) prepared in step 1A) using a peristaltic pump, the rest is the same as step 3C) of Example 3.

[0159] 1D. Except for weighing 10 mg of coumarin, that is, using coumarin to replace puerarin, the rest is the same as step 3D) of Example 3 to prepare a coumarin-TKDA solution (50 μL), where the mass ratio of coumarin to the volume of the crosslinking agent TKDA is 1:5, that is, 10 mg of coumarin is dissolved in every 50 μL of the DMSO solution of the crosslinking agent TKDA; the mass ratio of coumarin to chitosan is 25:100 (usually (20 - 25):100).

[0160] 1E. After the oil phase and the aqueous phase are fully mixed,

[0161] Use a peristaltic pump to slowly drip the coumarin-TKDA solution (50 μL) into the oil phase-aqueous phase mixture system (40 mL), and stir while dripping, where:

[0162] The molar ratio of the amino group in chitosan in the aqueous phase to the N-hydroxysuccinimide group in TKDA is 10:1 (usually 1:9 - 10); the mass ratio of chitosan in the aqueous phase to coumarin is 100:25; control the flow rate of adding the ROS-responsive crosslinking agent TKDA to be 0.15 mL / min; the stirring speed is 1000 rpm, and continue to stir for 3 h after dripping to carry out the third crosslinking reaction to make chitosan react fully with the TKDA crosslinking agent to form nanoparticles.

[0163] 1F. Transfer the third crosslinking reaction mixture liquid in the three-necked flask to a 50 mL centrifuge tube, carry out centrifugation, where centrifuge at 3800 rpm for 10 min to separate the aqueous phase and the oil phase, discard the oil phase, and collect the lower aqueous phase to obtain the Cou6 NP solution.

[0164] (2) Prepare coumarin-6-SS-31 peptide-loaded nanoparticles (Cou6-SS31 NP): Except that puerarin in step 3) is replaced by coumarin, the rest is the same as Example 3 to obtain a coumarin-SS-31 peptide-ROS-responsive nanoparticle solution (fragrant-peptide-ROS nanoparticle solution, abbreviated as Cou6-SS-31NP solution, 4 mL).

[0165] Replace puerarin with coumarin 6, which is a fluorescent substance and can be used to observe the intracellular distribution of nanoparticles under a confocal microscope.

[0166] (3) Take SH-SY5Y cells in the logarithmic growth phase, dissociate them with trypsin, and then prepare a single-cell suspension with complete medium containing 10% heat-inactivated FBS. Seed the cells into 35-mm confocal dishes at an inoculation density of 5×10 5 cells / dish.

[0167] (4) Take out the culture dishes and set up 6 replicate wells for each of the blank control group, chitosan nanoparticles loaded with coumarin-6 (Cou6 NP), and SS-31 modified chitosan nanoparticles loaded with coumarin-6 (Cou6-SS31 NP). Add the corresponding DMEM high-glucose medium containing the above drugs (the dosing concentration of coumarin is 1 μg / mL) to each well. The blank control group only adds DMEM high-glucose medium, and then place them in a cell incubator at a constant temperature and continue to incubate for 2 h.

[0168] (5) After incubating for 12 h respectively, take out the confocal culture dishes, aspirate the medium, wash the cells 2 times with PBS, add mitochondrial Mito-Tracker Red CMXRos for staining, and place them in a cell incubator at a constant temperature for 40 min. Then wash 2 times with PBS according to the above steps. Add Hochest 33342 solution and continue to incubate for 15 min. Then wash 2 times with PBS again. Observe the distribution of the two kinds of nanoparticles in SH-SY5Y cells with a laser confocal microscope. The fluorescence of Hoechst33342 stain is excited at 405 nm, the fluorescence of coumarin-6 stain is excited at 458 nm, and the fluorescence of Mito-Tracker Red is excited at 543 nm for observation. The distribution of nanoparticles in SH-SY5Y cells observed by laser confocal microscope is as Figure 4 shown.

[0169] Figure 4 In the figure, the red fluorescence is mitochondria labeled by Mito-tracker Red, the green fluorescence is nanoparticles loaded with coumarin, and the blue fluorescence is the nucleus labeled by Hoechst 33342. The scale bar is 10 μm. Compared with the Cou6 NP group, the Cou6-SS31 NP group showed stronger fluorescence at the mitochondrial localization site, indicating that SS-31 peptide-modified chitosan nanoparticles can increase the distribution of drugs in the mitochondria of SH-SY5Y cells, thereby enhancing the mitochondrial protection effect of drugs.

