CN113198042A

CN113198042A - Growth factor loaded injectable nanocomposite hydrogel material and construction method and application thereof

Info

Publication number: CN113198042A
Application number: CN202110445726.6A
Authority: CN
Inventors: 王迎军; 苗雅丽; 陈云华
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2021-04-25
Filing date: 2021-04-25
Publication date: 2021-08-03

Abstract

The invention discloses an injectable nano composite hydrogel material loaded with growth factors and a construction method and application thereof. The method utilizes the multi-hydrogen bond interaction among hydrogel structures to carry out physical crosslinking, and combines the black phosphorus nanosheet loaded growth factor to construct the osteoinductive active nanocomposite hydrogel material for promoting angiogenesis. The nano composite hydrogel material has obvious dynamic mechanical properties including self-healing, injectability and the like; the nano composite hydrogel material effectively improves the biological stability of the growth factor and realizes the slow release of the growth factor; the synergistic effect of the black phosphorus nanosheet and the growth factor endows the hydrogel material with excellent osteogenesis and angiogenisis activity, and is expected to be applied to clinical bone defect treatment.

Description

Growth factor loaded injectable nanocomposite hydrogel material and construction method and application thereof

Technical Field

The invention belongs to the field of preparation of biomedical bone repair materials, and particularly relates to an injectable nano composite hydrogel material loaded with growth factors, and a construction method and application thereof.

Background

The damage of the vascular structure prevents the free transport of nutrients, eventually leading to the failure of new bone formation. The blood vessels in the bone play an important role in the transport of nutrients and in circulating paracrine factors. Thus, in addition to osteoinductive factors, Vascular Endothelial Growth Factor (VEGF), which mediates vascular systems, has received considerable attention from many researchers. VEGF can directly stimulate migration and differentiation of human osteoblasts [ Mayr-Wohlfart U, Waltenberger J, Hausser H, et al, vascular endothial growth factor stimulation chemistry of primary human osteograms [ J ]. Bone,2002,30(3):472-7 ]. Although VEGF activity is directly related to bone formation, its use is more likely to be delivered by association with osteoinductive factors to promote regenerative repair of bone tissue.

Two-dimensional Black Phosphorus Nanomaterials (BPNSs) as novel two-dimensional nano active Materials can provide good nucleation sites for in-vivo biomineralization, and biomineralization has important significance for Regeneration and repair of Bone tissues [ Raucci M G, Fassoino I, Caporoli M, et al. For example, Yang et al propose the in situ conversion of degradation products of BPNSs into Phosphorus-based formulations capable of enhancing bone regeneration processes, which may be applied in the medical field of bone regeneration [ Yang B, Yin J, Chen Y, et al, 2D-Black-phosphorous-Reinforced 3D-Printed Scaffolds: A Stepwise Counterminacusupply for osteoaracoma [ J ]. Adv Mater,2018,30(10):1705611 ]. In addition, BPNSs can bind to active molecules or drugs by non-covalent forces such as electrostatic interactions, van der Waals forces, hydrophobic interactions, and pi-pi stacking [ Zeng X, Luo M, Liu G, et al, Polydopamine-Modified Black phosphorus nanocapsules with Enhanced Stability and Photothermal Performance for Tumor Multimodal Treatments [ J ]. Adv Sci (Weinh),2018,5(10):1800510 ]. There has been no report on the research of using BPNSs as VEGF carrier and applying it in the field of bone regeneration repair.

Disclosure of Invention

In order to solve the problems that the growth factors can not exist in the in-vivo environment for a long time and simultaneously give consideration to biological properties such as bone differentiation promoting performance and the like, the invention provides an injectable nano-composite hydrogel material loaded with the growth factors and a construction method and application thereof. After the physical crosslinking type hydrogel is subjected to high-temperature treatment, the nano material loaded with the growth factor is embedded in the physical crosslinking network, so that the self-healing injectable nano composite hydrogel material loaded with the growth factor is constructed.

The object of the invention is achieved by one of the following solutions.

A construction method of an injectable nanocomposite hydrogel material loaded with growth factors comprises the following steps:

(1) adding the growth factor into the nano material dispersion liquid, uniformly mixing, and then incubating to obtain a growth factor-nano material dispersion liquid; the nano material is nano Black Phosphorus (BPNSs);

(2) dissolving deoxyribonucleic acid (DNA), then carrying out high-temperature treatment, and adding the growth factor-nano material dispersion liquid to obtain a prepolymer;

(3) and fully crosslinking the prepolymer to obtain the injectable nanocomposite hydrogel material loaded with the growth factor.

