CN111978405A

CN111978405A - Functional polypeptide, erythrocyte drug-carrying system capable of specifically binding collagen and application thereof

Info

Publication number: CN111978405A
Application number: CN201910421440.7A
Authority: CN
Inventors: 戴建武; 陈艳艳; 胡玲玲
Original assignee: Suzhou Institute of Nano Tech and Nano Bionics of CAS
Current assignee: Suzhou Institute of Nano Tech and Nano Bionics of CAS
Priority date: 2019-05-21
Filing date: 2019-05-21
Publication date: 2020-11-24
Anticipated expiration: 2039-05-21
Also published as: CN111978405B

Abstract

The invention discloses a functional polypeptide, an erythrocyte medicine carrying system capable of specifically combining collagen and application thereof. The functional polypeptide comprises a collagen binding sequence and an erythrocyte binding peptide sequence linked to the collagen binding sequence. The functional polypeptide has an amino acid sequence shown in SEQ ID NO. 1. The erythrocyte medicine carrying system capable of being specifically combined with collagen comprises medicine carrying erythrocytes and functional polypeptide specifically combined with the medicine carrying erythrocytes. The erythrocyte medicine carrying system capable of specifically combining collagen constructed by the invention can effectively anchor medicine carrying erythrocyte microspheres on collagen materials, reduces the rapid loss of medicines after transplantation, has the capacity of realizing the concentration maintenance and slow release of medicines at the damaged part, can prolong the half life of the medicines, promotes more neural stem cells to differentiate towards neurons, promotes more axon regeneration, and has the function of promoting better movement recovery of spinal cord injury.

Description

Functional polypeptide, erythrocyte drug-carrying system capable of specifically binding collagen and application thereof

Technical Field

The invention particularly relates to an erythrocyte drug loading system capable of specifically binding collagen, and particularly relates to a functional polypeptide, an erythrocyte drug loading system capable of specifically binding collagen and prepared by adding the functional polypeptide, and application of the erythrocyte drug loading system.

Background

The use of a combination of a biological scaffold material and a drug has been widely used in the treatment of spinal cord injury. On one hand, the biomaterial collagen scaffold can provide a structural support to fill up the damaged cavity and provide a certain guiding function for the growth of the nerve axon. On the other hand, various drug molecules are combined on the surface of the spinal cord to more effectively improve or reconstruct the local inhibitory microenvironment formed after spinal cord injury.

A microtubule stabilizer, paclitaxel, has good promoting effect on axon regeneration and motor function recovery of injured nerve at low dose. However, the short half-life of the drug itself severely hinders the efficacy of the drug. In addition, the low-dose paclitaxel is applied in vivo due to the action of various factors such as rapid flushing of local cerebrospinal fluid, which causes uncontrollable rapid release and body side effects, and is difficult to maintain effective drug concentration.

Drug delivery systems have achieved effective sustained release of drugs in recent years. Research shows that the erythrocyte medicine carrier plays an important role in enhancing the utilization rate of the medicine, prolonging the release time of the medicine and improving the dynamic property of the medicine. Therefore, when in-vivo treatment is carried out, the drug is coated by the erythrocyte carrier and then is connected to the collagen scaffold, and the slow release effect of the erythrocyte is exerted on the basis of maintaining the local drug concentration of spinal cord injury.

The existing method is to simply physically compound red blood cells after drug loading with a stent, the red blood cells and collagen only depend on low-affinity nonspecific adsorption, although the effect of prolonging the half-life period of the drug due to red blood cell loading exists, the uncontrollable loss of the drug-loaded balloon at the damaged part still exists, and the drug at the damaged part and the exerted effect are very limited.

Another approach uses chemical cross-linking agents to cross-link red blood cells to the scaffold, but this may affect the stability of the red blood cell carrier and the scaffold structure.

Disclosure of Invention

The invention mainly aims to provide a functional polypeptide and an erythrocyte drug carrier system which is prepared by adding the functional polypeptide and can be specifically combined with collagen, so as to overcome the defects in the prior art.

The invention also aims to provide application of the erythrocyte medicine carrier system capable of specifically binding the collagen.

In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:

the embodiments provide a functional polypeptide comprising a collagen binding sequence and an erythrocyte binding peptide sequence linked to the collagen binding sequence.