[0170] Experimental Example 7 Study on the Content of ROS in H2O2-Induced SH-SY5Y Cells by Puerarin-SS-31 Peptide-ROS Responsive Nanoparticles

[0171] H2O2 can induce mitochondria in cells to produce excessive ROS. The content of ROS in cells is measured using a fluorescent probe. DCFH-DA (i.e., the ROS fluorescent probe) can freely cross the cell membrane and then be hydrolyzed by intracellular esterase into DCFH. In contrast, DCFH cannot cross the cell membrane. Therefore, DCFH is accumulated in cells. Although DCFH has no fluorescence, it can be oxidized by intracellular ROS into fluorescent DCF. Therefore, the content of ROS in SH-SY5Y cells can be reflected by detecting the fluorescence intensity of DCF.

[0172] (1) SH-SY5Y cells are seeded in 24-well plates at a cell density of 2×10 5 cells / well and incubated in a constant-temperature cell incubator for 24 hours.

[0173] (2) Take out the culture dishes and set up experimental groups as follows: blank control group, DCFH-DA group, H2O2 group, PU group, SS-31NP group, NP / PU group, SS-31NP / PU group. Each group has 3 parallels, where:

[0174] Blank control group (Control group): Cultured in complete medium without adding DCFH-DA reagent

[0175] DCFH-DA group: Cultured in complete medium with DCFH-DA reagent added

[0176] H2O2 group: Cultured in medium containing 50 μM H2O2 with DCFH-DA reagent added

[0177] PU group: Cultured in medium containing PU and 50 μM H2O2 with DCFH-DA reagent added

[0178] SS-31NP group: Cultured in medium containing SS-31NP and 50 μM H2O2 with DCFH-DA reagent added

[0179] NP / PU group: Cultured in medium containing NP / PU and 50 μM H2O2 with DCFH-DA reagent added

[0180] SS-31NP / PU group: Cultured in medium containing SS-31NP / PU and 50 μM H2O2 with DCFH-DA reagent added

[0181] The corresponding media are added to each of the above groups and cultured for 12 h, and the puerarin concentration in each group is 100 μM.

[0182] (3) Dissolve the ROS fluorescent probe (DCFH-DA) with DMSO and dilute it to a final concentration of 5 μg / mL. Add 400 μL of the fluorescent probe to each group and incubate it in a cell culture incubator at 37 °C for 30 min. Then wash it 3 times with PBS to fully remove the DCFH-DA that has not entered the cells.

[0183] (4) Dissociate the cells with trypsin and centrifuge them at 1000 rpm for 5 min. Resuspend the collected bottom cells with 500 μL of PBS. After filtering through a 200-mesh cell sieve, transfer the cell suspension to a flow tube and detect it using the FL1 channel of a flow cytometer. The excitation wavelength is 488 nm. The intracellular ROS level is expressed by the mean of fluorescence intensity (MFI). The test results are as Figure 5 , where A is the analysis of the intracellular ROS level by flow cytometry and B is the quantitative value of the intracellular ROS level. Figure 5 It is the influence diagram of SS-31NP / PU on intracellular ROS in H2O2-induced SH-SY5Y cells

[0184] It can be seen from the experimental results that compared with the blank control group (Control), the intracellular ROS content in the H2O2-induced group increased significantly; after drug administration, the increase in the intracellular ROS content induced by H2O2 was inhibited. Among them, the ROS contents in the SS-31NP / PU group and the NP / PU group were both lower than that in the PU group, and the ROS content in the SS-31NP / PU group was the lowest, indicating that the SS-31NP / PU nanoparticles had the strongest ability to scavenge ROS. This is because SS-31 enhanced the mitochondrial targeting of the nanoparticles, and mitochondria are the main sites for intracellular ROS production. The nanoparticles modified with SS-31 can transport more puerarin to the mitochondria, play a role in scavenging free radicals, inhibit the production of ROS from the source, and reduce the intracellular ROS level. Compared with the H2O2 group, the ROS level in the SS-31NP group also decreased slightly, probably because the chitosan modified with SS-31 peptide itself has certain antioxidant properties.

