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

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

Thioketal-containing polymer and its application in bone tissue repair

technical field

The application belongs to the technical field of stomatology, and specifically relates to a polymer containing a thioketal bond and its application in bone tissue repair.

Background technique

Reactive oxygen species (ROS) are chemically reactive chemicals containing oxygen, small molecules derived from oxygen, such as peroxides, superoxides, hydroxyl radicals, singlet oxygen, and alpha-oxygen. These small molecules can react with various chemicals (such as proteins, lipids, sugars and nucleic acids) and participate in normal life activities. There is an oxidative/antioxidant system in the body to maintain a stable level of ROS in the body. Abnormal concentrations of ROS can disrupt the homeostasis of redox in organisms, damage the intracellular antioxidant defense system, and generate oxidative stress. If exposed to oxygen free radicals for a long time, the biofilm, DNA, proteins and lipids of organisms will be damaged, seriously destroying the structure and function of intracellular proteins, lipids, nucleic acids, and then causing diseases, such as cancer, stroke , arteriosclerosis, inflammation, etc. However, too low ROS can affect intracellular signal transduction and osteogenic differentiation of stem cells. Therefore, regulating the level of ROS can also be used as a target for disease treatment.

In diseases with high concentrations of ROS, a potential solution to repair damage caused by oxidative stress is the use of exogenous ROS scavengers such as N-acetyl cysteine (NAC), resveratrol, vitamin C However, these compounds usually have obvious cytotoxicity, and because of their low molecular weight, they are easily metabolized rapidly by the kidneys. In addition, low molecular weight compounds are not localized in specific regions, but are distributed throughout the body, resulting in their low availability. These shortcomings limit its therapeutic effect on some oxidative stress-related diseases. And removing all ROS in cells is not conducive to tissue repair.

A promising strategy to address the above drawbacks is the use of drug delivery systems to deliver low molecular weight drugs. To date, many promising drug delivery systems using drug-loaded liposomes and polymeric micelles are under clinical and preclinical studies. However, at this stage, a smart drug response system that can respond to ROS and accurately deliver drugs is needed to cope with the oxidative stress environment, and can regulate ROS at an appropriate level. Hence the invention.

SUMMARY OF THE INVENTION

The present application provides a polymer containing a thioketal bond, which can be loaded with active pharmaceutical ingredients, the surface of the polymer is a hydrophilic PEG segment, which can effectively prolong the in vivo storage time of nanoparticles, and the inner core of the polymer is a hydrophobic Under the high level of ROS in the inflammatory environment, the thioketal bond is broken, and the drug-loaded microspheres change from a hydrophobic curled state to a stretched state, and the active pharmaceutical ingredients in the microspheres are released accordingly. The targeted accumulation at the site of inflammation, so it can maintain appropriate ROS levels and promote the osteogenic differentiation of stem cells, has a significant application prospect in bone tissue repair.

In order to solve the technical problems in the prior art, a technical solution adopted in the present application is to provide a sulfur-containing polymer, wherein the structural formula of the sulfur-containing polymer is shown in the formula (t):

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

In the preferred sulfur-containing polymer, the value of m ranges from 20 to 70, and the value of n ranges from 30 to 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 kind of preparation method of described sulfur-containing polymer, it is characterized in that, comprises the following steps:

(S1) Compound of formula (f) with amino group protected

Acylation with functional group-modified polycaprolactone to produce polymers of formula (i)

, where n is a natural number, and the value of n ranges from 10 to 180;

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

(S3) reacting the polymer of formula (j) with polyethylene glycol modified with functional groups to generate the polymer of formula (t);

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

In a preferred technical solution, the reaction in step (S1) is carried out at 25° C., the reaction solvent is dichloromethane (DCM), and the solvent contains DIEA.

In a preferred technical scheme, in the method, the compound of formula (f) is obtained by the following steps:

(S11) 2-aminoethanethiol and ethyl trifluoroacetate are subjected to an acylation reaction under basic conditions to generate a compound of formula (c)

(S12) The compound of formula (c) is reacted with 2-methoxypropene to generate the compound of formula (e)

(S13) hydrolyzing the compound of formula (e) under alkaline conditions to generate the compound of formula (f)

In a preferred technical solution, amino protection is performed using di-tert-butyl carbonic anhydride under basic conditions.

