A dual-targeting liposome and its application

Abstract

The present invention provides a dual-targeting liposome drug delivery system and its application. The dual-targeting liposome can not only better encapsulate and slowly release basic fibroblast growth factor (bFGF), but more importantly, the dual-targeting liposome can also carry bFGF across the blood-spinal cord barrier and accumulate at the spinal cord injury lesion site. The results of in vitro transwell simulation experiments and in vivo small animal imaging experiments both show that the dual-targeting liposome can better cross the barrier and target to the chondroitin sulfate proteoglycan expression region. At the same time, intravenous injection of the bFGF-loaded dual-targeting liposome can promote the repair of the blood-spinal cord barrier, improve the spinal cord microenvironment, promote the transformation of macrophages into the M2 type and inhibit their transformation into the M1 type, and also show good effects in promoting the repair of the hind limb motor function after spinal cord injury. Based on the above results, we believe that such dual-targeting drug delivery systems are expected to be translated into clinical practice for the treatment of spinal cord injury.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceuticals, and more specifically, to a dual-targeting liposome and its application. Background Art

[0002] Severe sequelae after spinal cord injury may prevent patients from standing and walking forever. There are currently many methods for treating spinal cord injury, including surgical decompression, rehabilitation training, cell implantation, polymer scaffold transplantation, etc., but all have inevitable defects. After spinal cord injury, whether in the acute stage or the secondary injury stage, drug treatment runs through the entire clinical treatment plan. Because it is relatively safe and highly operable. There are a variety of drugs for treating spinal cord injury. Among them, basic fibroblast growth factor (bFGF) is widely present in tissues throughout the body, especially in the nervous system, and has a strong promoting effect on the proliferation and mitosis of nerve cells. Another study found that bFGF can also repair the blood-spinal cord barrier (BSCB) and maintain the stability of the spinal cord internal environment, and it has important potential application value in the treatment of spinal cord injury. However, the defects of bFGF, such as short half-life, poor stability, complex structure, and enzyme-caused inactivation of biomacromolecules, limit their use. How to overcome the defects of bFGF and achieve efficient delivery of bFGF for the treatment of spinal cord injury is still an urgent problem to be solved.

[0003] Many drug carriers and polymer materials for spinal cord injury repair have been explored and also shown excellent therapeutic effects, but most of them use in-situ drug administration, which leads to the problem that the later treatment plan cannot be flexibly adjusted, and this is also a problem often encountered in clinical practice. As a nano-carrier, liposomes have been shown in a large number of studies to have good biocompatibility, and can also increase the drug circulation time and improve the bioavailability of drugs by adjusting the synthetic materials. Encapsulating drugs with liposomes shows extremely superior effects. However, encapsulating bFGF with liposomes by single in-situ administration cannot well repair the long-term chronic injury of spinal cord injury. Compared with in-situ administration, tail vein administration is a more flexible administration method. However, systemic tail vein administration has problems such as low bioavailability and causing toxic and side effects to surrounding tissues and organs. How to overcome the problems of low bioavailability and large toxic and side effects of liposome intravenous administration will be a challenge. In addition, due to the existence of the blood-spinal cord barrier, it will be more challenging for the drug bFGF injected into the tail vein to cross the BSCB and accumulate at the spinal cord injury lesion site. Summary of the Invention

[0004] In order to overcome the defects existing in the prior art, the present invention discloses a bFGF-loaded double-modified liposome that can efficiently penetrate the blood-spinal cord barrier and target and retain at the injury site to promote injury repair. Liposomes are used to efficiently load bFGF, improve the defects of short half-life, poor stability and easy degradation of bFGF, and improve the bioavailability of growth factors. At the same time, taking advantage of the overexpression of chondroitin sulfate proteoglycans (CSPGs) at the injury site after spinal cord injury, and the property that the CAQK tetrapeptide (Cp) can bind to CSPGs in the injury area, the CAQK tetrapeptide is attached to the liposome to achieve the effect of targeting the spinal cord injury site. At the same time, inspired by the mitochondrial targeting peptide (arginine-lysine, G2R) and the arginine-rich polypeptide that can promote the penetration of the BSCB, the R2KC tetrapeptide (Rp) is attached to the liposome to better cross the blood-spinal cord barrier. In order to achieve the above technical effects, the present invention provides the following technical solutions:

[0005] In the first aspect of the present invention, there is provided a double-polypeptide composite-modified liposome, which is obtained by grafting a targeting polypeptide and a transmembrane polypeptide onto the liposome.