[0185] Figure 5 In it: SS-31NP / PU: nanoparticles loaded with puerarin prepared from chitosan modified with SS-31 peptide, prepared in Example 3; NP / PU: nanoparticles loaded with puerarin prepared from unmodified chitosan, prepared in Test Example 5; SS-31NP: drug-free nanoparticles prepared from chitosan modified with SS-31 peptide, prepared in Test Example 5; PU: puerarin monomer. n = 6, Mean±SD. Compared with the H2O2 group, ##P < 0.01; compared with the Control group, &&P < 0.01; compared with the PU group and the SS-31NP group at the same concentration, **P < 0.01.

[0186] Experimental Example 8: Effect of puerarin-SS-31 peptide-ROS-responsive nanoparticles-thermosensitive gel on cerebral infarction volume in cerebral ischemia-reperfusion injury.

[0187] 1 Experimental grouping

[0188] The experiment was divided into 7 groups as follows: (1) blank control group (Control), (2) model group (MCAO), (3) puerarin-SS-31 peptide-ROS-responsive nano-thermosensitive gel (SS-31NP / PU Gel), (4) puerarin nanoparticles thermosensitive gel (NP / PU Gel), (5) SS-31 modified blank nanoparticles thermosensitive gel (SS-31NP Gel), (6) free puerarin (PU), (7) edaravone intravenous injection group (3 mg / kg).

[0189] The experiment was carried out with an effective concentration of puerarin in SS-31NP / PU Gel of 0.5 mg / kg. In subsequent experiments, the dosage of puerarin in each group of nanoparticles was kept consistent at 0.5 mg / kg. A unilateral middle cerebral artery occlusion reperfusion model was made by the suture method, and reperfusion was carried out 2 h after infarction. According to the puerarin content, administration was given at intervals through both nostrils, 20 μL for each nostril, and administration was given 3 times at 5 min, 24 h, and 48 h after cerebral ischemia, respectively. Samples were taken 72 h after cerebral ischemia. The positive drug edaravone group was given intravenous injection, and the other groups were given nasal administration.

[0190] 2 Experimental procedure

[0191] 72 h after cerebral ischemia, the rats in each group were anesthetized with 10% chloral hydrate (0.35 mL / 100 g), and perfused systemically through the heart with PBS buffer (pH 7.4). The brain was taken after the buffer fluid flowing out of the heart (about 300 mL) was almost clear. The brain was taken out and placed in a special brain trough for rats, and coronal sections were made at a thickness of 2 mm before and after the optic chiasm, and about 7 slices were cut. The brain slices were carefully taken out of the brain trough, immersed in 2% 2,3,5-triphenyltetrazolium chloride (TTC) solution (Sigma, St Louis, MO), and placed in the dark at room temperature for 10 min. Then the brain slices were taken out and arranged in a culture dish in turn, a pure white scale was placed beside the brain slices, and the camera was fixed 12 cm directly above the culture dish for photography. After photography, the brain slices were placed in formalin solution for soaking and stored in the dark.

[0192] After photography of the brain slices, the infarct area was measured by the indirect method using Image J software, avoiding the influence of brain edema on increasing the infarct volume. The infarct volume was calculated according to the following formula (1A):

[0193] LI = RT - LN(1A)

[0194] In formula (1A), LI: infarction volume of the left cerebral hemisphere; RT: total volume of the right cerebral hemisphere; LN: non-infarcted volume of the left cerebral hemisphere

[0195] To investigate the therapeutic effect of SS-31NP / PU Gel on cerebral ischemia-reperfusion injury, the effects of SS-31NP / PU Gel, NP / PU Gel, SS-31NP Gel, and PU on the infarction volume caused by cerebral ischemia-reperfusion injury were compared after 3 days of nasal administration. The results are as follows Figure 6 , where A is a photograph of brain slices of rats in different groups, and B is a quantitative analysis chart of the infarct area of brain slices

[0196] It can be seen from the experimental results that compared with the MCAO model group, the infarction volumes of the SS-31NP / PU Gel, NP / PU Gel, and SS-31NP Gel groups were significantly reduced. Moreover, the effect of SS-31NP / PU Gel in reducing the infarction volume was better than that of NP / PU Gel and SS-31NP Gel, with significant differences (P<0.01). The positive drug edaravone (3 mg / kg) could also effectively reduce the infarction volume. However, the raw material drug PU group failed to effectively reduce the infarction volume. It is worth noting that SS-31NP Gel also has good brain protection, which may be because SS-31NP Gel contains the antioxidant SS-31 peptide