In a preferred technical solution, the alkaline environment is selected from the group consisting of diethylamine, triethylamine, diisopropylethylamine, pyridine, quinoline, potassium carbonate, sodium carbonate, cesium carbonate, potassium hydroxide, in the presence of sodium hydroxide.

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

Another object of the present invention is to provide an application of the sulfur-containing polymer as a drug carrier.

Another object of the present invention is to provide a nano-drug-loaded particle carrier, and the material of the nano-drug-loaded particle carrier is the sulfur-containing polymer.

Another object of the present invention is to provide a pharmaceutical composition, characterized in that the pharmaceutical composition comprises:

Suitable said nano-drug-loaded particle carrier;

A pharmaceutically effective amount of a pharmaceutical active ingredient.

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

Another object of the present invention is to provide an application of the pharmaceutical composition for preparing an antioxidant therapeutic drug in an oxidative stress environment.

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

Different from the prior art situation, the beneficial effects of the present application are:

The sulfur-containing polymer obtained in the present application can be used as a good carrier for active pharmaceutical ingredients, and has the advantages of good biocompatibility, good biodegradability, low toxicity, good stability, long circulation time in vivo, and strong targeting. When it was loaded with PCL drugs, it could maintain the appropriate ROS level and promote the osteogenic differentiation of stem cells compared with the simple use of antioxidants. When loaded with PCL drugs, it can respond to ROS in the environment to release drugs on demand and store drugs when not needed.

Description of drawings

In order to illustrate the technical solutions in the embodiments of the present application more clearly, the following briefly introduces the drawings that are used in the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, under the premise of no creative work, other drawings can also be obtained from these drawings, wherein:

Figure 1 is the characterization of the 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 image of PssL NPs. (D) TEM image of PssL-NAC NPs. (E) TEM image of PssL-NAC NPs after ROS treatment. (F) GPC image of PssL NPs; (G) GPC image of PssL NPs treated with ROS. (H) DLS image.

FIG. 2 is a schematic diagram of the results of CCK-8 of the present application.

Figure 3 shows the regulation of ROS and the determination of apoptosis rate by PssL-NAC treatment of the present application. (A) CLSM images of hPDLSCs incubated with three groups (Control, NAC, PssL-NAC NPs) and different LPS concentrations (0, 1, 5, 10 and 20 μg/ml), the scale bar is 100 μm. The excitation wavelength was 488 nm. (B) The mean fluorescence intensity (MFI) of the three groups was detected by flow cytometry. . (C) Apoptotic rates of hPDLSCs treated with different concentrations of LPS (0, 5, 10 μg/ml) in the presence or absence of NAC and PssL-NAC.

Figure 4 shows that different concentrations of LPS (0, 5, 10 μg/ml) of the present application induce osteogenic differentiation of hPDLSCs in the absence of NAC and PssL-NAC. (A) ALP activity. (B) BMP-2 mRNA expression. (C) Runx2 mRNA expression. (D) Expression of PKA mRNA. (E) BMP-2, Runx2, PKA protein expression. (F) Alizarin red S staining image, scale bar: 100 μm. (G) Quantitative mineralization analysis of group F. NAC had no significant effect on the number of mineralized nodules, while PssL-NAC promoted the formation of mineralized nodules.

Figure 5 is the Micro-CT image of the rat after 1 week and 4 weeks of NAC and PssL-NAC treatment of the application. ((A-H)-1) The buccal-palatal section image of the maxillary second molar. ((A-H)-2) Three-dimensional reconstruction of digitized images.

FIG. 6 is the application of Micro-CT to analyze the distance between CEJ-ABC of 6 parts of maxillary second molars in different groups.

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

Figure 8 is the histological analysis of the maxillary second molar of the 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. A-D scale bar: 500 μm, A-D (1-3): 100 μm.

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

Figure 10 is the histopathological images of the heart, liver, spleen, lung, and kidney of the present application. Scale bar: 50 μm.

Figure 11 is a synthetic route diagram of the sulfur-containing polymer of the present application.

