A tet1 peptide-modified peripheral nerve-targeting liposome nanoparticle and a preparation method and application thereof
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SUN YAT SEN MEMORIAL HOSPITAL SUN YAT SEN UNIV
- Filing Date
- 2024-04-03
- Publication Date
- 2026-06-12
Smart Images
Figure CN118576562B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of nanomedicine technology, and in particular to a Tet1 peptide-modified liposome nanoparticle targeting peripheral nerves, its preparation method, and its application. Background Technology
[0002] Opioid analgesics such as morphine and fentanyl are commonly used clinical drugs for the treatment of moderate to severe pain and cancer pain, and they play an important role in pain management. However, due to their abuse, addiction problems and the increasing number of fatal overdose incidents, the medical and economic burden is constantly increasing, seriously endangering human health. At present, there is still a lack of potent analgesics that can replace opioids.
[0003] Opioid receptors can be mainly classified into four subtypes: μ, κ, δ receptors, and hyperalgesic peptide receptors. Each subtype mediates different effects and can also influence each other. For example, various adverse reactions caused by μ-opioid receptor activation can be inhibited or weakened by some κ-opioid receptor agonists. Activation of δ-opioid receptors can alleviate respiratory depression and withdrawal reactions induced by μ-receptor agonists, and conversely, activation of μ-opioid receptors can improve δ-receptor-induced seizures. Hyperalgesic peptide receptors, discovered in 1994 after μ, κ, and δ receptors, are a new type of opioid receptor. In addition to analgesia, they also have a dual effect of hyperalgesia, meaning that above the spinal cord level, they antagonize the analgesic effects of other activated opioid receptors. Only at the dorsal root ganglion and spinal cord level do they truly exert analgesic effects and synergize with other opioid receptors. Therefore, by activating receptors of different subtypes and at different sites, new opioid drugs can be developed to separate analgesia from adverse reactions. Examples include biased μ-receptor agonists and mixed opioid receptor agonists such as dezocine. Dezocine, a κ-receptor agonist / μ-receptor antagonist, has been widely used in clinical analgesia in my country since 2009. However, its high price and high incidence of nausea and vomiting have significantly reduced its cost-effectiveness.
[0004] Opioids exert their effects by binding to opioid receptors, which are widely distributed in the central and peripheral nervous systems. Opioids have many adverse central nervous system side effects caused by the non-selective activation of opioid receptors in the central nervous system (CNS). The leading cause of death from opioid overdose is respiratory depression, which is primarily caused by the activation of μ-opioid receptors in the pons and medulla oblongata, leading to a prolonged respiratory cycle, decreased respiratory rate, and even respiratory arrest. It also reduces sensitivity to high CO2 levels, ultimately resulting in hypoxia and death.
[0005] Since central nervous system effects account for the majority of opioid-related adverse reactions, selectively activating peripheral opioid receptors could reduce central side effects while maintaining analgesic efficacy. Increasing evidence suggests that peripheral sensory neurons can mediate analgesia; it has been reported that intra-articular morphine injections can reduce analgesia through local opioid administration. Pharmacological, genetic, animal, and clinical studies have shown that most analgesic effects from systemic opioid administration are mediated by peripheral opioid receptors. Existing peripheral opioid receptor agonists, such as cimadorine, are κ-receptor agonists that do not cross the blood-brain barrier and only activate peripheral opioid receptors. This avoids the adverse reactions associated with central opioid receptor activation, such as potentially fatal respiratory depression; however, as analgesics, their analgesic effect cannot meet the clinical needs of most patients.
[0006] Researchers are exploring various methods to mitigate the side effects of opioids, including splice variants of the μ-opioid receptor (MOR), agonist-biased approaches, and heteromers targeting other receptors, such as MOR. Despite significant progress in current research, clinical translation remains challenging. Previous studies have attempted to reduce opioid side effects by modulating the transient receptor potential vanillic acid 1 (TRPV1) receptor. However, TRPV1 antagonists may increase the thermal pain threshold in humans, potentially increasing the risk of burns.
[0007] Although some opioid alternatives exist, they still have drawbacks and limitations. The main drawback is their relatively limited analgesic effect, especially in managing severe chronic pain or postoperative pain. The effectiveness of these alternatives varies considerably among individuals. Some alternatives may be effective for some patients but less so for others, resulting in unsatisfactory treatment outcomes. Some chronic pain conditions still require opioids for effective symptom relief.
