Nanocomposite for targeted therapy of neuropathic pain as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of nano-agents, and discloses a nano-composite for targeted therapy of neuropathic pain as well as a preparation method and application of the nano-composite. The preparation method comprises the following steps: dissolving 1, 2-dioleoylglycerol-3-phosphoethanolamine, cholesterol, BK-1361 and PEG (Polyethylene Glycol) modified distearoyl phosphatidyl ethanolamine and the analgesic in absolute ethyl alcohol to obtain a phospholipid ethanol solution; and rapidly mixing the phospholipid ethanol solution with pure water, incubating at room temperature, and dialyzing to obtain the nano-composite. According to the nanocomposite prepared by the preparation method disclosed by the invention, a lipophilic shell is formed by single-layer phospholipid and wraps the analgesic drug; the blood circulation time of the medicine in the body can be prolonged, and the bioavailability of the medicine is improved; the compound can inhibit neuropathic pain in a targeted manner in vivo, and can be applied to treatment of diseases related to chronic pain.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanopharmaceuticals, and in particular relates to a nanocomposite for targeted treatment of neuralgia, and a preparation method and application thereof. Background Art

[0002] Pain can be classified as acute (less than 3 months) or chronic (more than 3 months) by duration. In daily life, acute pain is the cornerstone of the body's defense against potentially harmful stimuli and plays an important role in preventing tissue damage. Chronic pain is a major health problem that leads to a decline in the quality of life of many people around the world. Chronic pain patients are often dissatisfied due to insufficient drug dosage and lack of appropriate drug treatment. Analgesics are very effective for acute inflammatory traumatic pain, but are not very helpful for the treatment of most chronic pain. In addition, due to the poor pharmacokinetic characteristics of drugs, the effectiveness level of any particular drug is only 5-30%. Therefore, people began to consider the development of nanocarrier preparations, which are nanoscale delivery systems with extremely wide applications and play a key role in nanomedicine fields such as multimodal imaging, drug delivery and targeted therapy of human diseases. Compared with free drug molecules, nanocarriers can improve the solubility of various types of drugs, reduce the toxic side effects of drugs, improve the therapeutic effect and reduce the number of dosing. According to the different nanocarrier materials, they can be divided into polymer nanocarriers, lipid nanocarriers, metal and inorganic nanocarriers. These different types of nanocarriers have been widely used in the study of drug delivery. Among them, lipid nanocarriers are a new type of drug delivery system that uses lipids with good biocompatibility as materials.

[0003] Common lipid nanocarriers used for drug delivery can be divided into: liposomes, nonionic surfactant liposomes (Niosomes), solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs). Compared with nanoemulsions, liposomes and inorganic nanoparticles, solid lipid nanoparticles have better release characteristics, higher drug bioavailability and long-term stability in vivo. Summary of the invention

[0004] The present invention aims to provide a nanocomposite for targeted treatment of neuropathic pain and its preparation method and application. The nanocomposite can inhibit neuropathic pain in vivo, prolong the blood circulation time of the drug in the body, improve the bioavailability of the drug, and can be used in the treatment of related chronic neuropathic pain diseases.

[0005] To solve the above problems, the technical solution provided by the present invention is as follows:

[0006] The first aspect of the present invention provides a method for preparing a nanocomposite for targeted treatment of neuropathic pain, the preparation method comprising the following steps in sequence:

[0007] S1. dissolving the phospholipid mixture and the analgesic drug in anhydrous ethanol and mixing them evenly to obtain a phospholipid ethanol solution;

[0008] The phospholipid mixture includes 1,2-dioleoylglycerol-3-phosphoethanolamine, modified distearoylphosphatidylethanolamine and cholesterol;

[0009] The modified distearoylphosphatidylethanolamine is distearoylphosphatidylethanolamine modified with BK-1361 and PEG;

[0010] S2. The phospholipid ethanol solution is quickly mixed with pure water and then incubated at room temperature, and then dialyzed with pure water to remove unencapsulated analgesics to obtain the nanocomplex for targeted treatment of neuralgia.

[0011] Furthermore, the analgesic drug is ropivacaine.

