CN111407888A

CN111407888A - BPNs (BPNs)/chitosan/PRP (pseudochitosan/PRP) temperature-sensitive hydrogel and preparation method and application thereof

Info

Publication number: CN111407888A
Application number: CN202010069594.7A
Authority: CN
Inventors: 高丰雷; 潘文振; 郭开金
Original assignee: Xuzhou Medical University
Current assignee: Xuzhou Medical University
Priority date: 2020-01-21
Filing date: 2020-01-21
Publication date: 2020-07-14
Anticipated expiration: 2040-01-21
Also published as: CN111407888B

Abstract

The invention relates to BPNs (bis-bromodiphenyl sulfone)/chitosan/PRP (pseudoephedrine hydrochloride/hydroxypropyl phthalate) temperature-sensitive hydrogel and a preparation method and application thereof. The BPNs/chitosan/PRP temperature-sensitive hydrogel is mainly prepared by mixing BPNs, chitosan temperature-sensitive hydrogel and activated PRP, and hyperplasia is carried out through phototherapy of the BPNs, biotherapy treatment of platelet-rich plasma (PRP) and phosphorus-driven calcium extraction biomineralization, so that the BPNs/chitosan/PRP temperature-sensitive hydrogel has good PTT and PDT effects, the cytotoxicity is low in different cells, and the survival rate is higher than 85%; the biocompatibility, the cell affinity and the proliferation capacity are good; ROS are easily generated under the irradiation of near infrared light, and the maximum value is reached within 8 minutes, so that the cell adhesion, the growth and the extracellular matrix generation can be promoted; can effectively remove hyperplastic synovial tissue, repair defective bone tissue and maintain the smoothness of joint surfaces, and can be used as a medicine for photo-thermal and/or photodynamic treatment of rheumatoid arthritis.

Description

BPNs (BPNs)/chitosan/PRP (pseudochitosan/PRP) temperature-sensitive hydrogel and preparation method and application thereof

Technical Field

The invention belongs to the technical field of hydrogel, and particularly relates to BPNs (BPNs)/chitosan/PRP (pseudochitosan/PRP) temperature-sensitive hydrogel and a preparation method and application thereof.

Background

Rheumatoid Arthritis (RA) is the most common chronic systemic inflammatory disease affecting about 1% of the population worldwide, resulting in long-term arthritis, cartilage degradation, bone defects, disability and premature death. Therapy for RA may cause a number of adverse side effects, including infection, interstitial lung disease and impaired glucose metabolism. The disadvantages of surgery are that the trauma is large, the adhesion is extensive after surgery and it cannot be reused. Although arthroscopic synovectomy offers advantages over traditional surgery, blind spots still exist. Chemical synovectomy and radiosurgery resection may damage normal tissue.

In recent years, there has been increasing interest in phototherapy, including photodynamic therapy (PDT) and photothermal therapy (PTT), which is a relatively non-invasive and less acute option. In PTT, Near Infrared (NIR) light is converted through a light-to-heat converter to convert heat to localized heat to treat a diseased area. At the same time, the photosensitizer in PDT is excited to a singlet state, and then Reactive Oxygen Species (ROS) are generated to control abnormal cells or tissues. To date, a rich therapeutic platform built around phototherapy has been used as a strategy to combat pathological abnormalities. RA is characterized by persistent, polyarticular proliferation of synovial cells, ultimately leading to damage to articular cartilage and bone. However, this limits the use of PDT and PTT in the treatment of RA, since the need for efficiency is the removal of the proliferating synovial tissue and the concomitant generation of bone. Therefore, there is an urgent need to find new materials with phototherapy action and osteogenic potential to achieve the desired therapeutic effect.

Disclosure of Invention

The invention aims to provide an application of BPNs/chitosan/PRP temperature-sensitive hydrogel in a medicine for treating rheumatoid arthritis by photothermal and/or photodynamic on the basis of the prior art.

Another object of the present invention is to provide BPNs/chitosan/PRP temperature-sensitive hydrogels prepared by the above preparation methods.

The third purpose of the invention is to provide a preparation method of the BPNs/chitosan/PRP temperature-sensitive hydrogel.

The technical scheme of the invention is as follows:

the BPNs/chitosan/PRP temperature-sensitive hydrogel provided by the invention has good PTT and PDT effects and good biocompatibility, easily generates ROS under the irradiation of near infrared light, and promotes cell adhesion, growth and extracellular matrix generation; can effectively remove hyperplastic synovial tissue, repair defective bone tissue and maintain the smoothness of joint surfaces, and can be used as a medicine for photo-thermal and/or photodynamic treatment of rheumatoid arthritis.

BPNs (black phosphorus nanoplates) have good biocompatibility, excellent phototherapy effect and potential osteogenesis characteristics, and can effectively convert light energy into heat energy under near-infrared illumination, thereby destroying disease areas. BPNs also tend to produce ROS to further treat abnormal cells or tissues. In addition, phosphorus is a necessity in the human body, especially bones, as part of natural resources, and degradation products of BPNs can be converted in situ into P-based agents that can enhance the osteogenic process.