[0006] In one embodiment, the targeting polypeptide and the transmembrane polypeptide are the CAQK tetrapeptide and the R2KC tetrapeptide respectively.

[0007] In one embodiment, the liposome is loaded with a drug.

[0008] In one embodiment, the loaded drug is the water-soluble drug bFGF.

[0009] In the second aspect of the present invention, there is provided the use of the above double-polypeptide composite-modified liposome in the preparation of a drug.

[0010] In one embodiment, the use is to use the above double-polypeptide composite-modified liposome as a drug carrier.

[0011] In one embodiment, the drug is a drug for treating spinal cord injury.

[0012] In the third aspect of the present invention, there is provided a pharmaceutical composition, and the active ingredient of the pharmaceutical composition is loaded in the above double-polypeptide composite-modified liposome. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The drawings are used to provide a further understanding of the present invention, and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention, and do not constitute a limitation to the present invention. In the drawings:

[0014] Figure 1: Schematic diagram of the preparation and application in spinal cord injury repair of double-modified liposomes encapsulating bFGF (bFGF@Lip-Cp&Rp). After intravenous injection of bFGF@Lip-Cp&Rp, it crosses the blood-spinal cord barrier, targets the spinal cord lesion area, promotes the repair of the blood-spinal cord barrier, and promotes the polarization of macrophages into the M2 type.

[0015] Figure 2 : Preparation and characterization of CAQK-modified lipid material (DSPE-PEG-CAQK) and R2KC-modified lipid material (DSPE-PEG-R2KC). a: Synthetic diagram of DSPE-PEG-R2KC and DSPE-PEG-CAQK. b: 1 1H-NMR spectra of DSPE-PEG-Mal, DSPE-PEG-CAQK and DSPE-PEG-R2KC in CDCl3.

[0016] Figure 3 : Hydrodynamic particle size distribution, polydispersity index (PDI) and TEM images of double-modified liposomes (Lip-Cp&Rp).

[0017] Figure 4 : Encapsulation efficiency of bFGF in blank liposomes (bFGF@Lip) and double-modified liposomes (bFGF@Lip-Cp&Rp).

[0018] Figure 5 : Drug release curves of BSA-FITC-loaded liposomes (BSA-FITC@Lip) and BSA-FITC-loaded double-modified liposomes (BSA-FITC@Lip-Cp&Rp).

[0019] Figure 6 : Biocompatibility and safety analysis of drug-loaded double-modified liposomes. a: CCK-8 assay for the effects of liposomes on the cell viability and cell proliferation of PC12 cells and bEnd.3 cells. b: Hemolysis assay of liposomes on blood cells.

[0020] Figure 7 : Detection of blood-spinal cord barrier penetration and targeting at the injury site of drug-loaded double-modified liposomes in vitro transwell and in rats with spinal cord injury. a: Schematic diagram of double-modified drug-loaded liposomes crossing the barrier and targeting CSPGs. b: Amount of BSA-FITC-loaded double-modified liposomes (BSA-FITC@Lip-Cp&Rp) entering the lower chamber through the upper chamber of transwell. c: In vivo fluorescence imaging of animals at 6 hours, 12 hours and 24 hours after intravenous injection of Dil-loaded liposomes (Dil@Lip) and Dil-loaded double-modified liposomes (Dil@Lip-Cp&Rp).