[0012] Furthermore, the preparation method of the modified distearoylphosphatidylethanolamine is:

[0013] S11. dissolving PEG-modified distearoylphosphatidylethanolamine and BK-1361 ((cyc lo(RLsKDK))) in N,N-dimethylformamide, stirring for 8-12 hours, adding triethylamine, and then continuing to stir until the reaction is complete to obtain a reaction solution;

[0014] S12. dialyzing the reaction solution and then freeze-drying it to obtain the modified distearoylphosphatidylethanolamine;

[0015] The synthetic route of the preparation method is shown below:

[0016]

[0017] Furthermore, in the phospholipid mixture, the mass ratio of 1,2-dioleoylglycerol-3-phosphoethanolamine, modified distearoylphosphatidylethanolamine and cholesterol is 71:34:10.

[0018] Furthermore, the mass ratio of the phospholipid mixture to the analgesic drug is 4:1.

[0019] Furthermore, in step S2, the molecular weight cut-off of the dialysis bag used for dialysis is 14,000.

[0020] The second aspect of the present invention provides a nanocomposite prepared by the above preparation method.

[0021] Furthermore, the hydrated particle size of the nanocomposite is 120-170 nm.

[0022] The third aspect of the present invention provides a drug, which comprises the above-mentioned nanocomposite and a pharmaceutically acceptable excipient.

[0023] The fourth aspect of the present invention provides a use of the above-mentioned nanocomplex in the preparation of analgesic drugs for targeted treatment of neuropathic pain.

[0024] Furthermore, in the above application, the neuropathic pain is nerve pain caused by nerve branch damage.

[0025] The present invention also provides an application of the above-mentioned lipid nanocarrier loaded with analgesic drugs in the preparation of drugs targeting neuropathic pain.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The nanocomplex prepared by the preparation method provided by the present invention can achieve targeted inhibition of neuropathic pain in vivo by connecting the polypeptide to the lipid and then encapsulating the analgesic drug in the lipid nanoparticle carrier, while prolonging the blood circulation time of the drug in the body and improving the bioavailability of the drug, and can be applied to the treatment of related chronic neuropathic pain diseases. In addition, the nanocomplex provided by the present invention also has the following beneficial effects:

[0028] (2) The lipid nanoparticles prepared by the present invention can prevent aggregation between particles, reduce the binding of particles to plasma proteins in the body, prolong the blood circulation time of the drug, and improve the bioavailability of the drug in the lipid nanocarrier.

[0029] (3) The nanocomposite prepared by the present invention has higher biocompatibility than other nanocarriers, can control drug release and targeting, has biodegradability and low immunogenicity. In addition, its preparation is simple and convenient, the surface is easy to modify, and it can be quickly mass-produced. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The hydrated particle sizes of the ropivacaine-loaded lipid nanoparticles of Example 2 and the ropivacaine-loaded nanocomplexes of Example 3 are shown;

[0031] Figure 2 The potential values ​​of the lipid nanoparticles loaded with ropivacaine in Example 2 and the nanocomplex loaded with ropivacaine in Example 3 are shown;

[0032] Figure 3 It shows that after ND7 / 23 cells were transfected with Adam8 plasmid in Experimental Example 1, the expression level of Adam8 in the cells was detected by qPCR;

[0033] Figure 4 The figure shows the fluorescence intensity of cells captured after lipid nanoparticles loaded with fluorescent dye rhodamine B and nanocomplexes modified with BK-1361 were added to cells not transfected with plasmids and cells transfected with plasmids for 3 hours, and then the cells were fixed;

[0034] Figure 5 The expression level of Adam8 in DRG dorsal root ganglia of SNI model mice was detected by qPCR;

[0035] Figure 6 The figure shows the immunofluorescence intensity of dorsal root ganglia captured after lipid nanoparticles loaded with fluorescent dye rhodamine B and nanocomplexes modified with BK-1361 were injected into unmodeled mice and SNI-modeled mice through the tail vein, respectively;

[0036] Figure 7 The figure shows the changes in mechanical pain threshold of mice at 12 hours and 24 hours after the lipid nanoparticles loaded with ropivacaine in Example 2 and the nanocomplex loaded with ropivacaine in Example 3 were injected into unmodeled mice and SNI modeled mice through the tail vein, respectively;

[0037] Figure 8 The graph shows the changes in the thermal pain threshold of mice at 12 hours and 24 hours after the lipid nanoparticles loaded with ropivacaine in Example 2 and the nanocomplex loaded with ropivacaine in Example 3 were injected into non-modeled mice and SNI modeled mice respectively through the tail vein. DETAILED DESCRIPTION

[0038] In order to further understand the present invention, preferred embodiments of the present invention are described in detail below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the present invention.

[0039] All raw materials of the present invention have no particular limitation on their sources, and can be purchased from the market or prepared according to conventional methods known to those skilled in the art.