The chitosan temperature-sensitive hydrogel as an injectable material has outstanding biocompatibility and degradability. The structure of the chitosan thermal response hydrogel is closer to organism tissues and is similar to extracellular matrix in nature, so that the friction of surrounding tissues can be reduced, and the biological characteristics of the material are remarkably improved. The chitosan temperature-sensitive hydrogel has biodegradability, good biocompatibility and temperature sensitivity to hydrogel formation at human body temperature, so that the chitosan temperature-sensitive hydrogel can be effectively used for delivery and sustained release of proteins/peptides, anti-inflammatory drugs and antibiotics.

Phototherapy with BPNs can be used to remove proliferating synovial cells under NIR light irradiation. PRP has many growth factors and cytokines and can be used to enhance cell adhesion and proliferation. The chitosan thermal response hydrogel has unique rheological characteristics and has excellent cartilage protection and lubrication effects in joint cavities. In addition, the good degradability and thermo-responsiveness of chitosan hydrogels offers the possibility to control drug release.

The pathogenesis of RA is very complex, the invention carries out hyperplasia by a large amount of research works through phototherapy of BPNs, biotherapy treatment of Platelet Rich Plasma (PRP) and phosphorus-driven calcium extraction biomineralization, has good PTT and PDT effects and good biocompatibility under the coordination of other conditions, is easy to generate ROS under the irradiation of near infrared light, and promotes cell adhesion, growth and extracellular matrix generation; can effectively remove hyperplastic synovial tissue, repair defective bone tissue and maintain the smoothness of joint surfaces, generates synergistic effect on RA, and can be used as a photo-thermal and/or photodynamic medicament for treating rheumatoid arthritis.

The BPNs/chitosan/PRP temperature-sensitive hydrogel comprises 10-40 mg/m L chitosan, 35-45 μ g/m L BPNs and 0.2-0.8 mg/m L PRP.

In one embodiment, the chitosan concentration is 10mg/m L, 15mg/m L, 16mg/m L, 18mg/m L, 20mg/m L, 30mg/m L, or 40mg/m L, preferably the chitosan concentration is 16mg/m L.

In one embodiment, the concentration of BPNs is 35 μ g/m L, 37 μ g/m L, 40 μ g/m L, 42 μ g/m L, 44 μ g/m L, or 45 μ g/m L, preferably the concentration of BPNs is 42 μ g/m L.

In one embodiment, the concentration of PRP is 0.2mg/m L, 0.3mg/m L, 0.4mg/m L, 0.5mg/m L, 0.6mg/m L, 0.7mg/m L, or 0.8mg/m L, preferably the concentration of PRP is 0.6mg/m L.

In a preferred embodiment, the chitosan has a degree of deacetylation of 85 to 95%, for example 90%.

The invention provides BPNs/chitosan/PRP temperature-sensitive hydrogel which is mainly prepared by mixing BPNs, chitosan temperature-sensitive hydrogel and activated PRP.

In a preferred embodiment, the chitosan concentration is 16mg/m L.

In a preferred embodiment, the concentration of BPNs is 42 μ g/m L.

In a preferred embodiment, the concentration of PRP is 0.6mg/m L.

The invention also provides a preparation method of the BPNs/chitosan/PRP temperature-sensitive hydrogel, which mainly comprises the following steps:

(1) carrying out ultrasonic treatment on the black phosphorus crystal, and then centrifuging to obtain BPNs;

(2) dissolving chitosan in an acetic acid solution, and mixing with β -GP aqueous solution to obtain chitosan temperature-sensitive hydrogel;

(3) mixing the BPNs prepared in the step (1) with the chitosan temperature-sensitive hydrogel prepared in the step (2), and adjusting the pH to 7.0-7.5 through β -GP to obtain the BPNs/chitosan temperature-sensitive hydrogel;

(4) and (4) mixing the BPNs/chitosan temperature-sensitive hydrogel prepared in the step (3) with the activated PRP to obtain the preparation of the BPNs/chitosan/PRP temperature-sensitive hydrogel.

In one scheme, in the step (1), the ultrasonic treatment conditions of the black phosphorus crystal are as follows: carrying out ultrasonic treatment for 1-6 h at the power of 400-1200W. For example, the ultrasonic power is 400W, 800W or 1200W, preferably 1200W. The time of the ultrasonic treatment is 1h, 2h, 3h or 6h, preferably 3 h. Note that: probe ultrasound outperforms water bath ultrasound at the same time.

In a preferred embodiment, the conditions of the ultrasonic treatment are as follows: sonicate for 3h at 1200W.

The conditions of centrifugation were: centrifuging at 3500-4500 rpm for 5-15 minutes. Preferably, the conditions of centrifugation are: centrifuge at 4000rpm for 10 minutes.

In one scheme, in the step (2), the concentration of the acetic acid solution is 0.1-0.5 mol/L, and preferably 0.1 mol/L.

Furthermore, the concentration of the β -GP aqueous solution is 0.4-0.8 g/m L, preferably 0.56g/m L.

In a preferable scheme, in the step (2), the mass-to-volume ratio of the chitosan to the acetic acid solution is 20-25 mg/m L, and is preferably 22.2mg/m L.

Further, the mass ratio of the chitosan to the β -GP is 1: 2-4, and preferably 1: 2.8.

In one scheme, in the step (3), the mass-to-volume ratio of the BPNs to the chitosan temperature-sensitive hydrogel prepared in the step (2) is 25 μ g/m L-75 μ g/m L, preferably 50 μ g/m L.