Preferably, the incubation in step (1) is carried out at a temperature of 4-37 ℃ for 6-24 hours.

Preferably, the temperature of the high-temperature treatment in the step (2) is 90-100 ℃ and the time is 1-10 min.

Preferably, in the injectable nanocomposite hydrogel material loaded with the growth factors, the concentration of DNA is 0.03-0.05g/mL, the concentration of the nanomaterial is 50ppm-200ppm, and the concentration of the growth factors is 0.5-3 μ g/mL.

Preferably, the incubation time is 12 hours, and the temperature of the high temperature treatment is 95 ℃.

Preferably, the growth factor in step (1) is Vascular Endothelial Growth Factor (VEGF).

Preferably, the mixing in step (1) is performed at room temperature for 30 to 120 minutes.

Preferably, the DNA of step (2) is derived from sodium salt of deoxyribonucleic acid of salmon testis, Sigma-Aldrich, and has a molecular weight of about 1.3X 10⁶g/mol。

Preferably, the DNA of step (2) is dissolved in Duchen Phosphate Buffer (DPBS) at a temperature of 35-55 ℃.

Preferably, in the step (2), the temperature for adding the growth factor-nano material dispersion liquid is 30-50 ℃; the stirring speed of the high-temperature treatment is 50-200 rpm.

The injectable nanocomposite hydrogel material loaded with the growth factors is obtained by the construction method.

The injectable nano composite hydrogel material loaded with the growth factors is applied to preparation of bone defect medicines.

The invention constructs a physical cross-linked hydrogel network by using hydrogen bond action between DNA polymer base pairs, takes BPNSs as an osteoinductive active component, takes VEGF as an angiogenetic active molecule, constructs a VEGF-loaded DNA nano-composite hydrogel material, and simultaneously considers biological performances of the hydrogel material, such as angiogenetic activity, osteogenic activity and the like.

Compared with the prior art, the invention has the following advantages:

(1) the invention constructs the nano composite hydrogel material with osteogenesis and angiogenisis activity by BPNSs non-covalent physical combination VEGF and embedding in DNA dynamic physical cross-linking network. Not only ensures the long-acting action of the growth factor in the physiological environment, but also considers the biological properties of the hydrogel material, such as osteogenic activity, and the like.

(2) According to the invention, the black phosphorus nanosheet is introduced, so that the effective controlled release of the DNA hydrogel network on the growth factor can be obviously enhanced.

Drawings

FIG. 1 is a TEM picture of BPNSs used in example 1.

FIG. 2 is a TEM picture of VEGF used in example 1.

FIG. 3 is a TEM picture of VEGF-BPNSs prepared in example 2.

FIG. 4 is a rheological diagram of DNA and BP/DNA hydrogel in example 2.

FIG. 5 is a diagram showing a real body of the VEGF-BP/DNA hydrogel prepared in example 3.

FIG. 6 is a photograph of an injection of the VEGF-BP/DNA hydrogel prepared in example 3.

FIG. 7 is a graph of VEGF release from the hydrogel of example 2.

FIG. 8 is a graph showing the results of quantifying cell migration 12 hours after culturing HUVECs in a hydrogel in example 2.

FIG. 9 is a graph showing ALP gene expression levels 7 days after BMSCs cells were cultured in a hydrogel in example 2.

FIG. 10 is a graph showing the expression level of VWF gene after 3 days of hydrogel culture of HUVECs in example 3.

Detailed Description

The invention will be further described with reference to the following examples for better understanding, but the scope of the invention as claimed is not limited to the scope shown in the examples.

Example 1

The method comprises the following steps: preparation of VEGF-loaded BPNSs (VEGF-BPNSs)

(1) Mixing 0.6. mu.g VEGF with 200. mu.L of a 300ppm BPNSs dispersion;

(2) stirring for 30 minutes at room temperature;

(3) incubation was carried out at 37 ℃ for 6 hours to obtain VEGF-BPNSs dispersion. FIG. 1 shows TEM images of the BPNSs used. FIG. 2 is a TEM picture of the VEGF used.