[0040] Example 1: Preparation of BK-1361 and PEG-modified distearoylphosphatidylethanolamine (DSPE-PEG-BK-1361)

[0041] 4.6 mg of PEG-modified distearoylphosphatidylethanolamine and 1 mg of BK-1361 were dissolved in 1 mL of DMF (N,N-dimethylformamide), stirred for 10 hours, and then 10 microliters of triethylamine were added, followed by continued stirring until the reaction was complete. The resulting product was then dialyzed for 48 hours using a dialysis bag with a molecular weight of 3500, and the synthesized substance BK-1361 and PEG-modified distearoylphosphatidylethanolamine were prepared into powder by freeze drying.

[0042] Example 2: Preparation of ropivacaine-loaded lipid nanoparticles (SLNs / Rop)

[0043] 6.5 mg 1,2-dioleoylglycerol-3-phosphoethanolamine (DOPE), 4 mg cholesterol (CHOL), 1 mg PEG-modified distearoylphosphatidylethanolamine (DSPE-PEG) and 2.875 mg ropivacaine were dissolved in an ethanol solution, then mixed evenly to obtain a phospholipid ethanol solution, which was then dialyzed for 6 hours to obtain lipid nanoparticles loaded with ropivacaine (SLNs / Rop).

[0044] Example 3: Ropivacaine-loaded nanocomposite (BK-1361SLNs / Rop)

[0045] 11.1 mg 1,2-dioleoylglycerol-3-phosphoethanolamine (DOPE), 4.5 mg cholesterol (CHOL), 1 mg BK-1361 and PEG-modified distearoylphosphatidylethanolamine (DSPE-PEG-BK-1361) and 4.15 mg ropivacaine were dissolved in an ethanol solution, then mixed evenly to obtain a phospholipid ethanol solution, which was then dialyzed for 6 hours to obtain a ropivacaine-loaded nanocomposite (BK-1361SLNs / Rop).

[0046] Example 4: Preparation of lipid nanoparticles loaded with rhodamine B

[0047] The ropivacaine in Example 2 was replaced by rogastrobin B, and the other process parameters were the same.

[0048] Example 5: Nanocomposite loaded with rhodamine B

[0049] The ropivacaine in Example 3 was replaced by rogastrobin B, and the other process parameters were the same.

[0050] Test Example 1

[0051] The particle size and potential value of the ropivacaine-loaded lipid nanoparticles (SLNs / Rop) obtained in Example 2 and the ropivacaine-loaded nanocomposite (BK-1361SLNs / Rop) prepared in Example 3 were tested using a particle size analyzer. The results are as follows: Figure 1 , Figure 2As shown. Figure 1 It can be seen that the particle sizes of SLNs / Rop and BK-1361SLNs / Rop are both between 90-160nm. Figure 2 It can be seen that the potentials of SLNs / Rop and BK-1361SLNs / Rop are negative.

[0052] Experimental Example 1: Changes in fluorescence intensity of cells after the nanocomplex loaded with rhodamine B and solid lipid nanoparticles loaded with rhodamine B were added to cells transfected with ADAM8 plasmid and cells not transfected with plasmid.

[0053] After ND7 / 23 cells were transfected with ADAM8 plasmid, the expression level of Adam8 in the cells was detected by qPCR. The qPCR results of the cells are as follows: Figure 3 Then, 20 μl of nanocomplex loaded with rhodamine B and solid lipid nanoparticles loaded with rhodamine B without polypeptide modification were added to the cells transfected with the plasmid and the cells not transfected with the plasmid, and the change of fluorescence intensity was detected after 3 hours.

[0054] Lipid nanoparticles encapsulating the fluorescent dye rhodamine B and nanocomplexes encapsulating the fluorescent dye rhodamine B (modified with BK-1361) were added to cells that were not transfected with the plasmid and cells that were transfected with the plasmid, respectively. After 3 hours, the cells were fixed and the cell fluorescence intensity was photographed, as shown in FIG. Figure 4 .

[0055] according to Figure 4 It can be seen that the cell fluorescence intensity of the nanocomplex encapsulating the fluorescent dye Rhodamine B (solid lipid nanoparticles modified with the polypeptide of Rhodamine B) was significantly increased, and it was statistically significant.

