CN114702681A

CN114702681A - Polymer containing thioketal bond and application thereof in bone tissue repair

Info

Publication number: CN114702681A
Application number: CN202111191563.XA
Authority: CN
Inventors: 苗雷英; 刘超; 禹怡君; 邱心一
Original assignee: NANJING STOMATOLOGICAL HOSPITAL; Nanjing University
Current assignee: NANJING STOMATOLOGICAL HOSPITAL; Nanjing University
Priority date: 2021-08-11
Filing date: 2021-10-13
Publication date: 2022-07-05
Anticipated expiration: 2041-10-13
Also published as: CN114702681B

Abstract

The application discloses a polymer containing a thioketal bond and application thereof in bone tissue repair, wherein the structural formula of the polymer is shown as the formula (t):

wherein m and n are natural numbers, the value range of m is 20-200, and the value range of n is 10-180. Compared with the simple use of antioxidant, the application usesThe polymer can maintain proper ROS level and promote osteogenic differentiation of stem cells after NAC drug loading.

Description

Polymer containing thioketal bond and application thereof in bone tissue repair

Technical Field

The application belongs to the technical field of oral medicine, and particularly relates to a polymer containing a thioketal bond and application thereof in bone tissue repair.

Background

Reactive Oxygen Species (ROS) are oxygen-containing chemically reactive chemicals, small molecules derived from oxygen, such as peroxides, superoxides, hydroxyl radicals, singlet oxygen, and alpha-oxygen. These small molecules can react with various chemicals (e.g., proteins, lipids, sugars, and nucleic acids) and participate in normal life activities. An oxidation/antioxidation system exists in the organism, and the ROS level of the organism is maintained to be stable. Abnormal concentrations of ROS can disrupt the redox homeostasis in the organism, causing damage to the intracellular antioxidant defense system, resulting in oxidative stress. When exposed to oxygen free radicals for a long time, biological membranes, DNA, proteins and lipids of organisms are damaged, and the structures and functions of intracellular proteins, lipids and nucleic acids are seriously damaged, so that diseases such as cancer, stroke, arteriosclerosis, inflammation and the like are generated. However, too low ROS will affect intracellular signal transduction and osteogenic differentiation of stem cells. Thus modulating ROS levels may also be targeted for disease treatment.

In diseases where there is a high concentration of ROS, one potential approach to remediating damage from oxidative stress is to use exogenous ROS scavengers such as N-acetylcysteine (NAC), resveratrol, vitamin C, etc., however these compounds are generally significantly cytotoxic and, due to their low molecular weight, are readily metabolized by the kidneys quickly. In addition, the low molecular weight compounds are not distributed in a specific region but spread throughout the body, resulting in low utilization thereof. These disadvantages limit their therapeutic effectiveness in some oxidative stress related diseases. And eliminating all intracellular ROS is detrimental to tissue repair.

One promising strategy to address the above drawbacks is to deliver low molecular weight drugs using drug delivery systems. To date, many promising drug delivery systems using drug-loaded liposomes and polymeric micelles are in clinical and preclinical research. However, an intelligent drug response system capable of responding to ROS and accurately delivering drugs is required in the present stage to cope with the environment of oxidative stress and to regulate ROS at an appropriate level. The invention is achieved accordingly.

Disclosure of Invention

The application provides a polymer containing a thioketal bond, which can load a drug active ingredient, the surface of the polymer is a hydrophilic PEG chain segment and can effectively prolong the in vivo storage time of nanoparticles, the inner core of the polymer is a hydrophobic PCL chain segment, the thioketal bond is broken under the high level of ROS in an inflammation environment, the drug-loaded microspheres are converted into an extended state from a hydrophobic crouched state, the drug active ingredient in the microspheres is released therewith, the targeted accumulation of the drug active ingredient at an inflammation part is realized, and therefore, the polymer can maintain the appropriate ROS level and promote the osteogenic differentiation of stem cells, and has a remarkable application prospect in the aspect of bone tissue repair.

In order to solve the technical problem in the prior art, the technical scheme adopted by the application is as follows: provided is a sulfur-containing polymer, which is characterized in that the structural formula of the sulfur-containing polymer is shown as the formula (t):

wherein m and n are natural numbers, the value range of m is 20-200, and the value range of n is 10-180.

In the preferable sulfur-containing polymer, the value range of m is 20-70, and the value range of n is 30-180.

Preferably, the weight average molecular weight of the sulfur-containing polymer is 3000-31000 Da.

Another object of the present invention is to provide a method for preparing the sulfur-containing polymer, which comprises the steps of:

(S1) amino-protected Compound of formula (f)

Acylation reaction with functional group modified polycaprolactone to produce polymer of formula (i)

Wherein n is a natural number, and the value range of n is 10-180;

(S2) reacting the compound of formula (i) to produce a polymer of formula (j)

(S3) reacting the polymer of formula (j) with a functional group-modified polyethylene glycol to produce a polymer of formula (t);

in the reaction, m and n are natural numbers, the value range of m is 20-200, and the value range of n is 10-180.

