CN112843336B - Nanoparticles and gel for promoting dentin regeneration and preparation method and application thereof - Google Patents
Nanoparticles and gel for promoting dentin regeneration and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a nano-particle and a gel for promoting dentin regeneration as well as a preparation method and application thereof, and relates to the technical field of biological medicines. The AZD 2858-CD-chitosan nanoparticle for promoting the regeneration of dentin is prepared by taking diluent of stock solution containing AZD2858 and chitosan as raw materials, and the prepared AZD 2858-CD-chitosan nanoparticle can promote the migration of deciduous papilla stem cells of apical teeth, the differentiation of odontoblasts and the development and regeneration of dental tissues. The invention also prepares AZD 2858-CD-chitosan gel based on the AZD 2858-CD-chitosan nano particles, is a sustained-release preparation, is filled in an experimental animal model, has the activity of promoting dentin regeneration, has obvious superiority in dental pulp tissue engineering, and has huge application market in the development and regeneration of dental tissues.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to nanoparticles and gel for promoting dentin regeneration, and a preparation method and application thereof.
Background
The dental pulp infectious diseases such as the dental pulp disease, the periapical disease and the like are the most common oral diseases, the prevalence rate is high, the affected age span is large, even the loss of teeth can be caused, and the oral cavity and the whole body health of a patient are seriously affected [1] . Currently, the main method for clinical treatment of endodontic and periapical diseases is root canal therapy. Because bacteria can be planted in dentinal tubules and anatomical structures of root canal systemsComplicated and the like, the method has the difficulty of completely removing bacteria in the root canal and completely and tightly filling the root canal. In recent years, the total clinical success rate of root canal therapy is 78-98% [2] . In addition, root canal therapy is an ablative therapy of dental pulp and its outer dentin, and the dental pulp and its outer dentin are closely related anatomically and functionally to constitute a dental pulp-dentin complex, and exert important physiological functions of responding to external stimuli, promoting restorative dentin formation, regulating root development, maintaining homeostasis of teeth, and the like [3] . After the endodontic treatment and the removal of the dental pulp and dentin complex, the tooth has no longer functions of immune protection, external stimulation sensing, self-restoration and the like, and the risk of secondary infection and dental fissure is increased [4] . The root apex of the young permanent tooth is not completely developed, the root canal orifice is open, the wall of the root canal is thin, the root canal therapy can not effectively carry out the root canal preparation and filling, can not promote the normal completion development of the root canal, and the probability of the complications such as too short root of the tooth, tooth breakage and the like is increased, and the long-term curative effect is not ideal [4-5] . Therefore, on the basis of fully understanding the development, regeneration and restoration processes and mechanisms of the dental pulp dentin complex, the dental pulp regeneration treatment aiming at in-situ regeneration of the dental pulp dentin complex with complete tissue structure and normal physiological function in the root canal with strict control of infection by using tissue engineering and regenerative medical means is the development direction and inevitable trend of the treatment of dental pulp infectious diseases, and has become one of the hot points of the oral medical research.
Reference documents:
[1] the material is plain, dental endodontics, fourth edition, beijing: people health press, 2010.
[2]Ng YL,Mann V,Gulabivala K.Tooth survival following non-surgical root canal treatment:a systematic review of the literature.Int Endo J,2010,43(3):171-189.
[3]Kim SG,Zhou J,Solomon C,et al.Effects of growth factors on dental stem/progenitor cells.Dent Clin North Am.2012,56,563-75.
[4] Liu Da Yong, humeilin, pulp regeneration strategy in microenvironment and research progress, journal of International biomedical engineering, 2019,42 (1): 71-77.
[5]Dammaschke T,Steven D,Kaup M,et al.Long-term survival of root-canal-treated teeth:a retrospective study over 10years.J Endod,2003,29(10):638-643.
Disclosure of Invention
In view of the above, the invention aims to provide nanoparticles and a gel for promoting dentin regeneration, and a preparation method and an application thereof, which can improve the accuracy, stability and operability of AZD2858 in inducing the homing of endogenous stem cells, prolong the acting time of AZD2858 in root canals and promote the exertion of biological effects.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of AZD 2858-CD-chitosan nanoparticles for promoting dentin regeneration, which comprises the following steps: mixing a diluent of a stock solution containing AZD2858 with chitosan to obtain the AZD 2858-CD-chitosan nano-particles;
the concentration of AZD2858 in the diluent was 50nM;
the stock solution also comprises a normal saline solution of DMSO and beta-cyclodextrin, and the volume ratio of the normal saline solution of DMSO to the normal saline solution of beta-cyclodextrin is 1 (9-19); the volume percentage of DMSO in the stock solution is 10-5%, and the mass percentage of beta-cyclodextrin in the stock solution is 5-25%;
the mass ratio of the volume of the diluent to the chitosan is 400 mul: 1 to 3mg.