[0021] Figure 8 : To examine the effect of bFGF@Lip-Cp&Rp on the permeability of the blood-spinal cord barrier (BSCB). a: Representative images of tissues injected with Evans blue 24 hours after spinal cord injury. b: Statistical results of the Evans blue content in the tissues.

[0022] Figure 9 : mRNA expression levels of neurotrophic factors (NGF, NT-3) and anti-inflammatory factors (TGF-β, IL-10) in different groups 3 days after spinal cord injury.

[0023] Figure 10 : bFGF@Lip-Cp&Rp promotes the transformation of macrophages into the M2 type and inhibits the transformation into the M1 type. a: Quantitative analysis of CD86 protein expression 3 days after spinal cord injury. b: Quantitative analysis of Arg-1 protein expression 3 days after spinal cord injury

[0024] Figure 11 : Basso-Beattie-Bresnahan (BBB) motor function scores of rats in the normal saline, bFGF-loaded liposome (bFGF@Lip), and bFGF-loaded double-modified liposome (bFGF@Lip-Cp&Rp) groups. Specific embodiments

[0025] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only for the purpose of illustrating and explaining the present invention, and are not used to limit the present invention.

[0026] Example 1 Preparation of DSPE-PEG-CAQK and DSPE-PEG-R2KC

[0027] Using the thiol-maleimide coupling method, DSPE-PEG-R2KC and DSPE-PEG-CAQK were synthesized by covalently coupling the terminal thiols of R2KC and CAQK (synthesized by GL Biochem (Shanghai) Ltd.) with DSPE-PEG-Mal (synthesized by Xi'an Ruixi Biotechnology Co., Ltd.). DSPE-PEG-Mal and CAQK peptide (1:1.5, mol / mol) were separately dissolved in a buffer solution (pH 7.4), gently mixed, and reacted at 4°C for 24 h under nitrogen protection. Residual CAQK peptide was removed through a 1 kDa dialysis membrane, and the prepared DSPE-PEG-CAQK was freeze-dried. The synthesis of DSPE-PEG-R2KC was the same as that of DSPE-PEG-CAQK. The products were characterized by 1 1H-NMR. The results showed that the targeting tetrapeptide (CAQK) and the transmembrane tetrapeptide (R2KC) were first coupled with DSPE-PEG-Mal to form DSPE-PEG-CAQK and DSPE-PEG-R2KC (attached Figure 2A). From the results attached Figure 2 As can be seen from B, the characteristic peak of the Mal group at 6.7 ppm in DSPE-PEG-Mal disappeared after the addition of CAQK or R2KC, while the characteristic peaks of DSPE and PEG (1.2 ppm and 3.7 ppm) still remained, indicating the successful synthesis of DSPE-PEG-CAQK and DSPE-PEG-R2KC.

[0028] Preparation and Characterization of Dual-Targeted Liposomes (Lip-Cp&Rp) in Example 2

[0029] bFGF@Lip-Cp&Rp was prepared by the previously reported thin film hydration and membrane filtration extrusion method. Briefly, soy lecithin, cholesterol, DSPE-PEG-CAQK, and DSPE-PEG-R2KC were mixed in CHCl3 at a molar ratio of 80:20:4:4 and rotary evaporated in a 50 °C water bath to form a thin film. The prepared thin film was hydrated with PBS (pH 7.4, 50 °C, 1 h). The mixture was sonicated using a probe sonicator (300 w, on / off: 2 s / 4 s, 10 minutes) to reduce the particle size and sequentially passed through microporous membranes with pore sizes from 0.45 μm to 0.22 μm. The morphology of different liposomes was determined by transmission electron microscopy. In addition, the hydrodynamic diameter and polydispersity index (PDI) of different liposomes were measured using a particle size analyzer after appropriate dilution. The results showed that the average particle size of the dual-modified liposomes (Lip-Cp&Rp) composed of cholesterol, lecithin, DSPE-PEG-CAQK, and DSPE-PEG-R2KC (molar ratio 80:20:4:4) was about 97.51 ± 0.78 nm (PDI: 0.191 ± 0.01). It can be seen from the transmission electron micrograph that the formation of the liposome bilayer (attached Figure 3 ).