In one scheme, in the step (4), the mass-to-volume ratio of the PRP to the BPNs/chitosan temperature-sensitive hydrogel is 0.2-0.8 mg/m L, and preferably 0.6mg/m L.

In a preferred embodiment, the preparation method of the activated PRP is as follows: PRP is activated with calcium chloride and made into lyophilized PRP powder.

Among them, some abbreviations presented in this application correspond to the full names Rheumatoid Arthritis (RA), Oxidative Stress (OS), Reactive Oxygen Species (ROS), collagen-induced arthritis (CIA), synovial fibroblasts (F L Ss), mesenchymal stem cells (BMSC), Chitosan (CS), β -sodium glycerophosphate (β -GP), Black Phosphorus Nanoplates (BPNs), platelet-rich plasma (PRP), hematoxylin-eosin staining (HE), Scanning Electron Microscope (SEM), Transmission Electron Microscope (TEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), Atomic Force Microscope (AFM), cell counting kit (CCK-8), calcein (AM), Propidium Iodide (PI), acid salt buffer (PBS), Complete Freund's Adjuvant (CFA), Freund's adjuvant (IFA), bovine collagen type II (bovine fetal collagen type II), bovine Fetal (FBS), Bovine Serum Albumin (BSA), extracellular matrix (ECM), GAPDH, ultraviolet-visible light spectrophotometer (UV-Vis-UV-I).

By adopting the technical scheme of the invention, the advantages are as follows:

the BPNs/chitosan/PRP temperature-sensitive hydrogel prepared by the invention proliferates by phototherapy of the BPNs, biotherapy treatment of Platelet Rich Plasma (PRP) and phosphorus-driven calcium extraction biomineralization, so that the BPNs/chitosan/PRP temperature-sensitive hydrogel has good PTT and PDT effects, has low cytotoxicity in different cells and has a survival rate higher than 85%; the biocompatibility, the cell affinity and the proliferation capacity are good; ROS are easily generated under the irradiation of near infrared light, and the maximum value is reached within 8 minutes, so that the cell adhesion, the growth and the extracellular matrix generation can be promoted; can effectively remove hyperplastic synovial tissue, repair defective bone tissue and maintain the smoothness of joint surfaces, and the components are mutually matched to generate a synergistic effect on RA, so that the traditional Chinese medicine composition can be used as a photo-thermal and/or photodynamic medicine for treating rheumatoid arthritis.

Drawings

FIG. 1 is a representation of BPNs/chitosan/PRP temperature sensitive hydrogel, A) is a TEM of BPNs, B) is a TEM of enlarged individual Black phosphor nanoplates of A), C) is an AFM image of BPNs, D) is an XRD spectrum of BPNs and BPNs/chitosan/PRP temperature sensitive hydrogels, E) is an XPS spectrum of BPNs and BPNs/chitosan/PRP temperature sensitive hydrogels, wherein (020), (040), (060) represents a characteristic peak pattern of BPNs, relevant literature supports Xianjun Zhu, Taiming Zhang, Zijun Sun, Huanglin Chen, Jian Guan, Xiang Chen, Hengxijii, Pingwu Du, and Shangfeng Yang.

FIG. 2 is a graph of photothermal property analysis; A) the NIR radiation power intensity of the BPNs solution, the BPNs/chitosan temperature-sensitive hydrogel and the BPNs/chitosan/PRP temperature-sensitive hydrogel is 1.0W cm^-2The time is 0 to 8 minutes; B) the BPNs aqueous solution, the BPNs/chitosan temperature-sensitive hydrogel and the BPNs/chitosan/PRP temperature-sensitive hydrogel are 1.0W cm^-2Photothermal conversion characteristics under the condition of NIR irradiation for 8min, and normal saline is used as a control; C) BPNs/chitosan/PRP temperature-sensitive hydrogel is continuously irradiated by continuous NIR laser for 4 cycles (808 nm for each cycle, 1.0W cm)^-2790s and 300s without laser);

FIG. 3 is a graph of degradability analysis; A) SEM of BPNs/chitosan/PRP temperature sensitive hydrogel at different time periods (0, 4 and 8D); B) photographs of BPNs/chitosan/PRP temperature sensitive hydrogels stored at different time periods (0, 2, 4, 6 and 8D); C) an absorption spectrum;

FIG. 4 shows the cytotoxicity detection of BPNs/chitosan/PRP temperature-sensitive hydrogel on different cells when the BPNs/chitosan/PRP temperature-sensitive hydrogel is incubated for 24h and 48 h;

FIG. 5 is an analysis of ROS production from BPNs/chitosan/PRP temperature sensitive hydrogels;

FIG. 6 is the ROS production during different NIR illumination periods;

FIG. 7 is the cell viability after treatment with PBS or BPNs/chitosan/PRP with or without radiation (statistical sense: p <0.05, p < 0.01);

FIG. 8 is calcein-AM staining and an automatic cell counter for assessing cell proliferation;

FIG. 9 is an assessment of cell adhesion by FITC-labeled phalloidin staining;

FIG. 10 is an inverted microscope observation of cell chemotactic properties of materials of different PRP content.

Detailed Description

The BPNs/chitosan/PRP temperature-sensitive hydrogel of the present invention is further illustrated by the following examples in combination with the accompanying drawings, but the examples do not limit the present invention in any way.