Detailed ways

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. Obviously, the described embodiments are only a part of the embodiments of the present application, but not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

In order to satisfy the regulation of ROS in the body at an appropriate level, the present invention provides a sulfur-containing polymer and nano-drug-loading particles thereof. The structural formula of the polymer is shown in formula (t):

Among them, m and n are both natural numbers, and the value range of m is 20-200, and the value range of n is 10-180. Among them, polycaprolactone (PCL) is a hydrophobic segment, and polyethylene glycol (PEG) is a hydrophilic segment.

Among them, the PEG-bind-PCL amphiphilic polymer design system is used. In view of the increase of ROS in the local environment during the disease process, this application designs a thioketal bond to connect PEG and PCL, and the hydrophobic thioketal bond is used to link PCL. end and the hydrophilic end of PEG. The nano-drug-loaded microspheres with the oil-soluble antioxidant NAC inside can be obtained by self-assembly of the water phase and the oil phase. Among them, under the low level of ROS in a non-inflammatory environment, the surface of the drug-loaded nanoparticles is a hydrophilic PEG segment, which can effectively prolong the in vivo storage time of the nanoparticles, and the inner core is a hydrophobic PCL segment. When the thioketal bond was broken, the nano-drug-loaded microspheres changed from a hydrophobic curled state to a stretched state, and the NAC in the microspheres was subsequently released, realizing the targeted accumulation of NAC at the site of inflammation. This nano-drug loading system with a PEG-protected outer layer and ROS responsiveness can not only prolong the storage time, but also enhance the NAC-targeted and selective delivery capability and effectively improve the delivery efficiency of nanoparticles. PssL-NAC microspheres can be used as a new type of drug delivery carrier with clear ROS responsiveness and superior biocompatibility. The system will release the drug in a third self-regulating activation mode to maintain the ROS level in the environment at a certain appropriate level. s level.

In the present invention, polyethylene glycol (PEG) is a pH-neutral, non-toxic, highly water-soluble, and extremely versatile polyether polymer compound. Its repeating unit is an oxyethylene group, which is linear. or branched chain structure. Polyethylene glycol polymer is the polymer with the lowest level of protein and cell absorption among the known polymers so far. In vivo, polyethylene glycol can dissolve in tissue fluid and can be quickly excreted by the body without any toxic side effects. Because polyethylene glycol is non-toxic and has good biocompatibility, it has been approved by the FDA as a pharmaceutical polymer for in vivo injection. Due to the above characteristics, as well as biocompatibility, safety, and almost no foreign body reaction, polyethylene glycol is widely used in biomedicine. 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, lubricity, sustained release, etc. In the synthesis and modification of new biomaterials, polyethylene glycol, as a part of the material, will endow the material with new properties and functions, such as hydrophilicity, flexibility, anticoagulation, and anti-phagocytic properties of macrophages.

Poly(-caprolactone, PCL) is a synthetic polyester biopolymer material, which is biodegradable and has good biocompatibility. It is a hydrophobic polymer with a long-term degradation mechanism. The final degradation product will be absorbed and excreted by the body, and is widely used in the control system of slow-release microspheres to obtain satisfactory drug release behavior. The crystallinity of polycaprolactone is strong and the degradation is slow. The degradation in the body is divided into two steps: the first step is that the molecular weight continues to decrease, but the material does not deform and lose weight; the second step is that after the molecular weight is reduced to a certain value, the material It begins to become fragments and loses weight at the same time, and will eventually be absorbed and excreted by the body without being accumulated in the body. Therefore, polycaprolactone can be used as a drug release carrier material in the body. Blending polycaprolactone with other materials can increase the embedding rate of hydrophilic substances, change the drug release rate, and significantly change the drug release mechanism. In addition, polycaprolactone has good flexibility, slow degradation rate, and good film-forming properties, mechanical properties, and biocompatibility. The raw materials are readily available, and can be made into polycaprolactone porous membranes for postoperative anti-adhesion membranes. , tissue engineering scaffold materials, etc., are also widely used in tissue engineering.

The particle size of the nanoparticles of the present invention is preferably 50-100 nm. The particle size of the nano-drug-loaded particles of the present invention is preferably 50-200 nm.