[0008] Meanwhile, alternative medications may also be accompanied by side effects and adverse reactions. Non-opioid drugs, such as nonsteroidal anti-inflammatory drugs (NSAIDs), may cause gastrointestinal bleeding and kidney problems; antidepressants and anticonvulsants may also cause a range of adverse reactions. Some alternative medications themselves may also carry the risk of abuse and dependence. Although the risk is lower than that of opioids, patients still need to be monitored for drug dependence during long-term use.
[0009] The cost and accessibility of alternative medications also present a challenge. Some newer treatments may be expensive and unavailable in certain regions, limiting patient choices. Most alternative medications still require further research evidence to support their efficacy and safety in specific conditions, necessitating large-scale randomized controlled clinical trials and long-term follow-up studies to ensure their clinical feasibility. Furthermore, clinicians need relevant training to better understand and apply alternative medications, ensuring their proper use and monitoring of patient treatment progress.
[0010] Overall, opioid alternatives still face a number of challenges, requiring further research and efforts to overcome these issues in order to provide safer and more effective alternatives.
[0011] Nanomedicine is an emerging drug formulation. By designing and modulating the properties of organic or inorganic nanomaterials, stable, functionally diverse, and biocompatible nanocarriers can be prepared, significantly prolonging drug half-life, improving drug targeting, reducing dosage, and enabling combination therapy. With the advancement and development of nanomedicine, targeted nanomedicines modified with specific ligands can accumulate in specific regions of the body via predetermined pathways, increasing drug concentration near the target site while minimizing drug toxicity and side effects. Currently, some studies have utilized nanoparticles as carriers, such as loading analgesics like nonsteroidal anti-inflammatory drugs (NSAIDs), successfully treating pain caused by neuropathy. In cancer patients, tumors can cause severe pain. Studies show that using nanoparticles as carriers to load analgesics can precisely deliver them to the tumor area, alleviating tumor-related pain. Some magnetic nanocarriers have also been designed to treat chronic pain. These carriers can release drugs at the pain site under the influence of an external magnetic field, increasing local drug concentration and reducing pain. The design and development of nanomedicine has enormous potential for improving therapeutic efficacy, reducing side effects, and improving patients' quality of life. However, related issues such as safety, manufacturing process, and large-scale production still need to be studied and resolved. Summary of the Invention
[0012] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a Tet1 peptide-modified lipid nanoparticle that targets peripheral nerves. Morphine is encapsulated in liposomes modified with the Tet1 peptide. Tet1 can be targeted and delivered to peripheral sensory nerves, activating peripheral opioid receptors and achieving effective analgesia.
[0013] To achieve the above objectives, the technical solution adopted by the present invention is as follows:
[0014] In a first aspect, the present invention provides a Tet1 peptide-modified liposome targeting peripheral nerves, comprising a core formed by morphine encapsulated by lecithin and a PEG-modified liposome.2000 The shell is composed of -DSPE; the surface of the shell is modified with Tet1 peptide.
[0015] Since the vast majority of opioid-related adverse reactions are caused by central effects, activating peripheral opioid receptors alone could limit central adverse reactions while maintaining analgesic efficacy. Increasing evidence suggests that peripheral sensory neurons can mediate analgesia. The cell bodies of primary sensory neurons are located within the dorsal root ganglion (DRG), projecting fibers to the terminals of peripheral sensory neurons. Opioid receptors are present in DRG neurons of varying diameters and in sensory neuropeptides. When a pain signal is present, these opioid receptors are upregulated and transported via axons of DRG neurons to the peripheral terminals of sensory neurons. Therefore, selectively delivering opioids to peripheral sensory neurons and activating peripheral opioid receptors can produce analgesia. However, delivering morphine specifically to the peripheral dorsal root ganglion during systemic injection remains challenging.