Example 1

1. Preparation of BPNs/chitosan/PRP temperature-sensitive hydrogel

The BPNs/chitosan/PRP temperature-sensitive hydrogel provided by the invention

(1) The black phosphorus crystals were ground to a powder in a grinder. The black phosphorus crystal powder was sonicated at 1200W for 3h and then centrifuged at 4000rpm for 10 min to obtain BPNs, which were stored and sealed at 4 ℃.

(2) 200mg of chitosan (the deacetylation degree is 90%) is dissolved in 9m L0.1 mol/L acetic acid solution to prepare chitosan acetic acid solution, 560mg of β -GP is dissolved in 1m L deionized water to prepare β -GP water solution, β -GP water solution is dropwise added into the chitosan acetic acid solution under uniform stirring to obtain clear and uniform chitosan temperature-sensitive hydrogel, and the clear and uniform chitosan temperature-sensitive hydrogel is stored and sealed at 4 ℃.

(3) Mixing the BPNs (200 mu g/m L, 1.25m L) prepared in the step (1) with the chitosan temperature-sensitive hydrogel (4.75m L) prepared in the step (2), adjusting the pH value to 7.2 by β -GP to obtain the BPNs/chitosan temperature-sensitive hydrogel, and storing and sealing at 4 ℃.

(4) Collecting platelet-rich plasma (PRP) according to a method reported previously by Kim et al, activating the collected PRP by calcium chloride to prepare freeze-dried PRP powder, and mixing the BPNs/chitosan temperature-sensitive hydrogel (5m L) prepared in the step (3) with the activated freeze-dried PRP powder (3mg) to obtain the preparation of the BPNs/chitosan/PRP temperature-sensitive hydrogel.

2. Determination of physical and chemical properties and photo-thermal properties of materials

2.2.1 SEM and TEM observations

The surface and the internal appearance of the prepared temperature-sensitive gel material are characterized by a field emission scanning electron microscope and a transmission electron microscope. Prior to SEM imaging, the samples were frozen with liquid nitrogen and treated in a freeze dryer for 6 hours to remove excess moisture from the gel material, and then sputter coated with a layer of gold of about 5nm (gold spray) to prevent abnormal discharge from the stent surface.

2.2.2 XRD analysis

And (3) diffracting the X rays of the prepared gel material by using an X-ray diffractometer to research the existence state and degradation condition of BNPs in the gel material. And (3) detection: and (3) pressing the sample, controlling the diffraction angle and measuring the lattice constant d of the sample.

2.2.3 XPS analysis

The surface chemistry of the scaffold samples was studied using an XSAM800 analyzer.

2.2.4 AFM analysis

The surface information and roughness of the material were evaluated with nanometer-scale resolution using atomic force microscopy.

2.2.5 photothermographic analysis

The in vitro photothermal effect of the gel material was studied using a photothermal imager. Selecting 1W/cm²The material is effectively irradiated for 8 min. Record and plot photothermal curves.

3. Results and analysis

3.1 TEM analysis of Black Phosphorus Nanoplates (BPNs)

FIG. 2 is a representation of Black Phosphorus Nanoplates (BPNs) and BPNs/chitosan/PRP temperature sensitive hydrogels prepared in example 1. The TEM analysis of the surface morphology of the black phosphorus nanosheets is shown in FIGS. 1A-1B, the black phosphorus nanosheets have an obvious lamellar structure, are relatively uniform in size, have an average particle size of 150 nm-200 nm, and are complete in structure.

3.2 AFM analysis of Black Phosphorus Nanoplates (BPNs)

As shown in FIG. 1C, the black phosphorus nanosheet is large in transverse size, about 200nm, relatively flat in surface, stable in structure, 2-5 nm in thickness and about 7 layers.

3.3 XRD and XPS analysis

XRD analysis is carried out on the black phosphorus nanosheet and the BPNs/chitosan/PRP temperature-sensitive hydrogel, as shown in figure 1D, characteristic diffraction peaks of the black phosphorus nanosheet are observed at 020, 040 and 060, and the successful preparation of the black phosphorus nanosheet is indicated. To reduce surface energy, the layers between the sheets and between the layers are stacked together in a planar fashion to form uniform diffraction peaks. And the BPNs/chitosan/PRP temperature-sensitive hydrogel has slightly reduced peak values at 20 degrees, 40 degrees and 60 degrees and still has obvious characteristic peaks, which indicates that the hydrogel does not obviously influence the structure of the black phosphorus nanosheet, and also indicates that the black phosphorus nanosheet is abundantly present in the BPNs/chitosan/PRP temperature-sensitive hydrogel.

XPS analysis is shown in figures 1E-1F, the P2P oxidation peak has an obvious peak value at 130eV, and the peak is considered as the peak of a P-O or P ═ O bond, which indicates that the black phosphorus nanosheet in the BPNs/chitosan/PRP temperature-sensitive hydrogel is degraded, and from another perspective, the good biocompatibility of the black phosphorus nanosheet in the BPNs/chitosan/PRP temperature-sensitive hydrogel is verified.