In a preferred embodiment, the reaction in step (S1) is carried out at 25 ℃, and the reaction solvent is Dichloromethane (DCM), which contains DIEA.

In a preferred embodiment, the process wherein the compound of formula (f) is obtained by:

(S11) acylation of 2-aminoethanethiol with ethyl trifluoroacetate under basic conditions to produce the compound of formula (c)

(S12) reacting the compound of formula (c) with 2-methoxypropene to produce a compound of formula (e)

(S13) subjecting the compound of formula (e) to a hydrolysis reaction under basic conditions to produce a compound of formula (f)

In the preferred technical scheme, amino protection is carried out by using di-tert-butyl carbonate under the alkaline condition.

In a preferred technical scheme, the alkaline environment is selected from the condition that appropriate amount of diethylamine, triethylamine, diisopropylethylamine, pyridine, quinoline, potassium carbonate, sodium carbonate, cesium carbonate, potassium hydroxide and sodium hydroxide exist.

Typically, the compound of formula (f) is subjected to an amino protection reaction with di-tert-butyl carbonate anhydride under basic conditions to give the compound of formula (h).

The invention also aims to provide the application of the sulfur-containing polymer as a drug carrier.

The invention also aims to provide a drug-loaded nanoparticle carrier, and the material of the drug-loaded nanoparticle carrier is the sulfur-containing polymer.

It is still another object of the present invention to provide a pharmaceutical composition, wherein the pharmaceutical composition comprises:

a suitable drug-loaded nanoparticle carrier;

a pharmaceutically effective amount of a pharmaceutically active ingredient.

Preferably, the pharmaceutical active ingredient is selected from one or more of N-acetylcysteine (NAC), resveratrol, and vitamin C.

The invention also aims to provide application of the pharmaceutical composition in preparing an anti-oxidation therapeutic drug for oxidative stress environments.

Preferably, the antioxidant therapeutic drug is a drug for bone tissue repair.

Being different from the prior art situation, the beneficial effect of this application is:

the sulfur-containing polymer obtained by the method can be used as a good carrier of active ingredients of medicines, and has the advantages of good biocompatibility, good biodegradability, low toxicity, good stability, long in-vivo circulation time, strong targeting and the like. When loaded with PCL drug, the PCL drug can maintain proper ROS level and promote osteogenic differentiation of stem cells compared with the antioxidant which is used singly. When the PCL drug is loaded, the drug can respond to ROS in the environment, and the purposes of releasing the drug as required and storing the drug when not required are achieved.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the description of the embodiments are briefly introduced below, it is obvious that the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without inventive efforts, wherein:

FIG. 1 is a representation of PssL NPs and PssL-NAC NPs of the present application. (A) 13C NMR spectrum of PEG-ss-PCL; (B) 1H NMR spectrum of PEG-ss-PCL. (C) TEM images of PssL NPs. (D) TEM images of PssL-NAC NPs. (E) TEM images of PssL-NAC NPs after ROS treatment. (F) GPC images of PssL NPs; (G) GPC images of PssL NPs treated with ROS. (H) A DLS image.

FIG. 2 is a graph showing the results of CCK-8 of the present application.

FIG. 3 is a ROS regulation and apoptosis rate assay for PssL-NAC treatment of the present application. (A) CLSM images of hPDLSCs were incubated in three groups (Control, NAC, PssL-NAC NPs) and at different LPS concentrations (0, 1, 5, 10 and 20. mu.g/ml), with a scale bar of 100. mu.m. The excitation wavelength was 488 nm. (B) Flow cytometry measured the Mean Fluorescence Intensity (MFI) of the three groups. . (C) LPS at various concentrations (0, 5, 10. mu.g/ml) treated the apoptotic rate of hPDSCs in the presence or absence of NAC and PssL-NAC.

FIG. 4 shows that different concentrations of LPS (0, 5, 10. mu.g/ml) induced osteogenic differentiation of hPDLSCs in the absence of NAC and PssL-NAC. (A) ALP activity. (B) BMP-2mRNA expression. (C) Runx 2mRNA expression. (D) Expression of PKA mRNA. (E) BMP-2, Runx2, PKA protein expression. (F) Alizarin red S staining image with scale of 100 μm. (G) And F, quantitative mineralization analysis. NAC had no significant effect on the number of mineralized nodules, whereas PssL-NAC promoted the generation of mineralized nodules.

FIG. 5 is a graph of rat Micro-CT images after 1 and 4 weeks of NAC and PssL-NAC treatment according to the present application. ((A-H) -1) maxillary second molar buccal palatal sectional image. ((A-H) -2) three-dimensional reconstruction of digitized images.

Fig. 6 shows the distance between the 6 sites CEJ-ABC of maxillary second molars of different groups analyzed by Micro-CT in the present application.