Step two: preparation of VEGF-loaded injectable nanocomposite hydrogel (VEGF-BP/DNA)

(1) 0.036g of DNA polymer (sodium salt of deoxyribonucleic acid from salmon testis, Sigma-Aldrich, molecular weight about 1.3X 10⁶g/mol) is dissolved in 1mL of DPBS, and the mixture is stirred at 50 ℃ until the DNA polymer is fully dissolved, so as to obtain a DNA prepolymer;

(2) carrying out 100 ℃ high-temperature water bath on the DNA prepolymer for 1 minute, quickly transferring the DNA prepolymer to a 40 ℃ constant-temperature water bath kettle, adding 200 mu L of VEGF-BPNSs dispersion liquid, and fully stirring to obtain VEGF-BP/DNA prepolymer;

(3) the prepolymer is transferred into a mold and is left to stand at room temperature for crosslinking for 6 hours to obtain VEGF-BP/DNA (wherein, the concentration of the DNA is 0.03g/mL, the concentration of BPNSs is 50ppm, and the concentration of VEGF is 0.5 mu g/mL), and the injectable nano-composite hydrogel material can be used for delivering VEGF.

Example 2

The method comprises the following steps: preparation of VEGF-loaded BPNSs (VEGF-BP NSs)

(1) 2.4. mu.g of VEGF was mixed with 200. mu.L of 600ppm BPNSs dispersion;

(2) stirring for 60 minutes at room temperature;

(3) incubation was carried out at 4 ℃ for 12 hours to obtain VEGF-BPNSs dispersion. FIG. 3 shows TEM images of the prepared VEGF-BPNSs.

(1) 0.06g of a DNA polymer (sodium salt of deoxyribonucleic acid derived from salmon testis, Sigma-Aldrich, molecular weight about 1.3X 10)⁶g/mol) is dissolved in 1mL of DPBS, and the mixture is stirred at 50 ℃ until the DNA polymer is fully dissolved, so as to obtain a DNA prepolymer;

(2) carrying out high-temperature water bath on the DNA prepolymer at 95 ℃ for 5 minutes, quickly transferring the DNA prepolymer to a constant-temperature water bath kettle at 40 ℃, adding 200 mu L of VEGF-BP NSs dispersion liquid, and fully stirring to obtain VEGF-BP/DNA prepolymer;

(3) the prepolymer is transferred into a mold and is left to stand at room temperature for crosslinking for 6 hours to obtain VEGF-BP/DNA (wherein, the concentration of the DNA is 0.05g/mL, the concentration of BPNSs is 100ppm, and the concentration of VEGF is 2 mu g/mL), and the VEGF-BP/DNA can be used for delivering VEGF injectable nano-composite hydrogel materials.

FIG. 4 is a strain scan curve of the DNA and BP/DNA hydrogel of this example. The storage modulus (G ') of the DNA and the storage modulus (G ') of the BP/DNA hydrogel are both larger than the loss modulus (G "), and the introduction of the BPNSs obviously increases the G ' of the DNA hydrogel, which indicates that the BPNSs can effectively improve the micromechanical property of the DNA hydrogel network.

The invention firstly applies the combination of BPNSs and growth factors to the DNA injectable hydrogel. BPNSs loaded with growth factors are embedded in the DNA dynamic physical cross-linked network, so that the angiogenisis activity and the osteogenesis inducibility of the DNA hydrogel are obviously enhanced, and the problem of short action time of the growth factors in a physiological environment is solved. The incorporation of BPNSs significantly enhanced the effective controlled release of VEGF by the DNA hydrogel network compared to the single component DNA hydrogel (labeled VEGF/DNA in FIG. 7, as opposed to VEGF-BP/DNA prepared in this example, in that no BPNSs were added) (see FIG. 7). FIG. 8 shows the quantitative results of cell migration of Human Umbilical Vein Endothelial Cells (HUVECs) after 12 hours of culture in the VEGF-BP/DNA hydrogel effect medium of this example, and compared with the BP/DNA hydrogel group (labeled as BP/DNA in the figure, the VEGF-BP/DNA prepared in this example is different from the VEGF-BP/DNA prepared in this example in that VEGF is not added), the VEGF-loaded BP/DNA hydrogel can significantly promote the migration of HUVECs, thereby indicating that the BP/DNA hydrogel network can effectively retain the biological activity of VEGF.

FIG. 9 shows the level of mRNA expression of alkaline phosphatase (ALP) gene after 7 days of hydrogel culture of bone marrow mesenchymal stem cells (BMSCs), compared to the DNA hydrogel group (labeled DNA in the figure, different from the VEGF-BP/DNA prepared in this example in that no BPNSs and VEGF are added), the BP/DNA hydrogel (labeled BP/DNA in the figure, different from the VEGF-BP/DNA prepared in this example in that no VEGF is added) can significantly promote the expression of ALP gene of cells, the loading of VEGF further enhances the up-regulation effect of BP/DNA hydrogel on the ALP gene expression of cells, and VEGF and BPNSs synergistically promote the osteogenic differentiation of BMSCs cells.