3.3 BPNs/chitosan/PRP temperature sensitive hydrogel SEM and TEM observation results

As shown in the figure 1G-1K, which is the SEM of the BPNs on the surface of the BPNs/chitosan/PRP temperature-sensitive hydrogel, the surface morphology of the gel is smooth, the black phosphorus nanosheets are uniformly dispersed on the surface of the gel, no obvious impurities are seen, the size of the dispersed nanosheets is uniform, and the particle size is about 200 nm. FIGS. 1M-1N are SEM images of the internal morphology characteristics of the BPNs/chitosan/PRP temperature-sensitive hydrogel after freeze-drying, and it can be known that the BPNs/chitosan/PRP temperature-sensitive hydrogel has a porous structure which is communicated with each other, the pore structure is complete, and the pore diameter is 2-5 μ M.

3.5 BPNs/chitosan/PRP temperature sensitive hydrogel image

As shown in FIG. 1L-1O, the BPNs/chitosan/PRP temperature-sensitive hydrogel has darker color, indicating that BPNs have been successfully encapsulated in the temperature-sensitive hydrogel, as shown in FIG. 1O, after NIR illumination, the hydrogel is transformed from a liquid state to a gel state, indicating the temperature sensitivity of the hydrogel.

3.6 photothermal Property analysis of Material

As shown in fig. 2A and 2B, the temperature of the physiological saline solution hardly changed even after the near-infrared irradiation for 8 minutes, indicating that the photothermal effect was not or hardly any. However, under the same conditions, the temperature of the BPNs/chitosan/PRP temperature-sensitive hydrogel rises by about 25 ℃, thereby indicating that the BPNs/chitosan/PRP temperature-sensitive hydrogel can rapidly and effectively convert NIR light energy into heat energy and can effectively treat RA. In the PTT field, efficacy depends on NIR conversion efficiency and material stability, which limits the clinical application of multiple PTT agents. In contrast, the present inventors further explored the cyclic photothermal conduction stability of BPNs/chitosan/PRP temperature sensitive hydrogels. The BPNs/chitosan/PRP temperature sensitive hydrogel was subjected to 4 repeated irradiation cycles (fig. 2C). The result shows that the temperature of the BPNs/Chitins/PRP temperature-sensitive hydrogel changes rapidly, reaches a platform after being irradiated for 8 minutes, and then is cooled to be close to the ambient temperature within 5 minutes by radiation. Heating and cooling tests show that the BPNs/chitosan/PRP temperature-sensitive hydrogel has photo-thermal conversion stability and repeatability.

3.7 analysis of degradation Capacity of BPNs/Chitosan/PRP temperature sensitive hydrogel

Considering that black phosphorus nanoplates are the main donor of phosphate in the subsequent bone regeneration step, whether they degrade in BPNs/chitosan/PRP temperature sensitive hydrogel and their degradation rate, evaluation is required. In short, the BPNs/chitosan/PRP temperature-sensitive hydrogel is placed in a horizontal oscillator for 8d, and a scanning electron microscope shows that, as time goes by, the BPNs attached to the surface of the hydrogel are gradually dissolved and fused together, on the 8 th day, the surfaces of the black phosphorus nanosheets are blurred, the lamellar morphology is basically disappeared, no obvious boundary exists between every two adjacent layers, and the BPNs have good time-dependent degradation property (fig. 3A). As shown in FIG. 3B, the BPNs/chitosan/PRP temperature-sensitive hydrogel gradually faded in color and appeared colorless and transparent after 8 days, indicating that it was completely degraded, as observed by scanning electron microscopyThe phenomena are identical. The absorption band data indicated that the absorption intensity decreased with the longer degradation time (fig. 3C), demonstrating the gradual degradation of BPNs in BPNs/chitosan/PRP temperature sensitive hydrogels. Irreversible reaction with oxygen and water results in degradation of BPNs, producing oxidized phosphorus species (P → PxOy), followed by PxOy and extremozal Products (PO)^43-) The ions react. Although, BPNs remain stable for only a few days in BPNs/chitosan/PRP temperature sensitive hydrogels. But from another perspective, it remains significantly degradable after binding to the hydrogel, which is useful for biological applications. In addition, degradation products of BPNs can be converted in situ into P-based agents that can promote bone regeneration. The result shows that the BPNs/chitosan/PRP temperature-sensitive hydrogel has good effect of treating RA and can be degraded by itself after the treatment is finished.

Example 2 treatment experiment of BPNs/chitosan/PRP temperature-sensitive hydrogel for RA at cellular level

1. Material

1.1 reagents

1.2 instruments

2. Experimental methods

In order to research the ROS generation condition of the PRP-chitosan temperature-sensitive hydrogel combined with the black phosphorus nanosheet injectable biomaterial and the scavenging effect on rheumatoid synovial cells, the inventor researches a series of properties of the PRP-chitosan temperature-sensitive hydrogel combined with the black phosphorus nanosheet injectable biomaterial from a cell level.

2.1 culture of Rheumatoid synoviocytes

(1) Preparation of a culture medium:

taking 1000m L DMEM culture medium, adding 10% fetal calf serum, 1% penicillin and streptomycin, and 1% L-glutamine, filtering, sterilizing, and packaging in a special 50ml centrifugal tube for cell.