Further, the functional polypeptide has a dual function of specifically binding the erythrocyte-binding peptide sequence at one end to the surface of the erythrocyte and specifically binding the collagen-binding sequence at the other end to the surface of the collagen.

Furthermore, the functional polypeptide has an amino acid sequence shown in SEQ ID NO. 1.

The embodiment of the invention also provides an erythrocyte medicine carrying system capable of being specifically combined with collagen, which comprises medicine carrying erythrocytes and any one of the functional polypeptides specifically combined with the medicine carrying erythrocytes.

The embodiment of the invention also provides a collagen-erythrocyte medicine carrying system which comprises medicine carrying erythrocytes and a collagen material, wherein the medicine carrying erythrocytes are connected to the collagen material through any one of the functional polypeptides.

The embodiment of the invention also provides a preparation method of the collagen-erythrocyte medicine carrying system, which comprises the following steps: incubating the functional polypeptide, the drug-loaded red blood cells and the collagen material together to ensure that the red blood cell binding peptide sequence is specifically combined with the drug-loaded red blood cells, and the collagen binding sequence is specifically combined with the collagen material to form the collagen-red blood cell drug-loaded system.

The embodiment of the invention also provides application of the erythrocyte drug-loaded system or the collagen-erythrocyte drug-loaded system capable of specifically binding the collagen in preparation of products, and the products at least have the functions of maintaining local drug concentration at an injury part and prolonging the half life of the drugs.

The embodiment of the invention also provides application of the erythrocyte drug-loaded system or the collagen-erythrocyte drug-loaded system which can be specifically combined with the collagen in preparation of products, and the products at least have the function of promoting the differentiation of neural stem cells to neurons.

The embodiment of the invention also provides application of the erythrocyte drug carrier system or the collagen-erythrocyte drug carrier system capable of specifically binding the collagen in preparation of products, and the products at least have the function of promoting axon regeneration.

The embodiment of the invention also provides application of the erythrocyte drug-loaded system or the collagen-erythrocyte drug-loaded system which can be specifically combined with the collagen in preparation of products, and the products at least have the function of repairing spinal cord injury.

The embodiment of the invention also provides a functional product for repairing spinal cord injury, which comprises the erythrocyte medicine-carrying system or the collagen-erythrocyte medicine-carrying system capable of specifically binding the collagen.

Compared with the prior art, the method has the advantages that the materials are economical and easy to obtain, the clinical operability is strong, the constructed erythrocyte medicine carrying system capable of specifically combining collagen can effectively anchor the medicine carrying erythrocyte microspheres on the collagen material, the problem of efficient connection of the medicine carrying erythrocyte and the collagen material is solved by adding functional polypeptide, the rapid loss of the medicine after transplantation is reduced, the medicine carrying system has the functions of maintaining the concentration of the medicine at the damaged part, prolonging the half-life period of the medicine, promoting the differentiation of more neural stem cells to neurons, promoting the regeneration of more axons and promoting the better movement recovery of spinal cord injury.

Drawings

FIG. 1 is a schematic diagram of the construction of a collagen-erythrocyte drug carrier system according to an embodiment of the present invention.

Fig. 2A and fig. 2B are scanning electron microscope surface topography images of control erythrocytes and prepared drug-loaded erythrocytes, respectively, according to an embodiment of the present invention.

Fig. 2C is a graphical representation of in vitro storage stability of drug-loaded red blood cells prepared in one embodiment of the present invention.

Fig. 2D is a graph of the osmotic fragility change of drug-loaded red blood cells prepared in one embodiment of the present invention.

FIG. 3A is a graph of mass spectrometric characterization of functional polypeptides provided in one embodiment of the present invention.

FIG. 3B is a diagram of liquid chromatography purification of a functional polypeptide provided in an embodiment of the present invention.

FIG. 3C is a graph comparing the binding capacity of functional polypeptides to leukocytes and erythrocytes, respectively, provided in one embodiment of the invention.

FIG. 3D is a schematic diagram of an affinity study of binding of functional polypeptides to red blood cells provided in one embodiment of the present invention.

FIGS. 4A-4D are graphs showing the binding capacity of functional polypeptides under SEM and confocal microscopy to erythrocyte and collagen according to one embodiment of the present invention.

FIG. 4E is a graph comparing the release of drugs from collagen material for each group in an embodiment of the present invention.