[0197] Figure 6 Among them: SS-31NP / PU: puerarin-loaded nanoparticles prepared from chitosan modified with SS-31 peptide, prepared in Example 3; NP / PU: puerarin-loaded nanoparticles prepared from unmodified chitosan, prepared in Test Example 5; SS-31NP: drug-free nanoparticles prepared from chitosan modified with SS-31 peptide, prepared in Test Example 5; PU: puerarin monomer; Edaravone, edaravone. n = 8, Mean±SE. Compared with the MCAO group, ##P<0.01; compared with the Control group, &&P<0.01; compared with the SS-31NP / PU Gel group at the same concentration, **P<0.01;

[0198] Study on the expression of mitochondrial-related apoptotic proteins in the brain tissue of rats with cerebral ischemia-reperfusion injury by puerarin-SS-31 peptide-ROS-responsive nanoparticles-thermosensitive gel

[0199] According to the experimental grouping and animal treatment methods in Test Example 8, the rats in each group after perfusion were craniotomized, the ischemic side of the brain was removed, and stored at -80 °C

[0200] Take the brain tissues of the Control group, SS-31NP / PU Gel group, NP / PU Gel group, SS-31NP Gel group, PU group and edaravone group (3 mg / kg) stored at -80 °C and place them in a liquid nitrogen tank. The brain tissues were lysed by the method of a tissue lysis kit. Among them, the lysis buffer was added according to the ratio of tissue weight: lysis buffer volume = 1:9. The tissue was disrupted by an ultrasonic cell disruptor on ice for 30 s each time, and repeated 3-4 times. Centrifuge at 12,000 r / min at 4 °C for 10 min, and take the supernatant and place it in a 1.5 ml EP tube. Then, the Western-Blot detection method was used to detect the protein expression.

[0201] In this experiment, the Western blot method was used to detect the effects of SS-31NP / PU Gel on the expression of proteins Bcl-2 and Bax related to the mitochondrial apoptosis pathway in the brain tissues of rats with cerebral ischemia-reperfusion injury. As Figure 7 shown, among them, Figure A is the protein band diagram of Bcl-2, Bax, and β-actin, Figure B is the quantitative analysis of the content of Bcl-2 / β-actin in different groups, Figure C is the quantitative analysis of the content of Bax / β-actin in different groups, and Figure D is the quantitative analysis of the content of Bcl-2 / Bax in different groups.

[0202] Figure 7 Among them: SS-31NP / PU: nanoparticles loaded with puerarin prepared from chitosan modified with SS-31 peptide, prepared in Example 3; NP / PU: nanoparticles loaded with puerarin prepared from unmodified chitosan, prepared in Test Example 5; SS-31NP: unloaded nanoparticles prepared from chitosan modified with SS-31 peptide, prepared in Test Example 5; PU: puerarin monomer; Edaravone, edaravone. n = 8, Mean±SE. Compared with the MCAO group, ##P < 0.01; compared with the Control group, &&P < 0.01; compared with the SS-31NP / PU Gel group at the same concentration, *P < 0.05, **P < 0.01.

[0203] It can be seen from Figure 7 that

[0204] Compared with the Control group, the expression of Bcl-2 protein in the ischemic brain tissue of the MCAO group was significantly decreased, indicating that cerebral ischemia-reperfusion injury caused a decrease in the expression of anti-apoptotic proteins. However, after intranasal administration of SS-31NP / PU Gel, NP / PU Gel, SS-31NP Gel, PU, and intravenous injection of edaravone, the expression level of Bcl-2 protein in the ischemic brain tissue could be significantly increased. Among them, compared with edaravone, SS-31NP Gel, and PU, SS-31NP / PU Gel could more significantly increase the expression level of Bcl-2 protein in the ischemic brain tissue.