Preferably, the mass percentage of the beta-cyclodextrin in the physiological saline solution of the beta-cyclodextrin is 15-25%.
Preferably, the concentration of AZD2858 in the stock solution is 2.5mM.
Preferably, the preparation method of the stock solution comprises the step of preparing AZD2858 into 2.5mM stock solution by taking a mixed solution as a solvent, wherein the mixed solution comprises DMSO with the volume percentage of 10% and a physiological saline solution of beta-cyclodextrin with the volume percentage of 90%, and the mass percentage of the beta-cyclodextrin in the physiological saline solution of the beta-cyclodextrin is 15-25%.
The invention also provides the AZD 2858-CD-chitosan nano-particles prepared by the preparation method.
The invention also provides AZD 2858-CD-chitosan gel prepared on the basis of the AZD 2858-CD-chitosan nano particles, wherein the mass ratio of the AZD 2858-CD-chitosan nano particles to the matrigel is (3-7): (7-3).
The invention also provides a preparation method of the AZD 2858-CD-chitosan gel, which comprises the following steps: mixing AZD 2858-CD-chitosan nano particles with matrigel to obtain AZD 2858-CD-chitosan gel; the mass ratio of the AZD 2858-CD-chitosan nano particles to the matrigel is (3-7): (7-3).
Preferably, the matrigel comprises Matrix Gel, shellac or pectin.
Preferably, the mass ratio of the AZD 2858-CD-chitosan nanoparticles to the matrigel is 5.
The invention also provides the application of the AZD 2858-CD-chitosan nano-particles or the AZD 2858-CD-chitosan gel in preparing dental tissue development and/or regeneration reagents.
The invention provides a preparation method of AZD 2858-CD-chitosan nanoparticles for promoting dentin regeneration, which comprises the following steps: and (3) mixing the diluent of the stock solution containing the AZD2858 with chitosan to obtain the AZD 2858-CD-chitosan nano-particles. The AZD 2858-CD-chitosan nano-particles prepared by the preparation method can promote the migration of deciduous cells of the root tip teeth, the differentiation of odontoblasts and the development and regeneration of dental tissues. The invention also prepares AZD 2858-CD-chitosan gel based on the AZD 2858-CD-chitosan nano particles, is a slow-release preparation, is filled in an experimental animal model, verifies the activity of promoting dentin regeneration in an animal test, has obvious superiority in dental pulp tissue engineering, and has huge application market in the development and regeneration of dental tissues.
Drawings
FIG. 1 shows the morphology and cumulative release of AZD 2858-CD-chitosan gel, wherein A represents the uniform particle structure of AZD 2858-CD-chitosan nanoparticles with the diameter range of 150-200 nm; b represents AZD 2858-CD-chitosan nanoparticles: the cumulative amount released of AZD2858 at a Matrix Gel ratio of 50;
FIG. 2 shows that the SCAP migration ability of AZD2858 and AZD 2858-CD-chitosan nanoparticles is remarkably improved;
FIG. 3 is a diagram of dental pulp tissue successfully regenerated by AZD 2858-CD-chitosan @ Matrix Gel and human dental pulp stem cells, wherein A represents that dental pulp tissue regeneration is not seen in a blank control group; b shows the result of H & E staining, and AZD 2858-CD-chitosan @ Matrix Gel and human dental pulp stem cells can successfully induce the regeneration of dental pulp tissues.
Detailed Description
The invention provides a preparation method of AZD 2858-CD-chitosan nanoparticles for promoting dentin regeneration, which comprises the following steps: the concentration of AZD2858 in the diluent is 50-150 nM;
the stock solution also comprises a normal saline solution of DMSO and beta-cyclodextrin, and the volume ratio of the normal saline solution of DMSO to the normal saline solution of beta-cyclodextrin is 1 (9-19); the volume percentage content of DMSO in the stock solution is 10-5%, and the mass percentage content of beta-cyclodextrin in the stock solution is 5-25%;
the mass ratio of the volume of the diluent to the chitosan is 400 mu l: 1-3 mg.