(2) Cryopreservation and recovery of cells

Freezing when the cells grow to be about 90% of the bottom of a culture bottle or dish, discarding supernatant, adding sterilized PBS, repeatedly washing for 2 times, removing residual culture medium, adding 1-2ml of pancreatin containing EDTA into the culture bottle, incubating in an incubator at 37 ℃ for 3-5 minutes to completely digest the cells, adding an equal amount of culture medium to stop digestion after cell gaps are enlarged and cells are rounded, blowing by using a 15ml pasteur pipette to completely shed the cells from the cell bottle wall, transferring corresponding cell suspension into a centrifuge tube of 15m L, centrifuging at the normal temperature of 800rpm/min for 3 minutes, discarding supernatant, taking 1m L cell frozen solution to resuspend the cells, taking 10u L cell suspension for proper dilution, counting under a counting plate mirror, calculating cell density, then transferring the resuspended solution into a freezing tube, sealing by a sealing film, sealing by using a refrigerator at minus 80 ℃ for later use.

When the cells are recovered, firstly adding prepared culture medium of about 4m L into a culture bottle or a culture dish in a super clean bench, then taking out the cryopreservation tube from a refrigerator at minus 80 ℃, quickly thawing in water bath at 37 ℃, moving into a centrifugal tube of 15m L after complete thawing, adding the culture medium preheated by 2-4 m L, centrifuging for 3 minutes at 800rpm, discarding supernatant, suspending the cells by the culture medium of 1-2m L, counting by a counting plate, and adding the counting plate into the culture bottle or the culture dish for culture.

(3) Subculturing of cells

When the cells grow to about 90% of the bottom of a culture bottle or a culture dish, passage can be carried out, supernatant cell culture solution is discarded, PBS is rinsed for 2 times gently, a proper amount (based on that all the cells are just completely covered) of 0.25% pancreatin is added, the cells are incubated in the culture box for 3-5 minutes to digest the cells, when cell gaps are completely opened and partial cells float or become round, digestion is stopped, the corresponding cell suspension is transferred to a centrifuge tube of 15m L, the cells are centrifuged at normal temperature of 800rpm/min for 3 minutes, supernatant is discarded, 2-3 m L growth culture medium is used for resuspending the cells, 10u L cell suspension is taken to be diluted properly, the cells are counted under a cell counting plate, the cell density is calculated, and 4 × 10 cells are used⁴Per cm²The cell density of (3) is plated. The inoculated cells were still left in 5% CO₂Culturing at 37 deg.C and relative humidity of 90%.

2.2 in vitro cytotoxicity detection of BPNs/chitosan/PRP temperature-sensitive hydrogel to fibroblast (L929)

Hydrogels containing different material components were added to the well plates at about 10ul per well, and cells were transferred to 96 well plates (1 × 10)⁴Cells/well), after the cells are completely attached to the wall and the shape is normal, after incubation for 24h and 48h respectively, the cells are washed by PBS, 100ul of cell culture solution (10%) containing CCK-8 which is prepared in advance is added into each well, and incubation is carried out for 2-4 hours in a dark place. Optical density values at 450nm were measured using a Bio-Rad model 680 enzyme calibrator to determine relative cell viability.

2.3 photodynamic effect of BPNs/chitosan/PRP temperature-sensitive hydrogel

DCFH and DA were used to detect ROS produced by NIR radiation, the green fluorescence signal was only detected when ROS were produced in the cells the main step was as follows, first, the cells (1 × 10)⁵Cell culture dish) was incubated in a confocal culture dish for 12 h. PBS and BPNs/chitosan/PRP temperature sensitive hydrogels were used to treat cells for 4 hours. After removal of material and washing with PBS, cells were incubated with 20. mu.M DCFH-DA for 40 minutes and washed with 1.0W cm^-2In addition, we further evaluated the production of ROS in BPNs/chitosan/PRP temperature sensitive hydrogels at various times during the near infrared irradiation in 6-well plates (5 × 10) implanted with BPNs/chitosan/PRP temperature sensitive hydrogels⁵/well) and incubated overnight, then the cells were incubated with DCFH-DA for a further 40 minutes followed by irradiation at 60 ℃. The intensity per well at different times (2, 4, 6, 8 minutes) was 1.0W cm^-2. Subsequently, the 6-well plate was washed with PBS, and then a fluorescence image was taken.

2.4 scavenging effect of BPNs/chitosan/PRP temperature-sensitive hydrogel on hyperplasia F L Ss

The present inventors studied the ability of BPNs/chitosan/PRP temperature sensitive hydrogel to clear proliferating synovial tissue in vitro.the fluorescent color generated by the induced live/dead assay was used to differentiate live/dead cells to evaluate the ability of the material to clear proliferating synovial tissue the main steps are as follows, mouse arthritic synovial cells were implanted in 12 well plates for 12h, then they were incubated with BPNs and BPNs/chitosan/PRP temperature sensitive hydrogel, respectively, and replaced with fresh medium after which the cells were divided into two groups.

The present inventors further demonstrated the effect of scavenging proliferating F L Ss by assays for apoptosis and necrosis Annexin V-FITC/PI apoptosis assay for detecting apoptosis and necrosis the procedure was generally as follows, mouse arthritic synovial cells were implanted in 12-well plates and maintained overnight at a density of 5.0x10 per well⁵Cell m L^-1. Subsequently, the cells were treated with BPNs/chitosan/PRP temperature sensitive hydrogel for 24 hours and washed 3 times. Finally, these cells were stained with annexin V-FITC and PI and assayed.