FIGS. 5A-5C are graphs of the effect of Tuj-1 and Map2 immunostaining on neural differentiation of each group of drug-loaded treatment groups in one embodiment of the present invention.

FIGS. 5D-5E are graphs of staining and quantification of NF-positive cells in spinal cord injury regions in an embodiment of the invention.

FIG. 6A is a BBB score graph of hindlimb behavioral recovery within 12 weeks of spinal cord injury in groups of rats according to an embodiment of the present invention.

FIG. 6B is a graph of hindlimb locomotor recovery within 12 weeks of spinal cord injury in groups of rats according to one embodiment of the present invention.

Detailed Description

In view of the defects in the prior art, the inventor of the invention has long studied and practiced a lot, and solves the problem of efficient connection between drug-loaded red blood cells and collagen materials by introducing a functional polypeptide. The functional polypeptide has the double functions of specifically binding to the surface of red blood cells at one end and specifically connecting collagen at the other end (shown in figure 1). The functional polypeptide, the drug-loaded red blood cells and the collagen material are incubated together, so that the drug-loaded red blood cells are anchored on the collagen material, and the purposes of maintaining the local concentration of the drug at the damaged part and slowly releasing the drug are achieved.

The technical solution, its implementation and principles, etc. will be further explained as follows.

One aspect of the embodiments provides a functional polypeptide comprising a collagen-binding sequence and an erythrocyte-binding peptide sequence linked to the collagen-binding sequence.

Further, the functional polypeptide (CBD-pep28) has an amino acid sequence shown in SEQ ID NO. 1, and the amino acid sequence is TKKTLRTKYTWYGYSLRANWMR.

Further, the collagen binding sequence (CBD) has an amino acid sequence shown in SEQ ID NO. 2, specifically an amino acid sequence TKKTLRT, and the erythrocyte binding peptide sequence (pep28) has an amino acid sequence shown in SEQ ID NO. 3, specifically an amino acid sequence KYTWYGYSLRANWMR.

Further, the collagen binding sequence is labeled by FITC, namely FITC-CBD-pep 28.

Another aspect of the embodiments of the present invention provides an erythrocyte drug-loading system capable of specifically binding collagen, which comprises a drug-loaded erythrocyte and any one of the functional polypeptides specifically binding to the drug-loaded erythrocyte.

Further, the erythrocyte-binding peptide sequence of the functional polypeptide is specifically bound with the drug-loaded erythrocyte, and the collagen-binding sequence has the function of being capable of being specifically bound with the collagen material.

In another aspect of the embodiments of the present invention, there is also provided a collagen-erythrocyte drug-loading system, which comprises a drug-loaded erythrocyte and a collagen material, wherein the drug-loaded erythrocyte is connected to the collagen material through any one of the functional polypeptides.

Further, the erythrocyte-binding peptide sequence of the functional polypeptide is specifically combined with the drug-loaded erythrocyte, and the collagen-binding sequence is specifically combined with the collagen material.

Further, the collagen material includes a pure collagen material or a composite material containing collagen, preferably a collagen scaffold, particularly preferably collagen fiber, and may be other collagen materials such as collagen sponge or collagen membrane, but is not limited thereto.

Further, the drug loaded on the drug-loaded red blood cells may be paclitaxel, but is not limited thereto.

In another aspect of the embodiments of the present invention, there is provided a method for preparing the collagen-erythrocyte drug-loading system, which includes: and (2) incubating the functional polypeptide, the drug-loaded red blood cells and the collagen material together, or incubating the functional polypeptide and the drug-loaded red blood cells in advance, and then incubating the functional polypeptide and the collagen material, or incubating the functional polypeptide and the collagen material in advance, and then incubating the functional polypeptide and the collagen material with the red blood cells, so that the red blood cell binding peptide sequence is specifically bound with the drug-loaded red blood cells, and the collagen binding sequence is specifically bound with the collagen material, thereby forming the collagen-red blood cell drug-loaded system.

In another aspect of the embodiments of the present invention, there is also provided a use of the above-mentioned erythrocyte drug-loading system or collagen-erythrocyte drug-loading system capable of specifically binding to collagen in preparing a product, wherein the product has at least functions of maintaining local drug concentration at a damaged part (especially a spinal cord damaged part) and prolonging drug half-life.

In another aspect of the embodiments of the present invention, an application of the above-mentioned erythrocyte drug-loading system or collagen-erythrocyte drug-loading system capable of specifically binding collagen in preparing a product is also provided, and the product at least has a function of promoting differentiation of neural stem cells to neurons.