[0016] The nanoparticles prepared in this invention have a structure typical of liposomes, including a phospholipid bilayer surrounding an aqueous morphine core, a hydrophilic polyethylene glycol (PEG) chain on the shell, and a Tet1 peptide that specifically targets peripheral nerves. This invention encapsulates morphine in liposomes modified with the Tet1 targeting peptide, restricting the passive diffusion of morphine into the central nervous system and reducing central nervous system-related side effects. The Tet1 targeting peptide is a 12-amino acid peptide with tetanus toxin binding properties to trisialic acid ganglioside (GT1b) receptors in neuronal cells, and exhibits strong affinity for differentiated PC-12 cells, primary motor neurons, and dorsal root ganglion cells. Liposomes encapsulating morphine modified with the Tet1 peptide possess peripheral nerve targeting properties, delivering them specifically to the vicinity of the dorsal root ganglion, a peripheral sensory nerve. With the release of morphine, pain is relieved while the ascending pain conduction pathway is blocked. The lipid layer of the nanoparticles in this invention uses a biocompatible lipid material to avoid rapid clearance by the reticuloendothelial system; it also prevents rapid drug leakage, thus hindering the entry of free drugs into the central nervous system and preventing adverse reactions, while achieving long-term circulation and targeted accumulation in the periphery; on the other hand, it is composed of Tet1-PEG 2000 -DSPE modification enables it to target peripheral nerves and specifically deliver opioids to them.
[0017] Preferably, the amino acid sequence of the Tet1 peptide is shown in SEQ ID NO.1.
[0018] SEQ ID NO.1 is HLNILSTLWKYRC.
[0019] Secondly, the present invention provides a method for preparing the above-mentioned liposome nanoparticles, comprising the following steps:
[0020] (1) PEG 2000 -DSPE, Tet1-PEG 2000 -DSPE and lecithin were dissolved in chloroform respectively, and after mixing, the chloroform was removed to obtain mixture A;
[0021] (3) Add morphine aqueous solution to mixture A and stir to obtain mixture B;
[0022] (4) Sonicate mixture B;
[0023] (5) Ultrafiltration and centrifugation to obtain the liposome nanoparticles.
[0024] The above-mentioned synthetic raw material Tet1-PEG 2000 The preparation process of DSPE is as follows:
[0025] (1) To DSPE-PEG 2000 - Add chloroform to Mal and dissolve by sonication; dissolve Tet1 in DMSO; immediately mix the two and stir at room temperature for 24 hours under nitrogen atmosphere; the Tet1 and DSPE-PEG... 2000 - The molar ratio of the Mal mixture is 1:2; the sequence of Tet1 is HLNILSTLWKYRC;
[0026] (2) The product solution obtained in step (1) was dialyzed with distilled water for 72 hours, placed at -80°C overnight, and then freeze-dried using a vacuum freeze dryer. After freezing, it was dried and stored at -20°C to obtain the Tet1-PEG. 2000 -DSPE.
[0027] The Tet1 peptide-modified liposome nanoparticles targeting peripheral nerves of this invention are synthesized by a thin-film hydration method. This involves forming a thin film on a solid surface by dissolving liposomes in chloroform, removing the solvent with a nitrogen stream, followed by hydration, and then using mechanical stirring to promote swelling and budding of the polymer film. Under mechanical stirring, an aqueous morphine solution permeates through defects in the polymer film, causing each layer to expand sequentially to form protrusions, which then separate from the surface to produce vesicles. Finally, the mixture is sonicated to reduce the vesicle size distribution and control vesicle size.
[0028] Preferably, in step (1), PEG 2000 -DSPE, Tet1-PEG 2000 - The mass ratio of DSPE to lecithin is (2-4):(3-5):(45-55).
[0029] More preferably, the PEG 2000 -DSPE, Tet1-PEG 2000The mass ratio of DSPE to lecithin is 3.3:4:50.
[0030] Preferably, in step (3), the mass ratio of morphine to lecithin is 1:2.
[0031] Preferably, in step (3), the stirring rate is 1000 rpm / min.
[0032] Preferably, in step (5), the centrifugation conditions are centrifugation at a speed of 2000-3000 rpm for 15-20 minutes.
[0033] Thirdly, the present invention provides the application of the above-mentioned liposome nanoparticles in the preparation of analgesic drugs.
[0034] Fourthly, the present invention provides an analgesic drug comprising the above-mentioned liposome nanoparticles in the preparation of the analgesic drug.
[0035] The beneficial effects of this invention are as follows:
[0036] This invention develops a novel liposome-based morphine nanoparticle with peripheral nerve targeting, which can target opioid receptors in peripheral sensory neurons to limit central adverse reactions while maintaining the analgesic effect of opioids.