[0205] Compared with the Control group, the expression of Bax protein in the ischemic brain tissue of the MCAO group was significantly increased, indicating that cerebral ischemia-reperfusion injury promoted the expression of the pro-apoptotic protein Bax. Compared with the MCAO group, after administration of SS-31NP / PU Gel, NP / PU Gel, SS-31NP Gel, PU group, and edaravone group, the expression level of Bax protein in the ischemic brain tissue could be significantly decreased. Among them, the effect of SS-31NP / PU Gel was the best, although there was no significant difference compared with other groups.

[0206] Compared with the Control group, the expression of Bcl-2 / Bax protein in the ischemic brain tissue of the MCAO group was significantly decreased, indicating that cerebral ischemia-reperfusion injury led to a decrease in the Bcl-2 / Bax protein ratio. Compared with the MCAO group, after administration of SS-31NP / PU Gel, NP / PU Gel, SS-31NP Gel, PU group, and edaravone group, the expression level of Bcl-2 / Bax protein in the ischemic brain tissue could be significantly increased. Compared with SS-31NP Gel and PU, SS-31NP / PU Gel could significantly increase the Bcl-2 / Bax protein ratio in the brain tissue.

Claims

1. A preparation method of nanoparticles for nasal administration, wherein the nanoparticles for nasal administration have ROS responsiveness, and are characterized in that, It includes the following steps: (1) Add 2,2'-[propane-2,2-diylbis(thio)]diacetic acid, EDC, and NHS into DMSO, stir under dark conditions, and carry out an esterification reaction at room temperature to obtain the ROS-responsive crosslinker TKDA; (2) Add sodium 4-(N-maleimidomethyl)cyclohexane-1-carboxylate sulfosuccinimide ester to the chitosan aqueous solution and stir to carry out the first crosslinking reaction to obtain the first crosslinking reaction solution; then add the SS-31 peptide to the first crosslinking reaction solution and stir to carry out the second crosslinking reaction to obtain SS-31-chitosan; (3) Mix liquid paraffin and an emulsifier to obtain a paraffin-emulsifier mixture; (4) Under stirring, add the SS-31-chitosan aqueous solution to the paraffin-emulsifier mixture, stir, and carry out emulsification treatment to form an emulsification system to obtain a paraffin-emulsifier-SS-31-chitosan mixture; (5) Add puerarin to the ROS-responsive crosslinker TKDA prepared in step (1), mix evenly, then add the paraffin-emulsifier-SS-31-chitosan mixture prepared in step (4), stir, and carry out the third crosslinking reaction to obtain the product.

2. The preparation method according to claim 1, characterized in that: In step (3), the volume ratio of the liquid paraffin to the emulsifier is 100:(3.5 - 5.2).

3. The preparation method according to claim 1, characterized in that: In step (3), the volume ratio of the liquid paraffin to the emulsifier is 100:3.

71.

4. The preparation method according to claim 2, characterized in that: In step (3), the emulsifier selected is Span85 and Tween80.

5. The preparation method according to claim 1, characterized in that: In step (4), the mass / volume ratio concentration of the SS-31-chitosan aqueous solution is 0.5% - 1.5% (w / v).

6. The preparation method according to claim 1, characterized in that: In step (4), the emulsification treatment temperature is 50 - 60 °C; the emulsification treatment time is 50 - 70 min.

7. A nanoparticle for nasal administration, characterized in that: Prepared according to the method described in any one of claims 1 - 6.

8. A preparation method of a nano-thermosensitive assembled gel for nasal administration, characterized in that, It includes the following steps: Add the high molecular materials poloxamer P407 and poloxamer P188 to the nasal administration nanoparticles described in claim 6, stir and mix evenly, and after the high molecular materials are dissolved, stand for at least 18 h to make the molecular chains of the high molecular materials stretch and form a stable colloidal solution, thus obtaining the mitochondrial-targeted puerarin thermosensitive gel.

9. The preparation method according to claim 8, characterized in that: The mass ratio of poloxamer P188 to P407 is (8 - 9):

1.

10. A nano-thermosensitive assembled gel for nasal administration, characterized in that: Prepared according to the method described in claim 8 or 9.

11. Use of the nanoparticle for nasal administration according to claim 7 in the preparation of a drug for treating a nervous system disease, wherein the nervous system disease is cerebral ischemia-reperfusion injury.

12. Use of the nano-thermosensitive assembled gel for nasal administration according to claim 10 in the preparation of a drug for treating a nervous system disease, wherein the nervous system disease is cerebral ischemia-reperfusion injury.

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