In another aspect of the embodiments of the present invention, there is also provided an application of the above-mentioned erythrocyte drug-loading system or collagen-erythrocyte drug-loading system capable of specifically binding to collagen in preparing a product, wherein the product at least has a function of promoting axon regeneration.

In another aspect of the embodiments of the present invention, an application of the above-mentioned erythrocyte drug-loading system or collagen-erythrocyte drug-loading system capable of specifically binding collagen in preparing a product is also provided, and the product at least has a function of repairing spinal cord injury.

In another aspect of the embodiments of the present invention, there is provided a functional product for repairing spinal cord injury, which comprises the above-mentioned erythrocyte drug carrier system or collagen-erythrocyte drug carrier system capable of specifically binding collagen.

In another aspect of the embodiments of the present invention, there is provided a functional product for repairing myocardial damage, which comprises the above-mentioned erythrocyte drug carrier system or collagen-erythrocyte drug carrier system capable of specifically binding collagen.

The invention evaluates the binding specificity of the introduced functional polypeptide to drug-loaded red blood cells and collagen materials and the drug slow-release capacity of the system through in vitro experiments, finally adopts a rat spinal cord T9 section full-transverse injury model, immediately transplants the drug-loaded red blood cell system connected with the specifically-combined collagen and the collagen materials to an injury part after injury, detects the quantity difference of various nerve cells in the injury area by an immunohistochemical method after three months of transplantation, and evaluates the recovery condition of the rat motor function by adopting BBB score, thereby proving that the drug-loaded system has the capacity of keeping the concentration of the drug in the injury part and slowly releasing the drug.

The technical solution of the present invention is further described in detail below with reference to the accompanying drawings and exemplary embodiments.

1. Preparation and characterization of drug-loaded erythrocytes

Whole blood from healthy persons is centrifuged and red blood cells are collected and drug-loaded using a modified hypotonic pre-expansion method. Hypotonic solution (0.6% NaCl solution) was added to the red blood cell suspension for 30min, followed by centrifugation at 2000rpm for 5 min to remove excess hemoglobin. Paclitaxel (PTX) was mixed with red blood cells 1: PTX was added for 30min at a volume ratio of 1, followed by addition of 1.5M KCl as a hypertonic solution and resealing by incubation in a water bath at 37 ℃ for 30 min. The mixture was centrifuged at 12,000rpm for 15 minutes, the supernatant containing free PTX was removed and washed 3 times with PBS.

After the preparation of the drug-loaded red blood cells is finished, a series of in vitro biological evaluation and drug-loaded index researches are carried out on the drug-loaded red blood cells, such as morphology and hematology researches, stability experiments, drug loading capacity, encapsulation efficiency, cell recovery rate and the like.

The results show that: the drug loading method based on permeation can be used for preparing human erythrocytes loaded with paclitaxel, the majority of the carrier erythrocytes are in a spherical structure, the hemoglobin content in the cells is reduced, the stability in vitro storage for 5 days is good, and the permeation stability is reduced (see fig. 2A-2D), wherein fig. 2A represents washed erythrocytes, and fig. 2B represents drug-loaded erythrocytes. The carrier erythrocyte has good drug loading parameters, the drug loading rate of the paclitaxel is 212.4 plus or minus 23.1ug, the encapsulation rate is 46.36 plus or minus 3.46 percent, and the cell recovery rate is 77.08 plus or minus 7.63 percent. The drug loading of paclitaxel can meet the treatment concentration (nanomolar) of spinal cord injury. The preparation method is simple, and can be used for existing preparation.

2. Functional verification of functional polypeptide and construction of composite vector

The functional polypeptide CBD-pep28 is synthesized by a chemical solid phase synthesis method, and FITC fluorescent modification is carried out on the functional polypeptide CBD-pep28, wherein the amino acid sequence is TKKTLRTKYTWYGYSLRANWMR. Purifying by High Performance Liquid Chromatography (HPLC) and Mass Spectrometry (MS) to obtain product with purity of 98% or more (see FIG. 3A and FIG. 3B). Simultaneously, 22 peptide (identical to the amino acid sequence of pep28, but in random order of combination) was synthesized as a control peptide. Since the binding of the collagen binding sequence CBD to collagen has been previously confirmed in a large number of documents, the inventors herein focused on the binding specificity and affinity of pep28 to erythrocytes.