Figure 7 is a histological analysis of the maxillary second molar treatment of the present application after 1 week. (A-D) -1 and (A-D) -2 correspond to the red and black boxes in A-D. ((A-D) -3) representative TRAP images. 500 μm for A-D scale bar and 100 μm for A-D (1-3).

Figure 8 is a histological analysis of the maxillary second molars of the present application after 4 weeks of NAC treatment. (A-D) -1 and (A-D) -2 correspond to the red and black boxes in A-D. ((A-D) -3) representative TRAP images. The green squares in B-2 represent abscesses. 500 μm for A-D scale bar and 100 μm for A-D (1-3).

FIG. 9 is Masson's trichrome staining of the maxillary second molars 4 weeks after NAC and PssL-NAC treatment of the present application. A-D was 500 μm and E-H was 100. mu.m.

FIG. 10 is a histopathological image of the heart, liver, spleen, lung, and kidney of the present application. Scale bar 50 μm.

FIG. 11 is a scheme showing the synthesis of sulfur-containing polymers of the present application.

Detailed Description

The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

In order to meet the requirement of regulating ROS in organisms to be at a proper level, the invention provides a sulfur-containing polymer and drug-loaded nanoparticles thereof. The structural formula of the polymer is shown as the formula (t):

wherein m and n are natural numbers, the value range of m is 20-200, and the value range of n is 10-180. Wherein Polycaprolactone (PCL) is a hydrophobic chain segment, and polyethylene glycol (PEG) is a hydrophilic chain segment.

The PEG-bind-PCL amphiphilic polymer design system is adopted, and aiming at ROS increase in a local environment in a disease process, the thioketal bond is designed to connect PEG and PCL, and the hydrophobic end of the PCL and the hydrophilic end of the PEG are connected through the thioketal bond. The nano drug-loaded microspheres internally coated with the oil-soluble antioxidant NAC can be obtained in a water-phase oil-phase self-assembly mode. Under the condition of no inflammation environment ROS low level, the surface of the drug-loaded nanoparticle is provided with a hydrophilic PEG chain segment, the in-vivo storage time of the nanoparticle can be effectively prolonged, the inner core of the drug-loaded nanoparticle is provided with a hydrophobic PCL chain segment, under the condition of inflammation environment ROS high level, the thioketal bond is broken, the drug-loaded nanoparticle is converted from a hydrophobic crouched state into an extended state, NAC in the microsphere is released, and the targeted accumulation of NAC at an inflammation part is realized. The nano drug-loading system with the PEG protective outer layer and ROS responsiveness can prolong the storage time, enhance the NAC targeting selective delivery capacity and effectively improve the delivery efficiency of nanoparticles. The PssL-NAC microspheres can be used as a novel drug delivery vehicle with definite ROS responsiveness and excellent biocompatibility, and a system can carry out drug release in a third self-regulation activation mode to maintain the ROS level in the environment at a certain proper level.

In the invention, polyethylene glycol (PEG) is a polyether high molecular compound which has neutral pH, no toxicity, high water solubility and wide application, and a repeating unit of the PEG is oxyethylene and is in a linear or branched chain structure. The polyethylene glycol polymer is the polymer with the lowest protein and cell absorption level in the known polymers so far, and can be dissolved in tissue fluid in vivo and quickly eliminated from the body without generating any toxic and side effects. Polyethylene glycol has been approved by the FDA as a polymer for in vivo injection due to its non-toxicity and good biocompatibility. Due to the characteristics, biocompatibility, safety, and few foreign body reactions, polyethylene glycol is widely used in biological medicine. In the pharmaceutical industry, polyethylene glycol can be used as a pharmaceutical excipient to improve various properties of the drug, such as dispersibility, film-forming properties, lubricity, sustained release, and the like. In the synthesis and modification of novel biomaterials, polyethylene glycol as part of the material will impart new properties and functions to the material, such as hydrophilicity, flexibility, anticoagulation, anti-macrophage phagocytosis, etc.

Polycaprolactone (Poly (-caprolone), PCL) is a synthetic polyester biopolymer material, is biodegradable, has good biocompatibility, is a hydrophobic polymer with a long-acting degradation mechanism, and the final degradation product of the polycaprolactone is absorbed by the organism and excreted to the outside of the body, is widely applied to a control system of the sustained-release microspheres, and can obtain satisfactory drug release behavior. The crystallinity of the polycaprolactone is strong, the degradation is slow, and the degradation in vivo is divided into 2 steps: step 1, the molecular weight is continuously reduced, but the material is not deformed and weightless; and step 2, after the molecular weight is reduced to a certain value, the material begins to be changed into fragments and weightlessness, and finally the fragments can be absorbed and excreted by the body without accumulating in the body, so that the polycaprolactone can be used as a drug release carrier material to be applied to the body. The embedding rate of hydrophilic substances can be increased by blending polycaprolactone with other materials, and the drug release speed is changed; obviously change the drug release mechanism and the like. In addition, the polycaprolactone has good flexibility, slow degradation speed, good film forming property, mechanical property and biocompatibility, and easily obtained raw materials, can be prepared into polycaprolactone porous films, can be used for postoperative anti-adhesion films, tissue engineering scaffold materials and the like, and has wide application in tissue engineering.