[0056] Experimental Example 2: Peptide-modified solid lipid nanoparticles loaded with rhodamine B and non-peptide-modified solid lipid nanoparticles loaded with rhodamine B were injected into SNI model mice and non-model mice through the tail vein, and their dorsal root ganglia were obtained to count the changes in the fluorescence intensity of rhodamine B in their neurons.

[0057] After the mice were SN I modeled, the expression of Adam8 in the dorsal root ganglia was significantly increased by qPCR. The results of qPCR detection were as follows: Figure 5 shown.

[0058] Subsequently, the peptide-modified solid lipid nanoparticles loaded with rhodanamine B and the unpeptide-modified solid lipid nanoparticles loaded with rhodanamine B were injected into the SNI model mice and the unmodeled mice through the tail vein. Immunofluorescence was used to capture the SNI model mice. Figure 6The fluorescence intensity of neurons in the dorsal root ganglion increased significantly, and the results were statistically significant.

[0059] Experimental Example 3: Peptide-modified solid lipid nanoparticles loaded with ropivacaine and non-peptide-modified solid lipid nanoparticles loaded with ropivacaine were injected into SNI model mice via the tail vein, and their analgesic effects were detected by pain sensitivity experiments such as mechanical pain and thermal pain.

[0060] After the mice were SN I modeled, their mechanical pain and thermal pain thresholds were tested. Peptide-modified solid lipid nanoparticles loaded with ropivacaine and solid lipid nanoparticles loaded with ropivacaine without peptide modification were injected into the SN I modeled mice through the tail vein. The mechanical pain and thermal pain thresholds of the mice were then counted 12 hours, 24 hours, and 48 hours after injection. Figure 7 , 8 The results showed that the pain threshold of SNI model mice was significantly increased at 12 hours and 24 hours after the tail vein injection of solid lipid nanoparticles modified with peptides loaded with ropivacaine, and the results were statistically significant, indicating that solid lipid nanoparticles modified with peptides loaded with ropivacaine have better analgesic effect.

[0061] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the attached claims rather than the above description, and it is intended to include all changes within the meaning and scope of the equivalent elements of the claims. Although the embodiments of the present invention have been shown and described, it is understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for preparing a nanocomposite for targeted treatment of neuralgia, characterized in that: The preparation method comprises the following steps in sequence: S1. dissolving the phospholipid mixture and the analgesic drug in anhydrous ethanol and mixing them evenly to obtain a phospholipid ethanol solution; The phospholipid mixture includes 1,2-dioleoylglycerol-3-phosphoethanolamine, modified distearoylphosphatidylethanolamine and cholesterol; The modified distearoylphosphatidylethanolamine is distearoylphosphatidylethanolamine modified with BK-1361 and PEG; S2. The phospholipid ethanol solution is quickly mixed with pure water and then incubated at room temperature, and then dialyzed with pure water to remove unencapsulated analgesics to obtain the nanocomplex for targeted treatment of neuralgia.

2. The preparation method according to claim 1, characterized in that: The analgesic drug is ropivacaine.

3. The preparation method according to claim 1, characterized in that: The preparation method of the modified distearoylphosphatidylethanolamine is: S11. dissolving PEG-modified distearoylphosphatidylethanolamine and BK-1361 in N,N-dimethylformamide, stirring for 8-12 hours, adding triethylamine, and then continuing to stir until the reaction is complete to obtain a reaction solution; S12. dialyzing the reaction solution and then freeze-drying it to obtain the modified distearoylphosphatidylethanolamine; The PEG-modified distearoylphosphatidylethanolamine is shown in the following formula: BK-1361 is shown in the following formula: The modified distearoylphosphatidylethanolamine is shown in the following formula:

4. The preparation method according to claim 1, characterized in that: In the phospholipid mixture, the mass ratio of 1,2-dioleoylglycerol-3-phosphoethanolamine, modified distearoylphosphatidylethanolamine and cholesterol is 71:34:

10.

5. The preparation method according to claim 1, characterized in that: The mass ratio of the phospholipid mixture to the analgesic drug is 4:

1.

6. The preparation method according to claim 1, characterized in that: In step S2, the molecular weight cut-off of the dialysis bag used for dialysis is 14,000.

7. A nanocomposite prepared by the preparation method according to any one of claims 1 to 6.

8. A drug comprising the nanocomposite according to claim 7 and a pharmaceutically acceptable excipient.

9. Use of the nanocomplex according to claim 7 in the preparation of analgesics for targeted treatment of neuropathic pain.

10. The use according to claim 9, wherein the neuropathic pain is nerve pain caused by nerve branch damage.

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