[0037] The Tet1 peptide-modified liposome nanoparticles targeting peripheral nerves prepared in this invention have the following characteristics:
[0038] (1) The Tet1 peptide-modified liposome nanoparticles targeting peripheral nerves prepared in this invention encapsulate morphine and have a large molecular size, making it difficult to cross the blood-brain barrier, thereby reducing the passive diffusion of free morphine into the brain and reducing the central side effects of opioid drugs.
[0039] (2) The nanoparticles prepared in this invention are surface-modified with Tet1 peptide, which can be targeted to the peripheral dorsal root ganglion. With the release of the loaded drug, the peripheral opioid receptors are activated, thereby achieving the effective peripheral analgesic effect of opioid drugs and blocking the ascending pathway of pain transmission.
[0040] (3) Encapsulated morphine nanoparticles exhibit strong and long-lasting analgesic effects due to their targeting and sustained release properties; they are expected to be applied in pain treatment, providing new therapeutic drugs and strategies to improve the quality of life for pain patients. Attached Figure Description
[0041] Figure 1 This is a schematic diagram of the structure of a Tet1 peptide-modified liposome nanoparticle targeting peripheral nerves (Tet1-LNP).
[0042] Figure 2The dimensions of the Tet1-LNP are as follows.
[0043] Figure 3 The potential test results are for Tet1-LNP.
[0044] Figure 4 The in vitro neural targeting ability of Tet1 peptide was detected; the left image shows confocal laser scanning microscopy images of PC-12 cells (A) and NIH3T3 cells (B); the right image shows the quantitative determination of the relative fluorescence density of coumarin-6 in PC-12 cells and NIH3T3 cells.
[0045] Figure 5 The left image shows the quantitative fluorescence detection of the in vitro neural targeting ability of Tet1-LNP; the right image shows the quantitative measurement of the relative fluorescence density of coumarin-6 in PC-12 cells; the left image shows the quantitative measurement of the relative fluorescence density of coumarin-6 in DRG cells.
[0046] Figure 6 The results of flow cytometry analysis of the in vitro neural targeting ability of Tet1-LNP are shown in the left figure. (A) Flow cytometry analysis of PC-12 cells after incubation with Scarmble-LNP (C6) or Tet1-LNP (C6) at 4°C for 15 min, 30 min, 1 h, and 2 h; (B) Mean fluorescence intensity (MFI) of flow cytometry cells after incubation of PC-12 cells with Scarmble-LNP (C6) or Tet1-LNP (C6) at 4°C for 15 min, 30 min, 1 h, and 2 h.
[0047] Figure 7 The results are from the in vitro toxicity test of Tet1-LNP.
[0048] Figure 8 The results are from the in vivo toxicity test of Tet1-LNP.
[0049] Figure 9 The left image shows the in vivo targeting effect of Tet1-LNP; the right image shows the fluorescence images of the major organs of mice 24 hours after intravenous injection of free coumarin-6, Scramble-LNP (C6), or Tet1-LNP (C6); the left image shows the quantitative analysis of the fluorescence data.
[0050] Figure 10 The results of a test on thermal hyperalgesia behavior in mice after injection of Tet1-LNP (morphine).
[0051] Figure 11The results of the mechanical hyperalgesia test in mice after injection of Tet1-LNP (morphine) are shown. The left figure shows the mechanical pain threshold of the hind limbs of mice in each group measured with 0.07g von Frey; the right figure shows the mechanical pain threshold of the hind limbs of mice in each group measured with 0.4g von Frey.
[0052] Figure 12 Results of in vivo concentration measurements of free morphine in mouse circulating blood and brain; A, Results of in vivo concentration measurements of free morphine in mouse circulating blood; B, Results of in vivo concentration measurements of free morphine in mouse brain. Detailed Implementation
[0053] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.
[0054] PEG 2000 -DSPE: Distearatelphosphatidylethanolamine-polyethylene glycol 2000
[0055] Tet1-PEG 2000 -DSPE: Distearate phosphatidylethanolamine-polyethylene glycol 2000-Tet1 Example 1: Preparation and characterization of Tet1 peptide-modified liposome nanoparticles targeting peripheral nerves.