2.5 determination of cell proliferation and activity of BPNs/chitosan/PRP temperature-sensitive hydrogel on MSCs

With the gradual degradation of the black phosphorus nanosheets, the phototherapy effect of the BPNs/chitosan/PRP temperature-sensitive hydrogel is gradually weakened, and oxidized phosphorus substances (P → PxOy) formed by degradation products of the black phosphorus nanosheets can be converted into P-based agents capable of promoting the bone regeneration process in situ, so that the repair of the defective bone tissues is promoted. In this regard, the material was evaluated for the proliferation-inducing ability of MSCs.

(1) Quantitative determination of cell proliferation by enzyme-linked immunosorbent assay

BPNs/chitosan/PRP temperature sensitive hydrogels were placed in 96-well plates and were sterilized by 75% ethanol and UV irradiation, followed by 3 washes with sterile PBS after which 100 μ L MSCs suspensions were implanted into the hydrogels, followed by culturing in an incubator. cell proliferation on the temperature sensitive hydrogels was recorded at predetermined time intervals using the CCK-8 assay.

(2) Confocal laser microscopy

After 12, 24 and 48 hours of culture, the cells were fixed by soaking in 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% triton for 5 minutes, and then stained with fluorescein isothiocyanate-phalloidin to label the cell actin separately. Finally, 1% bovine serum albumin is subjected to sealing treatment for 20 minutes, and the morphology of cells cultured on the BPNs/chitosan/PRP temperature-sensitive hydrogel is observed by using a laser confocal microscope.

2.6 statistical analysis

All numerical data are expressed as mean ± standard deviation. Statistical software SPSS 22.0 was used for the analysis, the two-group comparisons were analyzed using the t-test, and the multi-group comparisons were analyzed using the one-way ANOVA test. A difference is statistically significant if p <0.01 or 0.05: denotes p <0.05, denotes p < 0.01.

3. Analysis of results

3.1 BPNs/chitosan/PRP temperature-sensitive hydrogel for in-vitro cytotoxicity detection of mesenchymal stem cells

The present inventors selected 3 different cells to evaluate biocompatibility in cells, as shown in fig. 4, no matter L929, BMSC or F L Ss cells, although the survival rate of the cells slightly changes with the change of material types, but is not significantly reduced, in the prepared target product BPNs/chitosan/PRP temperature sensitive hydrogel, the survival rate of the different cells is still higher than 85% after 48h of co-culture, thereby indicating that the BPNs/chitosan/PRP temperature sensitive hydrogel has negligible biotoxicity, and providing possibility for further in vivo experiments.

3.2 analysis of ROS production of BPNs/Chitosan/PRP temperature-sensitive hydrogels

DCFH and DA were used to detect ROS production following NIR irradiation. DCFH-DA entering cells will become DCFH, and DCFH can be oxidized by ROS in tissues, giving off green fluorescence. By the fluorescence intensity, we can intuitively judge the generation of ROS. As shown in fig. 5, after exposing the cells to NIR for 8 minutes, the cells treated with BPNs/chitosan/PRP temperature sensitive hydrogel showed strong green fluorescence indicating the presence of large amounts of interstitial ROS. However, the control group did not show significant green fluorescence under the uniform treatment conditions, indicating that no or only a small amount of ROS was produced in the cells. These results indicate that BPNs/chitosan/PRP temperature sensitive hydrogels interact with NIR to produce excellent photodynamic effects.

In order to more accurately verify the influence of different illumination time on the generation of the ROS, the generation condition of the ROS in different NIR illumination time periods is detected. As shown in fig. 6, the green fluorescence intensity increased with the increase of the illumination time, indicating that the amount of ROS produced gradually increased and reached a maximum within 8 minutes.

To assess the effects of photodynamic therapy, cells exposed to NIR light were subjected to CCK-8 detection to assess cell survival. As shown in FIG. 7, the results further illustrate the potential proliferative cell scavenging ability of BPNs/chitosan/PRP temperature sensitive hydrogels.

3.3 cell proliferation of mesenchymal Stem cells in BPNs/Chitosan/PRP temperature sensitive hydrogel

calcein-AM staining and an automatic cell counter were used to assess cell proliferation. As shown in FIG. 8, there was no significant difference in cell proliferation rate between the control group and the BPNs solution group at various times after the calcein AM staining assay. Most notably, however, the density of viable cells in BPNs/chitosan/PRP temperature sensitive hydrogels increased significantly over time. The material type and framework structure of biological materials generally determine the cellular behavior of the material surface. The BPNs/chitosan/PRP temperature-sensitive hydrogel shows more suitable cell adhesion and proliferation, and the cell proliferation is enhanced by adding the PRP component, which is beneficial to the regeneration of bone at the defect part. The above results indicate that the BPNs/chitosan/PRP temperature-sensitive hydrogel shows satisfactory cell affinity and proliferation ability compared with other groups. Furthermore, this further illustrates that moderate photo-thermal does not irreversibly affect the effectiveness of the PRP component in the material.

For in vitro cell adhesion analysis, cell culture studies were performed on BPNs/chitosan/PRP temperature sensitive hydrogels. Fluorescence imaging of cultured cells at 12h, 24h and 48h is shown in FIG. 9, the lower 3 panels are partial enlarged views of the corresponding upper panel, and the cells are uniformly dispersed on BPNs/chitosan/PRP temperature-sensitive hydrogel. Cells on the BPNs/chitosan/PRP temperature-sensitive hydrogel extend out of the pseudopodia and show the potential to extend in all directions, and cells growing in the hydrogel environment can be observed to have a large number of pseudopodia appearing and extending and anchoring on the gel surface. CCK-8 determination results show that the number of BPNs/chitosan/PRP temperature-sensitive hydrogel group cells is in an increasing trend, and the BPNs/chitosan/PRP temperature-sensitive hydrogel has an obvious cell proliferation effect.