Example 3

(1) 3.6. mu.g of VEGF was mixed with 400. mu.L of 600ppm BPNSs dispersion;

(2) stirring for 120 minutes at room temperature;

(3) incubating for 24 hours at 25 ℃ to obtain VEGF-BP NSs dispersion liquid;

(1) 0.048g of DNA polymer (sodium salt of deoxyribonucleic acid derived from salmon testis, Sigma-Aldrich, molecular weight of about 1.3X 10)⁶g/mol) is dissolved in 0.8mL of DPBS, and the mixture is stirred at 50 ℃ until the DNA polymer is fully dissolved, so as to obtain a DNA prepolymer;

(2) carrying out high-temperature water bath on the DNA prepolymer at 90 ℃ for 10 minutes, quickly transferring the DNA prepolymer to a constant-temperature water bath kettle at 40 ℃, adding 400 mu L of VEGF-BP NSs dispersion liquid, and fully stirring to obtain VEGF-BP/DNA prepolymer;

(3) the prepolymer is transferred into a mold and is left to stand at room temperature for crosslinking for 6 hours to obtain VEGF-BP/DNA (wherein, the concentration of the DNA is 0.04g/mL, the concentration of BPNSs is 200ppm, and the concentration of VEGF is 3 mu g/mL), and the VEGF-BP/DNA can be used for delivering VEGF injectable nano-composite hydrogel materials.

FIG. 5 is a diagram showing a sample of the VEGF-BP/DNA hydrogel prepared in this example.

FIG. 6 is a photograph showing the injection of the VEGF-BP/DNA hydrogel prepared in this example.

FIG. 10 shows the mRNA expression level of Von Willebrand Factor (VWF) gene after 3 days of hydrogel culture of HUVECs, compared to DNA hydrogel (labeled DNA in the figure, which is different from VEGF-BP/DNA prepared in this example in that BPNSs and VEGF are not added) and BP/DNA hydrogel (labeled BP/DNA in the figure, and is different from VEGF-BP/DNA prepared in this example in that VEGF is not added), the VEGF-BP/DNA hydrogel group of this example significantly up-regulates the expression level of the VWF gene of the cells, and the loading of VEGF significantly enhances the angiogenic differentiation performance of DNA and BP/DNA hydrogel.

Claims

1. A method for constructing an injectable nanocomposite hydrogel material loaded with growth factors is characterized by comprising the following steps:

(1) adding the growth factor into the nano material dispersion liquid, uniformly mixing, and then incubating to obtain a growth factor-nano material dispersion liquid; the nano material is nano black phosphorus;

(2) dissolving DNA, then carrying out high-temperature treatment, and then adding the growth factor-nano material dispersion liquid to obtain a prepolymer;

2. The method of claim 1, wherein the incubation in step (1) is performed at 4-37 ℃ for 6-24 hours.

3. The construction method according to claim 2, wherein the high temperature treatment in step (2) is performed at 90-100 ℃ for 1-10 min.

4. The construction method according to claim 3, wherein the injectable nanocomposite hydrogel material loaded with the growth factor has a DNA concentration of 0.03-0.05g/mL, a nanomaterial concentration of 50ppm-200ppm, and a growth factor concentration of 0.5-3 μ g/mL.

5. The method of claim 4, wherein the incubation time is 12 hours and the temperature of the hyperthermia treatment is 95 ℃.

6. The method according to any one of claims 1 to 5, wherein the growth factor of step (1) is vascular endothelial growth factor.

7. The method for constructing a recombinant DNA construct according to claim 6, wherein the DNA of step (2) is dissolved in Duchen phosphate buffer at a temperature of 35-55 ℃; the DNA is derived from sodium deoxyribonucleate of salmon testis, and has molecular weight of 1.3 × 10⁶g/mol。

8. The method of claim 7, wherein the temperature of the growth factor-nanomaterial dispersion added in step (2) is 30 to 50 ℃.

9. An injectable nanocomposite hydrogel material loaded with growth factors obtained by the construction method according to any one of claims 1 to 8.

10. Use of an injectable nanocomposite hydrogel material loaded with growth factors according to claim 9 for the preparation of a medicament for bone defects.