Firstly, preparing AZD2858 (3-amino-6- [4- [ (4-methyl-1-piperazinyl) sulfonyl ] phenyl ] -N-3-pyridyl-pyrazinecarboxamide) into a stock solution of 2.5-7.5 mM, wherein the solvent of the stock solution is a mixed solution of a normal saline solution containing DMSO and beta-cyclodextrin; the volume ratio of DMSO to beta-cyclodextrin in the solvent is 1 (9-19), and preferably 1. According to the invention, AZD2858 is dissolved by using the mixed solution and the mixed solution is prepared into 2.5-7.5 mM stock solution, in the embodiment of the invention, the mixed solution is preferably formed by mixing 10% (v/v) DMSO and 90% (v/v) physiological saline solution of beta-cyclodextrin, and the mass percentage of the beta-cyclodextrin in the physiological saline solution of the beta-cyclodextrin is preferably 5-25%. The method for producing the physiological saline solution of β -cyclodextrin of the present invention is not particularly limited, but in the examples, the ratio of β -cyclodextrin to physiological saline is preferably set to 1g:5mL of the beta-cyclodextrin solution is mixed according to the mass-volume ratio to prepare the physiological saline solution of 20 percent of beta-cyclodextrin. The AZD2858 of the present invention is preferably available from MCE reagents (MedChemExpress LLC 50mg package) and the beta-cyclodextrin is preferably available from MCE reagents (MedChemExpress LLC 1g package).
After the stock solution is obtained, diluting the stock solution to 50-150 nM by using the normal saline solution of the beta-cyclodextrin, and mixing the diluted solution with chitosan to obtain the AZD 2858-CD-chitosan nano particles; the volume mass ratio of the diluted solution to the chitosan is 400 mul: 1-3 mg. The preparation method of the diluted solution is not particularly limited in the present invention, and in the present embodiment, 25 μ l of the stock solution is preferably mixed with 975 μ l of the 20% cyclodextrin physiological saline solution to obtain a 50nM diluted solution. The present invention mixes the diluted solution with chitosan, preferably uniformly dispersed every 1.5mg of chitosan in 400. Mu.l of 50nM solution, to obtain a solution containing AZD 2858-CD-chitosan nanoparticles.
The invention also provides the AZD 2858-CD-chitosan nano-particles prepared by the preparation method. According to the AZD 2858-CD-chitosan nanoparticle, AZD2858 is wrapped in CD by utilizing intermolecular hydrogen bonds and intermolecular van der Waals force, and then the AZD 2858-CD-chitosan nanoparticle is loaded into a chitosan particle.
The invention also provides AZD 2858-CD-chitosan gel prepared on the basis of the AZD 2858-CD-chitosan nano particles, wherein the mass ratio of the AZD 2858-CD-chitosan nano particles to the matrigel is (3-7): (7-3).
The AZD 2858-CD-chitosan gel is in a solution state at 4 ℃ and in a gel state at 20-37 ℃, the concentration of AZD2858 in the AZD 2858-CD-chitosan gel is preferably 50nM, and the concentration of chitosan is preferably 3mg/ml.
The invention also provides a preparation method of the AZD 2858-CD-chitosan gel, which comprises the following steps: mixing AZD 2858-CD-chitosan nano particles with matrigel to obtain AZD 2858-CD-chitosan gel; the mass ratio of the AZD 2858-CD-chitosan nano particles to the matrigel is (3-7): (7-3).
The matrigel provided by the invention preferably comprises Matrix Gel, shellac or pectin, and the matrigel is used as a support, and the combined action of the Matrix Gel and a nano drug delivery system is utilized, so that the pharmacokinetic effect of the matrigel can be optimized, and the AZD2858 can be controlled and released. The mass ratio of the AZD 2858-CD-chitosan nanoparticles to the matrigel is preferably 5, and at the moment, the AZD2858 can realize the optimal linear release effect within 3 weeks, and the accumulated release amount exceeds 92%. The source of the matrigel of the present invention is not particularly limited, and conventional commercially available reagents in the art may be used.
The following examples are provided to illustrate the nanoparticles and gel for promoting regeneration of dentin, and the preparation method and application thereof, but they should not be construed as limiting the scope of the present invention.
Example 1
1. Preparation of AZD 2858-CD-chitosan nano-particles
1. 2g of beta-cyclodextrin was added to 10ml of physiological saline to prepare a 20% beta-cyclodextrin physiological saline solution.
2. 2.12mgAZD2858 was weighed and prepared into a 2.5mM stock solution using a mixed solution (10% DMSO +90% physiological saline solution containing 20% beta-cyclodextrin).
3. Mu.l of stock solution + 975. Mu.l of 20% beta-cyclodextrin in physiological saline was diluted to 50nM.
4. 1.5mg of chitosan was weighed and dispersed in 400. Mu.l of 50nM solution to obtain a solution containing AZD 2858-CD-chitosan nanoparticles. Dropping the solution on a mica sheet, volatilizing the solvent, and detecting under a scanning electron microscope, wherein the AZD 2858-CD-chitosan nano particles can be seen to be uniform particle structures with the diameter range of 150-200 nm under the scanning electron microscope (A in figure 1).