[0056] The synthesis is performed through the following steps:
[0057] (1) PEG 2000 -DSPE, Tet1-PEG 2000 -DSPE and lecithin are dissolved in chloroform;
[0058] (2) Add PEG to lecithin 2000 -DSPE, Tet1-PEG 2000 -DSPE, mixed, chloroform removed by rotary evaporation; the PEG 2000 -DSPE, Tet1-PEG 2000 - The mass ratio of DSPE to lecithin is 3.3:4:50;
[0059] (3) Add double-distilled water containing morphine and stir at 1000 rpm / min to obtain a mixture; the mass ratio of morphine to lecithin is 1:2;
[0060] (4) The mixture obtained in step (3) was ultrasonically treated for 1 min to accelerate the assembly of morphine-loaded nanomaterials;
[0061] (5) The mixture was transferred to an ultrafiltration device (EMD Millipore, MWCO 100K) and centrifuged at 3000 rpm for 20 min to remove free compounds, resulting in Tet1 peptide-modified liposome nanoparticles targeting peripheral nerves.
[0062] The above-mentioned synthetic raw material Tet1-PEG 2000 The preparation process of DSPE is as follows:
[0063] (1) To DSPE-PEG 2000 - Add chloroform to Mal and dissolve by sonication; dissolve Tet1 in DMSO; immediately mix the two and stir at room temperature for 24 hours under nitrogen atmosphere; the Tet1 and DSPE-PEG... 2000 - The molar ratio of the Mal mixture is 1:2; the sequence of Tet1 is HLNILSTLWKYRC;
[0064] (2) The product solution obtained in step (1) was dialyzed with distilled water for 72 hours, placed at -80°C overnight, and then freeze-dried using a vacuum freeze dryer. After freezing, it was dried and stored at -20°C to obtain the Tet1-PEG. 2000 -DSPE.
[0065] The structure of the Tet1 peptide-modified peripheral nerve-targeting liposome nanoparticles (hereinafter referred to as Tet1-LNP) prepared in this invention is as follows: Figure 1 As shown.
[0066] The prepared Tet1 peptide-modified liposome nanoparticles targeting peripheral nerves were dissolved in 1 mL of phosphate-buffered saline (PBS) solution to obtain Tet1 peptide-modified liposome nanomedicine targeting peripheral nerves. The size and potential of the nanoparticles were measured using a Nano-ZSZEN3600 particle size analyzer.
[0067] The results show that the nanoparticle size in the Tet1 peptide-modified liposome nanomedicine targeting peripheral nerves prepared in this invention is approximately 131 nm. Figure 2 The potential is -27.4mV ± 1.84mV. Figure 3 ).
[0068] Example 2: In vitro neural targeting ability of Tet1 peptide
[0069] Experimental methods: PC-12 cells and NIH3T3 cells were used at a concentration of 1×10⁶ cells per well. 5 Cells were seeded in 24-well plates. Cells were then coupled with Alexa Fluor. TMWheat germ lectin (WGA) stained with 488 fluorescent dye was incubated at 37°C (5 μg / mL) for 10 min to label the cell membrane, and then washed three times with 1×PBS to remove all unbound WGA. Cy5.5-labeled Tet1 was incubated with both cell types at 4°C for 15 min, 30 min, 1 h, and 2 h, respectively, followed by washing three times with PBS for fixation. Cell nuclei were observed using DAPI staining.
[0070] The results are as follows Figure 4 As shown, compared with the non-neural cell line mouse embryonic fibroblast cell line NIH3T3, Cy5.5-Tet1 showed significantly stronger red fluorescence in the neuronal cell-like PC-12 cells, demonstrating that the Tet1 peptide has in vitro neural targeting capabilities.
[0071] Example 3: In vitro neural targeting capability of Tet1-LNP
[0072] Since the structure of morphine cannot be coupled with fluorescent dyes, we used coumarin-6 (C6) instead of morphine to complete some of the verification of nanoparticle properties. Coumarin-6 is a fluorescent molecule with a molecular weight similar to that of morphine.
[0073] Experimental Sample: Tet1-LNP(C6) - Morphine encapsulated in Tet1 peptide-modified peripheral nerve-targeting liposome nanomaterials was replaced with coumarin-6. A control group and a blank group were also included. Control group: Non-targeting peptide-modified coumarin-6-encapsulated nanoparticles, Scramble-LNP(C6), with the Scramble peptide as a control (sequence: KRWYTNILHSLL). Blank group: Double-distilled water.