Polyethylene glycol (PEG) and polycaprolactone (PCL) have good biocompatibility and are usually designed as the hydrophilic segment and hydrophobic end of amphiphilic diblock copolymers, respectively. The thioketal bond obtained in the present 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 according to DIN 55672-1 of ≥500 to ≤20000 g/mol, more preferably ≥2000 to ≤15000 g/mol, even more preferably ≥3000 to ≤12000 g/mol, most preferably ≥4000 to ≤10000 g/mol, especially ≥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 according to the present invention as a drug carrier. In some embodiments of such pharmaceutical compositions, the pharmaceutical compositions are formulated for intravenous, intravitreal, intramuscular, oral, rectal inhalation, intranasal, topical Administration, ocular administration or intra-auricular administration. In other embodiments, the pharmaceutical compositions are in the form of tablets, pills, capsules, liquids, inhalants, nasal spray solutions, suppositories, solutions, emulsions, ointments, eye drops, or ear drops. In other embodiments, the pharmaceutical composition further comprises one or more other therapeutic agents.

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

The effective dose of the pharmaceutically active ingredient employed may vary with the drug employed, the mode of administration and the severity of the disease to be treated. Generally, however, satisfactory results are obtained when the pharmaceutically active ingredient of the present invention is administered in a daily dose of about 0.5-500 mg/kg of animal body weight, preferably in 2-4 divided doses per day, or in slow administered in release form. For most large mammals, the total daily dose is about 1-100 mg, preferably about 2-80 mg. Dosage forms suitable for oral administration contain about 0.5-500 mg of the active pharmaceutical ingredient in intimate admixture with a solid or liquid pharmaceutically acceptable carrier. This dosage regimen can be adjusted to provide optimal therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced, as dictated by the exigencies of the therapeutic situation.

These pharmaceutically active ingredients can be administered orally as well as intravenously, intramuscularly or subcutaneously. Solid carriers include: starch, lactose, dicalcium phosphate, microcrystalline cellulose, sucrose and kaolin, while liquid carriers include: sterile water, nonionic surfactants and edible oils (such as corn oil, peanut oil and sesame oil), as appropriate to the characteristics 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, such as flavors, colors, preservatives and antioxidants such as vitamin E, vitamin C, BHT and BHA.

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

These pharmaceutically active ingredients can also be administered parenterally or intraperitoneally. Solutions or suspensions of these pharmaceutically active ingredients (as 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 ordinary 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 for easy syringe expelling. It must be stable under the conditions of manufacture and storage and must be resistant to the contaminating influence of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, alcohol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

The purpose of the present invention is to prepare a nano-drug-loaded particle capable of responding to ROS for antioxidant treatment in an oxidative stress environment.

The present invention comprises unloaded PEG-ss-PCL nanoparticles (abbreviated as PssL NPs) and NAC-loaded PssL-NAC nanoparticles (abbreviated as PssL-NAC NPs). Compared with the use of antioxidants alone, PssL-NAC was more able to maintain appropriate ROS levels and promote the osteogenic differentiation of stem cells. Compared with other drug-loading systems, PssL-NAC can respond to ROS in the environment to release drugs on demand and store drugs when not needed.

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

Example 1 Preparation of PEG-ss-PCL

Under 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) , stirring 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 (3×100ml), and the combined organic layer was dried with anhydrous sodium sulfate under reduced pressure . Purification by silica gel column chromatography gave compound c (12.00 g, 69.30 mmol, 67%). Nuclear magnetic resonance detection results: ¹ H NMR (500MHz, CDCl ₃ )δ: 7.06(s, 1H), 3.53(q, 2H), 2.73(m, 2H), 1.42(t, 1H). ¹³ C NMR(126MHz, CDCl ) ₃ ) δ: 157.60, CF ₃ (119.19, 116.90, 114.61, 112.33), 42.54, 23.67. ¹⁹ F NMR (471 MHz, CDCl ₃ ) δ: -75.96.