2.1 specificity and affinity assays for CBD-pep28 binding to erythrocytes

The specificity of pep28 for erythrocyte binding was studied in comparison to leukocytes, which are also a component of blood cells. FITC-CBD-pep28, FITC-randomised peptide were incubated with Red Blood Cells (RBC), White Blood Cells (WBC), centrifuged after 2h, washed with PBS, and the fluorescence intensity on the cells was measured by flow cytometry. The affinity of the polypeptide to RBC is studied by the same method, the polypeptide with different concentrations is incubated with RBC, and the fluorescence intensity of the cells is analyzed by flow cytometry.

The results show that: the fluorescence intensity of pep28-RBC was significantly higher than the other three groups, indicating that pep28 was able to bind to RBCs without binding to White Blood Cells (WBCs) (fig. 3C), indicating that binding was specific. Furthermore, the functional polypeptide bound to erythrocytes with a certain concentration dependence, and at concentrations above 200nM, the binding tended to saturate (FIG. 3D).

2.2 construction of collagen-erythrocyte drug Carrier System

(1) Functional polypeptide (FITC-CBD-pep28, FITC-random peptide) is dissolved with PBS to a concentration of 200nM, and the concentration of drug-loaded erythrocytes is adjusted to 1X10⁶One per ml.

(2) Prior to performing the collagen scaffold binding study, the collagen scaffold was blocked with 5% BSA and washed three times with PBS.

(3) The functional polypeptide is mixed with the drug-loaded red blood cells by using 100-fold excess, slowly dropped on the collagen scaffold, and incubated for 2h at 37 ℃.

(4) PBS was washed three times and the retention of drug-loaded erythrocytes on collagen scaffolds was observed by scanning electron microscopy and fluorescence microscopy.

Scanning electron microscopy results show that the control group contains a few RBCs sporadically, which may be caused by physical adsorption of the collagen scaffold itself. The fluorescence results were consistent with the much higher number of RBCs remaining on the collagen scaffold with CBD-pep28 compared to the control (fig. 4A-4D), where fig. 4A and 4C represent the control peptide-RBC and fig. 4B and 4D represent CBD-pep28-RBC, indicating that the functional polypeptide had dual specific affinity for both red blood cells and collagen.

2.3 in vitro drug Release study on vehicle

The release of different treatment groups, i.e. the red blood cell loading group (RBC-PTX) and the polypeptide-added red blood cell loading group (PEP-RBC-PTX), on the collagen scaffold was examined. 20ul of the drug-loaded erythrocytes were added to the collagen scaffold with PBS or CBD-pep28 for sufficient absorption, incubated at 37 degrees for 30min and washed three times, and then the collagen scaffold containing the drug-loaded erythrocytes was placed in the well plate. Meanwhile, an additional naked drug control group was placed directly on the scaffold in a well plate, added to 5mL of release medium (PBS containing 0.5% Tween 80, pH7.4), at 37 ℃ and shaken at 200 rpm. At each time point of 2h, 4h, 6h, 12h, 24h, 36h, 48h, a volume of 200ul of release medium was subsequently withdrawn every 24 hours, extending over up to 7 days. An equal volume of fresh release medium was replenished after each sampling. Corresponding samples were collected for 4 degree storage, and the paclitaxel content was measured using ultra high performance liquid chromatography, and the cumulative release was calculated.

3. Application of erythrocyte medicine carrying system capable of specifically combining collagen in rat spinal cord injury repair

3.1 establishment of rat spinal cord Total transection model

60 healthy adult female SD rats (Sprague-Dawley) weighing 180-200g were raised for one week after purchase and then operated 24h before surgery with no water deprivation. Randomized into 4 groups: control group, PTX, RBC-PTX, PEP-RBC-PTX group. When 10% chloral hydrate is injected into abdominal cavity, the rats have loose muscles and weak limbs, which indicates that the anesthesia is successful. Fixing the rat in prone position, wiping skin with iodophor, and taking a proper position to scrape off excessive hair. The skin was dissected with a scalpel above segment T7-T10, the muscles on both sides of the spine were dissected away, and the spinal cord was exposed by opening the vertebral plate and transecting the spinal cord at segment T9. After hemostasis is performed by pressing with collagen sponge, the drug-loaded materials of each group are placed in the transverse section, and the muscle and the skin are sequentially sutured by a suture needle.