2. Preparation of AZD 2858-CD-Chitosan @ Matrix Gel
To the above solution of 400. Mu. LAZD 2858-CD-chitosan nanoparticles (containing AZD 2858-CD-chitosan 60 mg) was added 100. Mu. LMatrix Gel (BD Co., 10 mL) to obtain AZD 2858-CD-chitosan @ Matrix Gel containing 50nM AZD2858 and 3mg/mL chitosan, AZD 2858-CD-chitosan: the mass ratio of Matrix Gel is 37.5: the solution is at 62.5,4 deg.C, and the gel is at 20-37 deg.C.
The formulations were prepared according to the proportions shown in table 1, with different AZD 2858-CD-chitosans: matrix Gel mass ratio, determining the release effect of AZD2858, and finding that the content of AZD 2858-CD-chitosan: at a Matrix Ge of 50, AZD2858 achieved the best linear release within 3 weeks, with cumulative release exceeding 92% (B in fig. 1).
TABLE 1 formulation of AZD2858-CD-chitosan to Matrix Gel
Example 2
Effect of AZD2858 and AZD-CD-Chitosan on the ability to migrate root apical papilla Stem Cells (SCAPs)
In vitro SCAP separation culture according to cell density of 1 × 10 4 The cells/wells are inoculated in the upper chamber of a transwell co-culture system, and nonspecific regulatory bioactive factors Amelogenin (AML), AZD2858 and AZD 2858-CD-chitosan nanoparticles are respectively added into the lower chambers of the co-culture systems of each group. After 24h of culture, the migrated SCAPs were digested, collected and counted, and the results showed that the AZD2858 and AZD-CD 2858-chitosan nanoparticles can significantly promote the migration ability of the SCAPs, and no significant difference was observed between the AZD2858 and AZD 2858-CD-chitosan nanoparticles (FIG. 2).
Example 3
AZD 2858-CD-Chitosan @ Matrix Gel was transplanted into immunodeficient mouse balb/c (purchased from Wintonlihua laboratory animals Co.). The regenerated pulp tissue activity was examined according to the method of Dental pulp engineering with stem cells from ex-folded devious tissue (M.M.Cordeiro, Z.Dong, T.Kaneko et al, "Dental pulp engineering with stem cells from ex-folded devious tissue," Journal of endodynamics, vol.34, no.8, pp.962-969, 2008):
AZD 2858-CD-chitosan @ Matrix Gel and human dental pulp stem cells were implanted back into the hypodermis of immunodeficient mice in a complex manner, and histological observation revealed that dental pulp tissue regeneration was locally visible (FIG. 3).
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (3)
1. An AZD 2858-CD-chitosan gel prepared from AZD 2858-CD-chitosan nanoparticles for promoting dentin regeneration, and a preparation method of the AZD 2858-CD-chitosan gel, comprising the following steps: mixing AZD 2858-CD-chitosan nano particles with matrigel to obtain AZD 2858-CD-chitosan gel; the mass ratio of the AZD 2858-CD-chitosan nano particles to the matrigel is (3 to 7): (7 to 3); the matrigel comprises Matrix Gel, shellac or pectin;
the preparation method of the AZD 2858-CD-chitosan nanoparticle comprises the following steps: mixing the diluent of the stock solution containing AZD2858 with chitosan to obtain the AZD 2858-CD-chitosan nano-particles;
the concentration of AZD2858 in the diluent is 50-150nM;
the stock solution also comprises a normal saline solution of DMSO and beta-cyclodextrin, and the volume ratio of the normal saline solution of DMSO to the normal saline solution of beta-cyclodextrin is 1 (9-19); the volume percentage of DMSO in the stock solution is 5-10%, and the mass percentage of beta-cyclodextrin in the stock solution is 5-25%;
the mass ratio of the volume of the diluent to the chitosan is 400 mu l:1 to 3mg;
the mass percentage of the beta-cyclodextrin in the physiological saline solution of the beta-cyclodextrin is 5 to 25 percent;
the concentration of the AZD2858 in the stock solution is 2.5 to 7.5 mM.
2. The AZD 2858-CD-chitosan gel according to claim 1, wherein the mass ratio of AZD 2858-CD-chitosan nanoparticles to matrigel is 5.
3. Use of the AZD 2858-CD-chitosan gel of claim 1 in the preparation of a dental tissue development and/or regeneration agent.
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