[0074] Experimental method: PC-12 cells and primary dorsal root nerve cells (DRG) were cultured at a density of 1 × 10⁶ cells per well. 5 Cells were seeded in 24-well plates. To label the cell membrane, the cells were coated with Texas Red. TM Wheat germ agglutinin (WGA) conjugated with the X-fluorescent dye (Molecular Probes, Inc.) was incubated at 37°C (5 μg / mL) for 10 minutes and then washed three times with 1×PBS to remove all unbound WGA.
[0075] The samples were then added to culture medium containing the experimental samples and incubated for 15 minutes, 30 minutes, 1 hour, and 2 hours, respectively. After incubation at 4°C for the specified time, the samples were washed three times with PBS and fixed with 4% (w / v) paraformaldehyde (PFA). The cell nuclei were counterstained with 1 μg / mL DAPI in the dark for 15 minutes.
[0076] Fluorescence intensity was measured using CytoFLEX flow cytometry to analyze the uptake of coumarin-6-encapsulated Tet1-LNP (C6) by the PC-12 cell line. The in vitro neural targeting ability of Tet1-LNP was evaluated by comparing the uptake of coumarin-6-encapsulated Tet1-LNP (C6) and coumarin-6-encapsulated Scramble-LNP (C6) by the PC-12 cell line and primary dorsal root ganglion (DRG) cells. Results are as follows... Figure 5 , 6 As shown.
[0077] The results showed that PC-12 cells and DRG cells had a stronger uptake capacity for the targeted nanomaterial Tet1-LNP(C6) than the control group Scramble-LNP(C6). Figure 5 ).
[0078] After modification with Tet1 peptide, the cellular uptake of nanomaterials increased by approximately 2-fold (p<0.001), indicating that Tet1-LNP exhibits good neural targeting ability in vitro. Figure 6 ).
[0079] Example 4: In vitro toxicity test of Tet1-LNP
[0080] To investigate the inhibitory effect of nanoparticles on nerve cell growth in vitro, PC-12 cells were seeded at a density of 5000 cells per well in 96-well plates. After incubation in 100 mL of culture medium for 24 hours, Scramble-LNP or Tet1-LNP containing absolute concentrations of 0, 0.005, 0.01, 0.05, 0.1, 0.15, 0.2, 0.3, 0.5, and 1 μM morphine were added to 100 mL of fresh culture medium, and incubation was carried out for 48 hours.
[0081] After replacing the medium with 100 mL of fresh medium, add the CCK-8 reagent to each well according to the manufacturer's instructions, incubate for 1 hour, and immediately read the absorbance at a wavelength of 450 nm. Calculate cell viability according to formula (1).
[0082] Cell viability (%) = [A(experimental wells) - A(blank wells)] / [A(control wells) - A(blank wells)]
[0083] ×100%——Equation (1)
[0084] In the above formula, A (experimental well): the absorbance value of the well containing cells, CCK8 and nanoparticles of different concentrations;
[0085] A (Control well): Absorbance value of wells containing cells and CCK8, but without nanoparticles;
[0086] A (Blank Well): Absorbance value of wells containing no cells, CCK8 and culture medium, and no nanoparticles (used to subtract background absorbance).
[0087] The results are as follows Figure 7 As shown, within the concentration range of 0.005 μM to 1 μM, there was no significant difference in the inhibition of PC-12 cell growth between free morphine in PBS solution, blank LNP, and morphine-loaded nanoparticles.
[0088] Example 5: In vivo toxicity test of Tet1-LNP
[0089] To further assess in vivo safety, the nanomedicine was intravenously injected into C57 mice at a dose of 3 mg / kg per mouse. After 24 hours, brain, dorsal root ganglia (DRGs), and major organs (heart, liver, spleen, lung, and kidney) were collected as experimental samples.
[0090] After fixation with 4% paraformaldehyde and paraffin embedding, the tissue sections were stained with hematoxylin and eosin (H&E) and then observed using an optical microscope.
[0091] The results are as follows Figure 8 The results showed that, compared with the control group, mouse tissues (including heart, liver, spleen, lung and kidney) treated with targeted or non-targeted nanomedicines did not show significant histological changes after HE staining, indicating that Tet1-LNP is safe in mice.
[0092] Example 6: In vivo targeting effect of Tet1-LNP
[0093] On day 7 post-CCI surgery and sham surgery in C57 mice, free coumarin-6, Scramble-LNP (C6), or Tet1-LNP (C6) were intravenously (iv) injected into C57BL / 6J mice. The injection dose was determined based on a coumarin-6 dosage of 1 nmol. Twenty-four hours later, the mice were sacrificed, and major organs, brain, and DRG were collected as experimental samples. The imaging system is used for observation.