[0030] Preparation of Drug-Loaded Dual-Targeted Liposomes (bFGF@Lip-Cp&Rp) in Example 3

[0031] The pH gradient method was used to encapsulate drugs (such as bFGF and BSA-FITC) into the drug-loaded dual-targeted liposomes. Specifically, the liposome thin film prepared in Example 2 was hydrated with citrate buffer (0.03 mol / L) instead of PBS for 1 h, bFGF or BSA-FITC was added, and the pH was adjusted to 6.8 with saturated disodium hydrogen phosphate solution and incubated at 35 °C for 15 min. For liposomes loaded with lipophilic lipids such as Dil, Dil was dissolved in CHCl3 and then prepared into liposomes together with cholesterol, lecithin, DSPE-PEG-CAQK, and DSPE-PEG-R2KC (80:20:4:4 mol / mol). The drug loading detection showed that the encapsulation efficiency of bFGF was 90.83 ± 2.81% (attached Figure 4 ).

[0032] Example 4 Drug Release Detection

[0033] The dialysis method was used to detect the in vitro drug release behavior of different liposomes. Considering that bFGF is a biologic macromolecule drug and is prone to inactivation during long-term release experiments, FITC-labeled bovine serum albumin (FITC-BSA) with a similar molecular weight was used to replace bFGF and loaded into liposomes. Then, the drug-loaded liposomes were placed in a dialysis bag, and the dialysis bag was put into 15 ml of PBS (pH 7.4) release medium. At 12 h, 1 d, 2 d, 3 d, 5 d, 7 d, 10 d, and 14 d, 500 mL of the liquid in the medium was taken for detection. After each sampling, 500 mL of fresh PBS was replenished. A multifunctional microplate reader was used to detect the fluorescence intensity of FITC-BSA in the samples, calculate the cumulative release rate, and plot the release curve. The results showed that BSA-FITC was continuously released for about two weeks, which was a great improvement compared to the easily degradable bFGF (attached Figure 5 ).

[0034] Example 5 Biocompatibility and Safety Analysis of Drug-Loaded Dual-Targeted Liposomes (bFGF@Lip-Cp&Rp)

[0035] We used the CCK-8 assay to determine the effects of the dual-modified liposomes on the cell viability and cell proliferation of PC12 cells and bEnd.3 cells. First, 100 μl of cell suspension was added to a 96-well plate, with 3000 cells seeded in each well. After 24 h, liposomes were added. The concentration of bFGF in the drug-loaded liposomes was 10 μg / mL. After 48 h of incubation, the culture medium was discarded, 100 μl of CCK-8 working solution was added, and the plate was further incubated in an incubator for 2 h. Then, the absorbance at 450 nm was measured using a microplate reader to calculate the cell viability. The results of the CCK-8 cell assay (attached Figure 6 a) showed that the blank liposomes (Lip), drug-loaded liposomes (bFGF@Lip), and dual-modified liposomes (bFGF@Lip-Cp&Rp) had good in vitro biocompatibility and did not produce cytotoxicity. A certain cell proliferation effect could be observed in the drug-loaded group, but it was not obvious.

[0036] The hemolytic performance of liposomes was evaluated by red blood cells. First, fresh blood was drawn from the hearts of female SD rats and placed in anticoagulant tubes. The obtained blood was washed with physiological saline and centrifuged repeatedly (2000 rpm, 10 minutes), and then a 2% red blood cell suspension was prepared. The 2% red blood cell suspension was mixed with liposomes at a volume ratio of 1:1. After incubation for 3 hours, the mixture was centrifuged (2000 rpm, 10 minutes). 200 μL of the supernatant was taken, and the hemoglobin content was detected with an enzyme-labeled instrument at 576 nm. Physiological saline and 1% Triton solution were used as negative and positive controls, respectively. The results of the red blood cell hemolysis experiment (attached Figure 6 b) showed that 1% Triton completely lysed the red blood cells, while the blank liposomes (Lip), drug-loaded liposomes (bFGF@Lip), and double-modified drug-loaded liposomes (bFGF@Lip-Cp&Rp) groups were similar to the physiological saline group, and the hemolysis phenomenon was not obvious. Therefore, it is relatively safe for tail vein injection in rats.