3.2 behavioral assessment

Hindlimb motor function was assessed weekly within three months post-surgery using the open field (120X 200cm) Basso-Beattie-Bresnahan (BBB) motor test. The score ranges from 0 to 21 points, where 0 points represents complete loss of hind limb motor function and 21 points represents substantially normal hind limb motor function.

3.3 immunohistochemistry

And after three-month observation, taking samples of animal injured part specimens, performing immunohistochemical staining, and evaluating nerve regeneration conditions of the injured part.

4. Technical effects

4.1 erythrocyte drug-carrying system capable of specifically binding collagen has the function of prolonging half-life period of drug

The release profile of naked drug PTX on stents showed a strong burst release of 31% over the first 2 hours and a cumulative amount of PTX released over 12 hours of about 73%. However, for the RBC-PTX group and pep-RBC-PTX group, approximately 4.8%, 21.29%, and 4%, 16.59% were released at 2h, 12h, respectively, and 100%, 80.17%, and 70.77% were released at 48 hours for the three groups, respectively (fig. 4E).

4.2 erythrocyte drug-carrying system capable of specifically binding collagen has the function of promoting more neural stem cells to differentiate towards neurons

The present inventors evaluated whether a functional scaffold that slowly releases PTX allows for the induction of more neuronal production at the site of injury. Tuj-1 is a tubulin specific to neurons and can be used as a marker for detecting early neurons. Map2 is a marker for mature neurons.

As the inventors have expected, the PEP-RBC-PTX group had the largest numbers of Tuj-1 positive neurons and Map2 positive neurons (fig. 5A-5C). Quantitative analysis showed that for PTX (b), RBC-PTX (c), PEP-RBC-PTX (d) groups, Tuj-1 positively stained neurons were 15.34%, 23.45% and 34.22%, respectively, and Map2 positively stained neurons were 17.3%, 28.45% and 36.22%, respectively, indicating that targeting of functional polypeptides resulted in the production of more early and mature neurons.

4.3 erythrocyte drug-carrying system capable of specifically binding collagen has the function of promoting more axon regeneration

Neurofilament (neurofilament NF) is the main framework structure that constitutes nerve cells, especially nerve axons, and immunochemical staining of neurofilament NF200 antibodies can reflect axonal repair.

The inventor finds that the other three groups have stronger promotion effects in axon regeneration compared with the control group, and compared with the PTX (a) group, in the RBC-PTX (b) group and the PEP-RBC-PTX (c) group, more NF positive nerve fibers appear in the damage central area, the coloring points are dense, the nerve axis processes are prolonged along the direction of the collagen fibers (fig. 5D-5E), and the scaffold has important effects of guiding the growth of nerve cells and promoting the reconstruction connection of a nerve network after damage.

4.4 erythrocyte drug-carrying system capable of specifically binding collagen has the function of promoting better exercise recovery of spinal cord injury

Hind limb locomotion was performed by BBB scoring during 12 postoperative periods. The rats had hardly any significant recovery in the control treated group with only the stent graft. In contrast, rats in the PEP-RBC-PTX group started to recover faster from week 3, showing significantly improved BBB score values compared to the other groups (fig. 6A). The highest BBB score of about 8 was shown at 12 weeks post-surgery, when the rat hind limbs could be grounded without load bearing and occasionally dragged along (shown as c in fig. 6B), while the PTX group (shown as B in fig. 6B) was only maintained on slight or extensive movement of several joints of the hind limbs and showed no supportive movement. These results demonstrate the enhanced therapeutic efficacy of the sustained release of PEP-RBC-PTX collagen scaffold in spinal cord repair, suggesting potential in clinical trials.

In conclusion, the beneficial effects of the invention are as follows: the method has the advantages of economical and easily obtained materials and strong clinical operability, and the constructed erythrocyte medicine carrying system specifically combined with the collagen can effectively anchor the medicine carrying erythrocyte microspheres on the collagen, thereby reducing the rapid loss of the medicine after transplantation and realizing the concentration maintenance of the damaged part. And in vitro release results show that the system has good effect of prolonging the release of the drug.