The nanoparticles of the present invention preferably have a particle size of 50 to 100 nm. The particle size of the drug-loaded nanoparticle is preferably 50-200 nm.

Polyethylene glycol (PEG) and Polycaprolactone (PCL) have good biocompatibility and are typically designed as the hydrophilic and hydrophobic ends, respectively, of an amphiphilic diblock copolymer. The thioketal bond obtained by the invention has ROS response characteristics and can be applied to ROS corresponding materials. The polyethylene glycols of the invention have a weight-average molecular weight Mw, determined in accordance with DIN55672-1, of from ≥ 500 to ≤ 20000g/mol, more preferably from ≥ 2000 to ≤ 15000g/mol, even more preferably from ≥ 3000 to ≤ 12000g/mol, most preferably from ≥ 4000 to ≤ 10000g/mol, in particular from ≥ 4000 to ≤ 8000 g/mol.

Another aspect provided herein is a pharmaceutical composition comprising a therapeutically effective amount of a pharmaceutically active ingredient and a sulfur-containing polymer of the invention as a drug-loaded carrier. In some embodiments of such pharmaceutical compositions, the pharmaceutical composition is formulated for intravenous administration, intravitreal administration, intramuscular administration, oral administration, rectal administration inhalation, intranasal administration, topical administration, ophthalmic administration, or otic administration. In other embodiments, the pharmaceutical composition is in the form of a tablet, pill, capsule, liquid, inhalant, nasal spray solution, suppository, solution, emulsion, ointment, eye drop, or ear drop. In other embodiments, the pharmaceutical composition further comprises one or more additional therapeutic agents.

An "effective amount" or "therapeutically effective amount" as used herein refers to an amount of a compound described herein administered sufficient to alleviate to some extent one or more of the symptoms of the disease or disorder being treated. The result can be a reduction and/or alleviation of the signs, symptoms, or causes of a disease or any other desired change in a biological system. For example, an "effective amount" for treatment is the amount of a composition comprising a compound disclosed herein that is required to provide a clinically significant reduction in the symptoms of a disease. In any individual case, techniques (e.g., dose escalation studies) may be employed to determine an appropriate "effective" amount.

The effective dosage of the pharmaceutically active ingredient employed may vary with the drug employed, the mode of administration and the severity of the condition being treated. However, in general, satisfactory results are obtained when the pharmaceutically active ingredient of the invention is administered at a dose of about 0.5-500mg/kg animal body weight per day, preferably 2-4 divided doses per day, or in a sustained release form. For most large mammals, the total daily dose is from about 1 to about 100mg, preferably from about 2 to about 80 mg. A dosage form suitable for oral administration comprising about 0.5 to 500mg of a pharmaceutically active ingredient in intimate admixture with a solid or liquid pharmaceutically acceptable carrier. This dosage regimen may be adjusted to provide the best therapeutic response. For example, divided doses may be administered several times per day, or the dose may be proportionally reduced, as may be required by the urgency of the condition being treated.

The pharmaceutically active ingredients can be administered orally, as well as intravenously, intramuscularly or subcutaneously. The solid support comprises: starch, lactose, dicalcium phosphate, microcrystalline cellulose, sucrose and kaolin, and liquid carriers include: sterile water, nonionic surfactants, and edible oils (e.g., corn, peanut and sesame oils) as are appropriate to the nature of the active ingredient and the particular mode of administration desired. Adjuvants commonly used in the preparation of pharmaceutical compositions may also advantageously be included, for example flavouring agents, colouring agents, preservatives and antioxidants such as vitamin E, vitamin C, BHT and BHA.

Preferred pharmaceutical compositions are solid compositions, especially tablets and solid-filled or liquid-filled capsules, from the standpoint of ease of preparation and administration. Oral administration of the compounds is preferred.

The pharmaceutically active ingredients may also be administered parenterally or intraperitoneally. Solutions or suspensions of these pharmaceutically active ingredients (as the free base or pharmaceutically acceptable salt) can also be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquids, polyethylene glycols and mixtures thereof in oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injection include: sterile aqueous solutions or dispersions and sterile powders (for the extemporaneous preparation of sterile injectable solutions or dispersions). In all cases, these forms must be sterile and must be fluid to facilitate the syringe to expel the fluid. Must be stable under the conditions of manufacture and storage and must be resistant to the contaminating effects of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, alcohols (for example, glycerol, propylene glycol and liquid polyethylene glycols), suitable mixtures thereof and vegetable oils.

The invention aims to prepare a drug-loaded nanoparticle capable of responding ROS for antioxidant treatment of oxidative stress environment.