[0037] Example 6 Targeting and Blood-Spinal Cord Barrier Penetration Test of Double-Modified Liposomes

[0038] In vitro, the Transwell system was used to construct an in vitro blood-spinal cord barrier to detect the targeting and barrier penetration effects of liposomes. The steps included plating 1×10 5 HUVECs cells on the upper chamber of Transwell, culturing for 48 hours, and starving for 1 day with hydrocortisone to increase the tightness. At the same time, a layer of chondroitin sulfate proteoglycan (CSPGs) was plated on the new lower chamber of Transwell. After fixing overnight, it was washed twice with PBS, and then the cells in the upper chamber of Transwell were transferred to the chamber fixed with CSPGs. 10 μL of double-modified liposomes loaded with fluorescent dye was added to the upper chamber. After 3 hours, 100 μL of the culture medium in the lower chamber was aspirated, and the fluorescence intensity was detected with a multifunctional enzyme-labeled instrument to evaluate the ability of the liposomes to cross the barrier. In vivo, Dil-loaded double-modified liposomes were injected into the tail veins of rats after spinal cord injury. At 6 h, 12 h, and 24 h after injection, the aggregation of liposomes at the spinal cord injury site was detected by in vivo animal imaging.

[0039] From the experimental results attached Figure 7 a, 7b showed that the double-modified liposomes significantly entered the lower chamber through the barrier more than the blank liposomes, indicating that the double-target modified liposomes had a more obvious effect of crossing the barrier and had the effect of targeting CSPGs. The experimental results of detecting the fluorescence intensity at the injury site by in vivo imaging of small animals are attached Figure 7 c showed that almost no obvious fluorescence aggregation effect could be seen for the liposomes without peptides. The gradually increasing fluorescence of the double-targeted liposomes in vivo also proved that the double-targeted liposomes could better aggregate and retain at the injury site.

[0040] Effect Detection of Drug-loaded Double-modified Liposomes (bFGF@Lip-Cp&Rp) in Repairing Blood-spinal Cord Barrier and Stabilizing Microenvironment

[0041] Forty adult female Sprague Dawley (SD) rats (200 - 220 g) were purchased from Huatong Lihua Laboratory Animal Technology Co., Ltd. and raised in Wenzhou Medical University. The feeding, care, and experiments of the animals conformed to the regulations of the Experimental Animal Ethics Committee of Wenzhou Medical University. The rats were fasted overnight before the operation. The rats were randomly divided into 4 groups, including Sham group, SCI+Lip group, SCI+bFGF@Lip group, and SCI+bFGF@Lip-Cp&Rp group. After anesthesia, T8 laminectomy was performed, and spinal cord contusion was caused by dropping from a height of 50 mm with a MASIS impactor. After the contusion, the skin was sutured layer by layer and placed in an incubator at 37 °C until waking up. The Sham group underwent laminectomy without contusion. Then, 0.5 mL of liposomes (equivalent to 10 μg / mL bFGF) was injected into the tail vein once a week until the end of the experiment, and manual bladder urination was performed twice a day until the urination function recovered.