The drug-loaded system is transplanted into a rat full-transection spinal cord injury model together with a collagen scaffold, and an immunofluorescence staining result shows that the PEP-RBC-PTX group has a significant difference in the differentiation number of neurons from the RBC-PTX group to the PTX group. The functional polypeptide-linked erythrocyte carrier promotes more neuron differentiation of endogenous neural stem cells through local retention and delayed release of the drug. In addition, the behavioral characteristics of the injured rats are further improved on a macroscopic level.

Furthermore, the erythrocyte medicine carrying system capable of specifically combining with collagen provides a new scheme for delivering medicines in spinal cord injury repair, and has a certain clinical application prospect.

It should be understood that the above-mentioned embodiments are merely illustrative of the technical concepts and features of the present invention, which are intended to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and therefore, the protection scope of the present invention is not limited thereby. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Sequence listing

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Claims

1. A functional polypeptide comprising a collagen binding sequence and an erythrocyte binding peptide sequence linked to said collagen binding sequence.

2. The functional polypeptide of claim 1, wherein: the functional polypeptide has the dual functions of specifically binding the erythrocyte-binding peptide sequence at one end to the surface of the erythrocyte and specifically binding the collagen-binding sequence at the other end to the surface of the collagen.

3. The functional polypeptide of claim 1 or 2, characterized in that: the functional polypeptide has an amino acid sequence shown in SEQ ID NO. 1.

4. The functional polypeptide of claim 1, wherein: the collagen binding sequence has an amino acid sequence shown as SEQ ID NO. 2, and the erythrocyte binding peptide sequence has an amino acid sequence shown as SEQ ID NO. 3.

5. An erythrocyte drug-loaded system capable of specifically binding collagen, which comprises a drug-loaded erythrocyte and the functional polypeptide of any one of claims 1 to 4 specifically binding to the drug-loaded erythrocyte.

6. The erythrocyte drug carrier system capable of specifically binding collagen according to claim 5, wherein: the erythrocyte-binding peptide sequence of the functional polypeptide is specifically bound with the drug-loaded erythrocyte, and the collagen-binding sequence has the function of being specifically bound with the collagen material.

7. A collagen-erythrocyte medicine carrying system comprises medicine carrying erythrocytes and a collagen material, and is characterized in that: the drug-loaded red blood cells are linked to the collagen material by the functional polypeptide of any one of claims 1 to 4.

8. The collagen-erythrocyte drug carrier system of claim 7, wherein: the erythrocyte-binding peptide sequence of the functional polypeptide is specifically combined with the drug-loaded erythrocyte, and the collagen-binding sequence is specifically combined with the collagen material; preferably, the collagen material comprises pure collagen raw material or collagen-containing composite material, preferably a collagen scaffold, and particularly preferably any one or a combination of two or more of collagen sponge, collagen fiber and collagen membrane.

9. The method of preparing a collagen-erythrocyte drug carrier system of any one of claims 7 to 8, which comprises: incubating the functional polypeptide of any one of claims 1-4, a drug-loaded erythrocyte, with a collagen material such that the erythrocyte-binding peptide sequence specifically binds to the drug-loaded erythrocyte and the collagen-binding sequence specifically binds to the collagen material to form the collagen-erythrocyte drug-loaded system.

10. Use of a collagen-specific binding erythrocyte drug carrier according to any of claims 5 to 6 or a collagen-erythrocyte drug carrier according to any of claims 7 to 8 for the preparation of a product having at least the function of maintaining a local drug concentration at the site of injury and prolonging the half-life of the drug.

11. Use of the collagen-specific erythrocyte drug carrier of any one of claims 5 to 6 or the collagen-erythrocyte drug carrier of any one of claims 7 to 8 for the preparation of a product having at least a function of promoting differentiation of neural stem cells into neurons.

12. Use of a collagen-specific binding erythrocyte drug carrier according to any of claims 5 to 6 or a collagen-erythrocyte drug carrier according to any of claims 7 to 8 for the preparation of a product having at least the function of promoting axon regeneration.

13. Use of the collagen-specific erythrocyte drug carrier of any one of claims 5 to 6 or the collagen-erythrocyte drug carrier of any one of claims 7 to 8 for the preparation of a product having at least the function of repairing spinal cord injuries.

14. A functional product for repairing spinal cord injury, comprising the collagen-specifically binding erythrocyte drug carrier of any one of claims 5 to 6 or the collagen-erythrocyte drug carrier of any one of claims 7 to 8.