[0094] The results are as follows Figure 9 As shown, the targeted nanomedicine Tet1-LNP(C6) accumulates more in the DRG and less in the brain with free fluorescence.
[0095] Example 7: Evaluation of the analgesic effect of Tet1-LNP in mice
[0096] To evaluate the analgesic effect, on day 7 after establishing a chronic sciatic nerve compression injury (CCI) model in healthy male C57 mice or after sham surgery, mice with CCI or sham surgery were intravenously injected with free morphine, Scramble-LNP, and Tet1-LNP, respectively. The amount of morphine in the nanoparticles was quantified by ultraviolet spectrophotometry to ensure that each group contained the same absolute amount of morphine (3 mg / kg). Thermo- and mechano-hyperalgesia behaviors were tested in mice at 1 h, 3 h, 5 h, 8 h, 24 h, 32 h, and 48 h after injection.
[0097] Results of thermal hyperalgesia Figure 10 As shown, the duration of analgesia in the Tet1-LNP (morphine) group was significantly longer than that in the free morphine group. The results for mechanical hyperalgesia are as follows: Figure 11 As shown, the Tet1-LNP (morphine) group exhibited good analgesic effect on mechanical pain, and the duration of analgesia was longer than that of the free morphine and Scramble-LNP (morphine) groups.
[0098] Example 8: Determination of in vivo concentrations of free morphine in circulating blood and brain of mice
[0099] Following intravenous injection of morphine (n=6), Scramble-LNP (n=8), or Tet1-LNP (n=8) (equivalent to 3 mg / kg free morphine), blood and brain samples were collected from CCI mice at specified time points (1 hour, 3 hours, 5 hours, 8 hours, 24 hours, and 48 hours) to detect the concentration of free morphine in the blood and brain, directly comparing the amount of morphine released by LNPs with the amount of free morphine. Morphine concentration was determined using enzyme-linked immunosorbent assay (ELISA). All samples were purified and processed according to the manufacturer's instructions.
[0100] The results are as follows Figure 12 As shown, the average peak concentration of morphine in the brains of mice in the morphine group was as high as 246 ng / mL, while that in the Scramble-LNP group was 32 ng / mL and that in the Tet1-LNP group was 28 ng / mL. Compared with free morphine, the concentration of morphine in the brains of the Tet1-LNP group was 8 times lower.
[0101] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.
Claims
1. A Tet1 peptide-modified liposome nanoparticle targeting peripheral nerves, characterized in that, Includes a core formed by morphine encapsulated by lecithin and a core composed of PEG. 2000 The shell is composed of -DSPE; the surface of the shell is modified with Tet1 peptide; the amino acid sequence of the Tet1 peptide is shown in SEQ ID NO.1; The preparation method of the liposome nanoparticles includes the following steps: (1) PEG 2000 -DSPE, Tet1-PEG 2000 -DSPE and lecithin are dissolved in chloroform respectively; (2) After mixing, remove chloroform to obtain mixture A; the PEG 2000 -DSPE, Tet1-PEG 2000 -The mass ratio of DSPE to lecithin is (2-4):(3-5):(45-55); (3) Add morphine aqueous solution to mixture A and stir to obtain mixture B; (4) Sonicate mixture B; (5) Ultrafiltration and centrifugation to obtain the liposome nanoparticles.
2. The liposome nanoparticles as described in claim 1, characterized in that, The PEG 2000 -DSPE, Tet1-PEG 2000 The mass ratio of DSPE to lecithin is 3.3:4:
50.
3. The liposome nanoparticles as described in claim 1, characterized in that, In step (3), the mass ratio of morphine to lecithin is 1:
2.
4. The liposome nanoparticles as described in claim 1, characterized in that, In step (3), the stirring rate is 1000 rpm / min.
5. The liposome nanoparticles as described in claim 1, characterized in that, In step (5), the centrifugation conditions are centrifugation at a speed of 2000-3000 rpm for 15-20 min.
6. The use of the liposome nanoparticles according to any one of claims 1-5 in the preparation of analgesic drugs.
7. An analgesic drug, characterized in that, Including liposome nanoparticles as described in any one of claims 1-5.