The invention comprises drug-free PEG-ss-PCL nanoparticles (PssL NPs for short) and NAC-loaded PssL-NAC nanoparticles (PssL-NAC NPs for short). PssL-NAC is better able to maintain appropriate ROS levels and promote osteogenic differentiation of stem cells than antioxidant alone. Compared with other medicine carrying systems, the PssL-NAC can respond to ROS in the environment, and the purposes of releasing the medicine as required and storing the medicine when not required are achieved.

The synthetic route for the sulfur-containing polymers of the present invention is shown in FIG. 11.

EXAMPLE 1PEG-ss-PCL preparation

In a rare gas (Ar) atmosphere, compound a (2-aminoethanethiol, 8.0g,103.69mmol,1.0equiv) and trimethylamine (TEA,15.74g,155.5mmol,1.5eq) were dissolved in methanol (100ml) and stirred at room temperature. Then, compound b (ethyl trifluoroacetate, 17.67g,124.42mmol,1.2eq) was added, stirred at room temperature overnight, extracted with ethyl acetate (3X 100ml), and the combined organic layers were dried over anhydrous sodium sulfate under reduced pressure. Purification by silica gel column chromatography gave compound c (12.00g,69.30mmol, 67%). Nuclear magnetic resonance detection results:¹H NMR(500MHz,CDCl₃)δ:7.06(s,1H),3.53(q,2H)2.73(m,2H),1.42(t,1H).¹³C NMR(126MHz,CDCl₃)δ:157.60,CF₃(119.19,116.90,114.61,112.33),42.54,23.67.¹⁹F NMR(471MHz,CDCl₃)δ:-75.96。

compound c (11.72g,67.65mmol,2.5 equivolume) and p-toluenesulfonic acid monohydrate (PTSA) (1.54g,8.12mmol,0.3eq) were dissolved in 100ml of Tol under Ar. Stirring for 10min, adding 100g molecular sieve

Stirring for another 10 min. After addition of compound d (2-methoxypropene, 1.95g,27.06mmol,1.0eq), the mixture was stirred at room temperature for 24 h. The crude product was purified by silica gel column chromatography to give compound e as a white solid (8.41g,21.77mmol, 80%). Nuclear magnetic resonance detection results:¹H NMR(500MHz,CDCl₃)δ:7.05(s,2H),3.59(q,4H),2.83(t,4H),1.61(s,6H).¹³C NMR(126MHz,CDCl₃)δ:157.66,157.36,CF₃(119.20,116.91,114.62,112.34),56.52,39.39,29.27.¹⁹F NMR(471MHz,CDCl₃):δ-75.94。

compound e (2.00g,5.17mmol) was dissolved in 20ml of 6M aqueous NaOH under Ar, stirred at room temperature for 4h, then extracted with dichloromethane (DCM, 4X 50 ml). The organic layer was dried over anhydrous sodium sulfate under reduced pressure. Compound f was an amber oil (0.95g,4.91mmol, 95%). Nuclear magnetic resonance detection results:¹H NMR(500MHz,CDCl₃)δ:2.88(t,4H),2.70(t,4H),1.58(s,6H),NH₂ 1.31(s,4H).¹³C NMR(126MHz,CDCl₃)δ:55.79,41.77,34.63,31.28。

compound f (0.90g,4.63mmol,1.2eq) was dissolved in chloroform (20 ml) under Ar and dried under stirring for 15 minutes, then in an ice bath, the solution of the above solution was added drop by drop compound g (di-tert-butyl carbonate anhydride, 0.84g,3.86mmol,1.0eq) in dry chloroform (10 ml) at 0 ℃. The reaction mixture was stirred at 0 ℃ for 4h and then at room temperature overnight. The solid was removed by filtration and the solvent was removed under reduced pressure. Purification by column chromatography gave compound h as butter (0.85g,2.89mmol, 75%).¹H NMR(500MHz,CDCl₃)δ:5.00(d,1H),3.32(t,3H),2.91(t,3H),2.74(q,4H),1.91(s,2H),1.59(s,3H),1.58(s,3H),1.42(s,9H).¹³C NMR(126MHz,CDCl₃)δ:155.77,79.34,41.33,40.13,34.04,31.16,31.10,30.77,28.40。

A dry Dichloromethane (DCM) (50ml) PCL-COOH (5.00g,0.33mmol,1.0eq) and N, N-Dipropropylamine (DIEA) (129.3mg,0.99mmol,3.0eq) were stirred at 0 deg.C for 10min under Ar. Then N-hydroxyuccinimide (nhs) (57.5mg,0.50mmol,1.5eq), and N- (3-methylenepropyl) -N' -ethyl carbonate diimide hydrochloride (EDC) (95.8mg,0.5mmol,1.5eq) and compound h (98.2mg,0.33mmol,1.0eq) were added to the reactor at 0 ℃. Stir at room temperature for 48 h, rinse with water (3X 20 ml). The organic layer was dried over anhydrous sodium sulfate under reduced pressure to give compound i (4.05g, 80%) as a white solid.¹H NMR(500MHz,CDCl₃)δ:4.03(t),2.28(t),1.62(m),1.36(m).¹³C NMR(126MHz,CDCl₃)δ:173.73,173.53,64.13,62.56,53.46,34.11,32.32,31.10,28.41,28.34,25.52,25.30,24.68,24.57。