In order to evaluate the effect of PRP in cell chemotaxis, PRP with different masses is added into BPNs/chitosan/PRP temperature-sensitive hydrogel, and the BPNs/chitosan/PRP temperature-sensitive hydrogel with the mass-volume ratios of 0.2mg/m L, 0.4mg/m L and 0.6mg/m L are prepared, and the BPNs/chitosan/PRP temperature-sensitive hydrogel is co-cultured with BMSC cells to evaluate whether the addition of PRP has an influence on the chemotaxis of the cells, and as shown in figure 10, the blank control group has a small cell content around the material, while the chemotaxis of the BPNs/chitosan/PRP temperature-sensitive hydrogel is obviously enhanced along with the increase of the PRP concentration, and the cell number is also obviously increased.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: modifications of the technical solutions described in the foregoing embodiments are still possible, or some technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

The application of BPNs/chitosan/PRP temperature-sensitive hydrogel in medicines for treating rheumatoid arthritis through photo-thermal and/or photodynamic.
2. The use of claim 1, wherein the BPNs/chitosan/PRP temperature sensitive hydrogel is made by mixing predominantly BPNs, chitosan temperature sensitive hydrogel and activated PRP.
3. The use of claim 1, wherein the BPNs/chitosan/PRP temperature-sensitive hydrogel comprises 10-40 mg/m L chitosan, 35-45 μ g/m L BPNs and 0.2-0.8 mg/m L PRP, preferably the concentration of chitosan is 16mg/m L, the concentration of BPNs is 42 μ g/m L, and the concentration of PRP is 0.6mg/m L.
4. Use according to claim 1, wherein the chitosan has a degree of deacetylation of 85 to 95%, preferably 90%.
5. A BPNs/chitosan/PRP temperature-sensitive hydrogel is characterized in that the BPNs/chitosan/PRP temperature-sensitive hydrogel is mainly prepared by mixing BPNs, chitosan temperature-sensitive hydrogel and activated PRP.
6. The BPNs/chitosan/PRP temperature-sensitive hydrogel according to claim 1, which comprises 10-40 mg/m L of chitosan, 35-45 μ g/m L of BPNs and 0.2-0.8 mg/m L of PRP, preferably the concentration of chitosan is 16mg/m L, the concentration of BPNs is 42 μ g/m L, and the concentration of PRP is 0.6mg/m L.
7. A preparation method of BPNs/chitosan/PRP temperature-sensitive hydrogel is characterized by mainly comprising the following steps:

(1) carrying out ultrasonic treatment on the black phosphorus crystal, and then centrifuging to obtain BPNs;

(2) dissolving chitosan in an acetic acid solution, and mixing with β -GP aqueous solution to obtain chitosan temperature-sensitive hydrogel;

(3) mixing the BPNs prepared in the step (1) with the chitosan temperature-sensitive hydrogel prepared in the step (2), and adjusting the pH to 7.0-7.5 through β -GP to obtain the BPNs/chitosan temperature-sensitive hydrogel;

(4) and (4) mixing the BPNs/chitosan temperature-sensitive hydrogel prepared in the step (3) with the activated PRP to obtain the preparation of the BPNs/chitosan/PRP temperature-sensitive hydrogel.
8. The method for preparing BPNs/chitosan/PRP temperature-sensitive hydrogel according to claim 7, wherein in the step (1), the ultrasonic treatment conditions are as follows: carrying out ultrasonic treatment for 1-6 h at the power of 400-1200W, preferably, carrying out ultrasonic treatment for 3h at the power of 1200W; the conditions of centrifugation were: centrifuging at 3500-4500 rpm for 5-15 minutes; preferably, centrifugation is carried out at 4000rpm for 10 minutes.
9. The preparation method of the BPNs/chitosan/PRP temperature-sensitive hydrogel as claimed in claim 7, wherein in the step (2), the concentration of the acetic acid solution is 0.1-0.5 mol/L, preferably 0.1 mol/L, the concentration of the β -GP aqueous solution is 0.4-0.8 g/m L, preferably 0.56g/m L, the mass-to-volume ratio of the chitosan to the acetic acid solution is 20-25 mg/m L, preferably 22.2mg/m L, and the mass ratio of the chitosan to β -GP is 1: 2-4, preferably 1: 2.8.
10. The method for preparing BPNs/chitosan/PRP temperature-sensitive hydrogel according to claim 7, wherein in the step (3), the mass-to-volume ratio of the BPNs to the chitosan temperature-sensitive hydrogel prepared in the step (2) is 25 μ g/m L-75 μ g/m L, preferably 50 μ g/m L, in the step (4), the mass-to-volume ratio of the PRP to the BPNs/chitosan temperature-sensitive hydrogel is 0.2-0.8 mg/m L, preferably 0.6mg/m L, and the method for preparing the activated PRP comprises the following steps of activating the PRP by calcium chloride and preparing freeze-dried PRP powder.