[0042] Immediately after spinal cord injury, the blood-spinal cord barrier is damaged, resulting in a large number of external factors such as macrophages entering the spinal cord internal environment, destroying the stability of the spinal cord internal environment, and making it difficult to repair the injury. After the blood-spinal cord barrier is damaged, Evans blue dye will penetrate through the barrier into the spinal cord. We injected Evans blue dye into the tail vein 1 day after spinal cord injury and detected the amount of Evans blue dye penetrating through the barrier into the spinal cord. As shown in Figure 8 Figures 8a and 8b, the blue color in the spinal cord of the blank liposome group was deeper after spinal cord injury. After encapsulating bFGF into liposomes, the blue color in the spinal cord became slightly lighter, but not significantly. After encapsulating bFGF into double-modified liposomes, the blue color at the spinal cord injury site decreased more significantly. The same result was obtained by detecting the Evans blue content in the spinal cord tissue ( Figure 8 Figure 8b). The damage of the blood-spinal cord barrier will lead to a large number of external factors entering the spinal cord internal environment and destroying the stability of the spinal cord internal microenvironment. Therefore, we detected the nutritional factors and anti-inflammatory factors in the spinal cord. The results showed ( Figure 9 Figures 9a, 9b, 9c, 9d) that double-modified liposomes could significantly increase the expression of nutritional factors and anti-inflammatory factors. The above results showed that double-modified liposomes could promote the repair of the blood-spinal cord barrier and maintain the stability of the spinal cord internal environment.

[0043] Example 8 Drug-loaded Double-modified Liposomes (bFGF@Lip-Cp&Rp) Promote the Transformation of Macrophages into M2 Type and Inhibit the Transformation into M1 Type

[0044] M1 macrophages are macrophages that promote the expression of inflammatory cytokines, while M2 macrophages are macrophages that suppress the expression of inflammatory cytokines. We detected the phenotypes of M1 macrophage factor (CD86) and M2 macrophage factor (Arg-1) in spinal cord injury tissues, attached Figure 10 a, 10b The results showed that the expression of the pro-inflammatory cytokine CD86 increased after spinal cord injury, while the expression of the anti-inflammatory cytokine (Arg-1) decreased. Administering liposomes loaded with bFGF (bFGF@Lip) could reduce the expression of the pro-inflammatory cytokine CD86 and promote the expression of the anti-inflammatory cytokine (Arg-1). The drug-loaded dual-modified liposomes (bFGF@Lip-Cp&Rp) had the best effect in inhibiting the expression of pro-inflammatory factors and increasing the expression of anti-inflammatory factors. This indicates that the dual-targeted liposomes we prepared can better deliver the drug bFGF to the injury site for treatment, achieving the effect of promoting the transformation of macrophages into M2 and inhibiting the transformation into M1 type.

[0045] Example 9 Drug-loaded dual-targeted liposomes (bFGF@Lip-Cp&Rp) promote motor function repair

[0046] The Basso-Beattie-Bresnahan (BBB) scoring system was used to evaluate the motor function of rats. Two observers evaluated and recorded the hind limb scores at 1, 3, 7, 14, 21, and 28 days after spinal cord injury in rats.

[0047] BBB score (attached Figure 11 ) The results showed that the hind limbs of the rats in the blank liposome group remained in a state that was difficult to recover. After administering bFGF-Lip, there was some improvement but the effect was not obvious. The dual-modified liposomes had the strongest effect (P < 0.01).

[0048] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and its equivalent technologies, the present invention is also intended to include these changes and modifications.

Claims

1. A dual-polypeptide complex-modified liposome, characterized in that, The double-polypeptide complex-modified liposome is obtained by grafting a targeting polypeptide and a transmembrane polypeptide onto the liposome. Among them, the targeting polypeptide and the transmembrane polypeptide are CAQK tetrapeptide and R2KC tetrapeptide respectively. The grafting method is as follows: CAQK and R2KC are respectively coupled with DSPE-PEG-Mal to form DSPE-PEG-CAQK and DSPE-PEG-R2KC, and bFGF is loaded on the double-polypeptide complex-modified liposome.

2. Use of the double-polypeptide complex-modified liposome according to claim 1 in the preparation of a drug for treating spinal cord injury.

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