Compound i (4.00g) was dissolved in dry DCM, stirred at RT, then trifluoroacetic acid (TFA) (2ml) was added, stirred at RT for 2h and the solvent removed under reduced pressure to give the crude product. The crude product was dissolved in DCM (50ml) and NaHCO₃(aq) (2X 20ml) and water (2X 20 ml). The organic layer was dried over anhydrous sodium sulfate under reduced pressure to give compound j as a white solid (3.20g, 80%).¹H NMR(400MHz,CDCl₃)δ:4.03(t),2.27(t),1.62(m),1.34(m).¹³C NMR(101MHz,CDCl₃)δ:173.51,64.12,34.10,28.33,25.51,24.56。

Dry DCM (50ml) PEG-COOH (2.00g,0.20mmol,1.0eq) and DIEA (78.4mg,0.99mmol,3.0eq) were stirred at 0 ℃ for 10min under Ar. Then NHS (46.0mg,0.40mmol,2.0eq), EDC (76.6mg,0.40mmol,2.0eq) and compound j (3.00g,0.20mmol,1.0eq) were added to the reactor at 0 ℃. Stir at room temperature for 48 hours, rinse with water (3 × 20 ml). Anhydrous Na for organic layer₂SO₄Drying under reduced pressure gave a terminal white solid (t) (3.90g, 78%).¹H NMR(500MHz,CDCl₃)δ:4.02(t),3.60(s),2.26(t),1.60(m),1.34(m).¹³C NMR(126MHz,CDCl₃) 173.50,70.55,64.11,34.09,28.32,25.51,24.55.(A) 13C NMR spectrum of PEG-ss-PCL; (B) 1H NMR spectrum of PEG-ss-PCL. The obtained polymer t was named PEG-ss-PCL amphiphilic polymer (abbreviated as PssL).

EXAMPLE 2 characterization of Sulfur-containing Polymer nanospheres (PssL NPs) and preparation of drug-loaded nanospheres (PssL-NAC NPs)

N-acetylcysteine (NAC, A7250, Sigma-Aldrich, China) was embedded in PssL polymer carrier obtained as described above. That is, PssL (50mg) was dissolved in deionized water (50 ml). To the above suspension was added a dichloromethane suspension (5ml) containing NAC (10mg), and the mixture was sonicated for 5 min. And (3) after 30min of ultrasonic treatment, centrifuging, washing for 5 times by using deionized water, removing residues, and collecting the nano drug-loaded microspheres (PssL-NAC NPs).

1H NMR and 13C NMR were performed on a Bruker AV400 spectrometer with CDCl3 as solvent and tetramethylsilane as internal standard. The particle size and particle size distribution of the PEG ss PCL and PssL NAC NPs in the whole cell culture media was evaluated by dynamic light scattering (brueck hein instruments, usa). The morphology of the PEG ss PCL and PssL NAC NP was examined using a transmission electron microscope (TEM, JEOL TEM-100). The molecular weight and polydispersity of the PEG-ss-PCL were determined by Gel Permeation Chromatography (GPC). The number average molecular weight (Mn), weight average molecular weight (Mw) and polydispersity index (PDI, Mw/Mn) were determined using polystyrene as a reference.

Cleavage of the thioketal bond in PEG-ss-PCL after hydrogen peroxide treatment was analyzed using a Malvern gel permeation chromatography (Viscotek GPC/SEC, Malvern, USA) system. Pure Tetrahydrofuran (THF) was used as the mobile phase. The temperature was fixed at 35 ℃ and the flow rate was set at 1.0 ml/min. PssL was injected through a Malvern autosampler and polystyrene (PSt) was used as a standard sample measurement. The relative molecular weights were analyzed by OmniSEC.

Transmission Electron Microscopy (TEM) was used to observe the size and morphology of the nanoparticles, Gel Permeation Chromatography (GPC) was used to observe the molecular weight of the nanoparticles, and dynamic light Diffraction (DLS) was used to observe the hydrated particle size of the nanoparticles.

As shown in FIG. 1, FIG. 1 is a representation of PssL NPs and PssL-NAC NPs. (A) 13C NMR spectrum of PEG-ss-PCL; (B) 1H NMR spectrum of PEG-ss-PCL. (C) TEM images of PssL NPs. (D) TEM images of PssL-NAC NPs. (E) TEM images of PssL-NAC NPs after ROS treatment. (F) GPC images of PssL NPs; (G) GPC images of PssL NPs treated with ROS. (H) A DLS image.

After the successful synthesis of PssL NPs, as an amphiphilic polymer, PssL can self-assemble into nanoparticles, which then serve as a powerful drug carrier for NAC. The morphology of PssL-NPs and PssL-NAC-NPs was observed by transmission electron microscopy. As shown in FIG. 1C, PssL NPs exhibit uniformly dispersed spherical morphology with dimensions of about 80 nm. After packaging the NAC, the PssL NAC NPs are slightly larger in size, about 120nm, as shown in FIG. 1D. However, upon exposure to ROS, the spherical morphology of PssL NAC NPs would be disrupted (fig. 1E). As shown in FIGS. 1F-G, the molecular weight decreased and the retention time on the gel column became longer after ROS treatment, indicating thioketal bond cleavage and polymer failure. In the Dynamic Light Scattering (DLS) results (PEG-PCL-NAC i.e., PssL NAC NPs, PEG-PCL i.e., PssL NPs in FIG. 1H), PssL NAC NPs (. about.155 nm) showed a slightly larger hydrodynamic diameter than PssL NPs (. about.102 nm), which showed similar results as TEM. There are three peaks for PssL-NAC-NPs after they participate in the ROS reaction, indicating the cleavage of ROS-reactive thioketal bonds and the cleavage of amphiphilic polymers.

Example 3 biocompatibility of PssL-NAC NPs

Cells were seeded overnight in 96-well plates. After incubation for 1,3, 5 and 7 days with medium containing 20. mu.g/ml NAC and the corresponding volume ratio (10% v/v) of PssL-NAC, the biocompatibility was determined by the CCK-8 method.

FIG. 2 shows the results of CCK-8. The results show that: compared with a control group, NAC and PssL-NAC have no obvious influence on cell proliferation and have better biocompatibility.

Example 4 ROS scavenging Capacity of PssL-NAC NPs

Intracellular ROS are marked by using a DCFH-DA fluorescent probe, and the condition of the intracellular ROS in each group is observed by a confocal microscope and a flow cytometer. Detecting the apoptosis condition of the cells by using an Annexin V-FITC/PI apoptosis detection kit, and observing by using a flow cytometer.

FIG. 3: modulation of ROS and determination of apoptosis rate by PssL-NAC treatment. (A) CLSM images of hPDLSCs were incubated in three groups (Control, NAC, PssL-NAC NPs) and at different LPS concentrations (0, 1, 5, 10 and 20. mu.g/ml), with a scale bar of 100. mu.m. The excitation wavelength was 488 nm. (B) Flow cytometry measured the Mean Fluorescence Intensity (MFI) of the three groups. . (C) LPS at various concentrations (0, 5, 10. mu.g/ml) treated the apoptotic rate of hPDSCs in the presence or absence of NAC and PssL-NAC.

The results show that: (1) intracellular ROS are significantly elevated after LPS stimulation, NAC regulates cellular ROS to control levels, and PssL-NAC NPs regulate intracellular ROS to twice control levels. (2) Both NAC and PssL-NAC reduced LPS-induced apoptosis, and PssL-NAC retained 2-fold control levels of ROS without promoting apoptosis.

Example 5 Effect of PssL-NAC NPs on osteogenic differentiation of Stem cells

Extracting cell RNA by a Trizol method, and analyzing the expression condition of an osteoblast marker gene in the cell by PCR. Extracting intracellular protein, and detecting the expression condition of the osteogenic marker protein by using a Western Blot method. After 7 days of cell culture, alkaline phosphatase expression was detected, alizarin red staining was performed, the number of mineralized nodules was observed, and semi-quantitative results were analyzed.

FIG. 4: different concentrations of LPS (0, 5, 10. mu.g/ml) induced osteogenic differentiation of hPDLSCs in the absence of NAC and PssL-NAC. (A) ALP activity. (B) BMP-2mRNA expression. (C) Runx 2mRNA expression. (D) Expression of PKA mRNA. (E) BMP-2, Runx2, PKA protein expression. (F) Alizarin red S staining image with scale of 100 μm. (G) And F, quantitative mineralization analysis. NAC had no significant effect on the number of mineralized nodules, whereas PssL-NAC promoted the generation of mineralized nodules.

The results show that: (1) PssL-NAC promoted ALP activity and osteogenic marker expression better than NAC. (2) NAC had no significant effect on the number of mineralized nodules, whereas PssL-NAC promoted the generation of mineralized nodules.

Example 6 in vivo experiments

Male SD rats 32, weighing about 240g, were divided into 4 groups of 8 rats each. Rats were anesthetized with 7% chloral hydrate (0.4ml/100g) and ligated buccally with a 0.2mm ligature across the mesial-distal interstice of the second molar of the upper jaw. And establishing a periodontitis model. After 1 week of ligation, 10. mu.l of 2mg/ml NAC or 2mg/ml PssL-NAC was injected every 3 days into the center of the submucosal palate using a microinjector, with the ligature wire remaining during treatment. The groups were a control group (non-ligated healthy rats), a periodontitis group (non-ligated treatment), an NAC group (NAC-treated group), and a PssL-NAC group (psl-NAC-treated group). One week later, 4 weeks treated (two weeks, five weeks after ligation) rats were sacrificed and intact maxilla containing maxillary dentition were collected for micro CT and histological analysis. Collecting the drug toxicity detection of heart, liver, spleen, lung and kidney.

FIG. 5 is a schematic view of: Micro-CT images of

rats

1 and 4 weeks after NAC and PssL-NAC treatment. ((A-H) -1) maxillary second molar buccal palatal sectional image. ((A-H) -2) three-dimensional reconstruction of digitized images. FIG. 6: Micro-CT was applied to analyze the distance between the 6 sites CEJ-ABC of the maxillary second molars of different groups. FIG. 7: histological analysis of maxilla after 1 week of second molar treatment. (A-D) -1 and (A-D) -2 correspond to the red and black boxes in A-D. ((A-D) -3) representative TRAP images. 500 μm for A-D scale bar and 100 μm for A-D (1-3). FIG. 8: histology of maxillary second molars after 4 weeks of NAC treatment. (A-D) -1 and (A-D) -2 correspond to the red and black boxes in A-D. ((A-D) -3) representative TRAP images. The green squares in B-2 represent abscesses. 500 μm for A-D scale bar and 100 μm for A-D (1-3). FIG. 9: masson trichrome staining of the maxillary second molars 4 weeks after NAC and PssL-NAC treatment. A-D was 500 μm and E-H was 100. mu.m.

FIG. 10: histopathological images of heart, liver, spleen, lung, kidney. Scale bar 50 μm.

The results show that:

(1) as the ligation time increases, the destruction around the alveolar bone after ligation of the second molar of the upper jaw worsens. Ligation significantly increased the loss of attachment (periodontitis group), indicating successful establishment of the periodontitis model.

(2) After NAC and PssL-NAC injection, PssL-NAC inhibited bone destruction by ligation and NAC was observed to exacerbate periodontal bone destruction at 4 weeks.

(3) The loss of attachment was shortened after NAC and PssL-NAC injections, whereas the PssL-NAC group was shorter, indicating less bone loss. The above results suggest that PssL-NAC improves bone loss in periodontitis caused by ligation.

(4) The periodontitis group showed that ligation resulted in destruction of the attachment site of the connective epithelium and infiltration of inflammatory cells. In addition, TRAP staining showed higher osteoclast activity after ligation.

(5) NAC and PssL-NAC reduced tissue damage compared to untreated ligation. After 1 week of injection, NAC group had better tissue repair effect and decreased osteoclast activity than PssL-NAC group. Over time, tissue destruction worsens, forming abscesses around the alveolar ridge (green square marks). The tissue structure of the PssL-NAC group is relatively restored, and the activity of osteoclasts is reduced. The effect of treating inflammatory cell infiltration by NAC is obvious.

(6)4 weeks Masson staining showed collagen fibrosis with disorganized, sparse after ligation, while the PssL-NAC group had densely and regularly arranged periodontal ligament fibers.

(7) No significant drug toxicity was seen with PssL-NAC.

The results prove that the PssL-NAC has the effect of promoting bone tissue repair under the periodontitis state, and the effect is better than that of NAC.

The above description is only an example of the present application and is not intended to limit the scope of the present application, and all modifications of equivalent structures and equivalent processes, which are made by the contents of the specification and the drawings, or which are directly or indirectly applied to other related technical fields, are intended to be included within the scope of the present application.

Claims

1. A sulfur-containing polymer having a formula (t):

2. The sulfur-containing polymer of claim 1, wherein m is in the range of 20 to 70 and n is in the range of 30 to 180.

3. The sulfur-containing polymer of claim 1, wherein the weight average molecular weight of the sulfur-containing polymer is 3000-31000 Da.

4. A process for producing the sulfur-containing polymer according to any one of claims 1 to 3, comprising the steps of:

(S1) amino-protected Compound of formula (f)

Acylation reaction with functional group-modified polycaprolactone to produce a polymer of formula (i)

Wherein n is a natural number, and the value range of n is 10-200;

(S2) reacting the compound of formula (i) to produce a polymer of formula (j)

in the reaction, m and n are natural numbers, the value range of m is 20-200, and the value range of n is 10-200.

5. The process according to claim 4, wherein the compound of formula (f) is obtained by:

(S11) acylation of 2-aminoethanethiol with ethyl trifluoroacetate under basic conditions to produce a compound of formula (c)

6. A drug-loaded nanoparticle carrier, wherein the material of the drug-loaded nanoparticle carrier is the sulfur-containing polymer in any one of claims 1-3.

7. A pharmaceutical composition, comprising:

(1) the drug-loaded nanoparticle carrier of claim;

(2) a pharmaceutically effective amount of a pharmaceutically active ingredient.

8. The pharmaceutical composition according to claim 7, wherein the pharmaceutically active ingredient is selected from one or more of N-acetylcysteine (NAC), resveratrol, vitamin C.

9. Use of a pharmaceutical composition according to claim 7 or 8 for the preparation of a medicament for the anti-oxidant treatment of oxidative stress conditions.

10. The use according to claim 9, wherein the antioxidant therapeutic is a medicament for bone tissue repair.