再搅拌10min。加入化合物d(2-甲氧基丙烯，1.95g,27.06mmol,1.0eq)后，在室温下搅拌24h。粗产物经硅胶柱层析纯化得到化合物e为白色固体(8.41g,21.77mmol,80％)。核磁共振检测结果：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 c (11.72 g, 67.65 mmol, 2.5 equiv) and p-toluenesulfonic acid monohydrate (PTSA) (1.54 g, 8.12 mmol, 0.3 eq) were dissolved in 100 ml Tol under Ar environment. After stirring for 10min, add 100g molecular sieve

Stir for another 10 min. After adding compound d (2-methoxypropene, 1.95 g, 27.06 mmol, 1.0 eq), the mixture was stirred at room temperature for 24 h. The crude product was purified by silica gel column chromatography to obtain compound e as a white solid (8.41 g, 21.77 mmol, 80%). Nuclear magnetic resonance detection results: ¹ H NMR (500MHz, CDCl ₃ )δ: 7.05(s, 2H), 3.59(q, 4H), 2.83(t, 4H), 1.61(s, 6H). ¹³ C NMR(126MHz, CDCl ₃ ) δ: 157.66, 157.36, CF ₃ (119.20, 116.91, 114.62, 112.34), 56.52, 39.39, 29.27. ¹⁹ F NMR (471 MHz, CDCl ₃ ): δ-75.94.

Under Ar atmosphere, compound e (2.00 g, 5.17 mmol) was dissolved in 20 ml of 6M aqueous NaOH solution, stirred at room temperature for 4 h, and then extracted with dichloromethane (DCM, 4×50 ml). The organic layer was dried over anhydrous sodium sulfate under reduced pressure. Compound f was an amber oil (0.95 g, 4.91 mmol, 95%). Nuclear magnetic resonance detection results: ¹ H NMR (500MHz, CDCl ₃ )δ: 2.88(t, 4H), 2.70(t, 4H), 1.58(s, 6H), NH ₂ 1.31(s, 4H). ¹³ C NMR( 126MHz, CDCl ₃ ) delta: 55.79, 41.77, 34.63, 31.28.

Under Ar environment, compound f (0.90 g, 4.63 mmol, 1.2 eq) was dissolved in chloroform (20 mL) and dried with stirring for 15 min, then in an ice bath, the solution of the above solution was added drop by drop of compound g (carbonic acid) Di-tert-butyl anhydride, 0.84 g, 3.86 mmol, 1.0 eq) in dry chloroform (10 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 4 h, then at room temperature overnight. The solids were removed by filtration and the solvent was removed under reduced pressure. Purification by column chromatography gave compound h as a butter (0.85 g, 2.89 mmol, 75%). ¹ H NMR (500MHz, CDCl ₃ )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). ¹³ C NMR (126 MHz, CDCl ₃ ) δ: 155.77, 79.34, 41.33, 40.13, 34.04, 31.16, 31.10, 30.77, 28.40.

Under Ar environment, a dry dichloromethane (DCM) (50ml) PCL-COOH (5.00g, 0.33mmol, 1.0eq) and N,N-Diisopropylethylamine (DIEA) (129.3mg, 0.99mmol, 3.0eq) were placed in Stir at 0°C for 10 minutes. Then N-hydroxysuccinimide (NHS) (57.5mg, 0.50mmol, 1.5eq), and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) (95.8mg, 0.5mmol, 1.5eq) and compound h (98.2 mg, 0.33 mmol, 1.0 eq) was added to the reactor at 0 °C. Stir at room temperature for 48 hours and rinse with water (3 x 20ml). The organic layer was dried over anhydrous sodium sulfate under reduced pressure to obtain compound i (4.05 g, 80%) as a white solid. ¹ H NMR (500 MHz, CDCl ₃ ) δ: 4.03(t), 2.28 (t), 1.62 (m), 1.36 (m). ¹³ C NMR (126 MHz, CDCl ₃ ) 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 (4.00 g) was dissolved in dry DCM, stirred at RT, then trifluoroacetic acid (TFA) (2 ml) was added, stirred at RT for 2 h, and the solvent was removed under reduced pressure to give the crude product. The crude product was dissolved in DCM (50ml), washed with _NaHCO3 (aq) (2x20ml) and water (2x20ml). The organic layer was dried over anhydrous sodium sulfate under reduced pressure to obtain compound j as a white solid (3.20 g, 80%). ¹ H NMR (400 MHz, CDCl ₃ ) δ: 4.03(t), 2.27 (t), 1.62 (m), 1.34 (m). ¹³ C NMR (101 MHz, CDCl ₃ ) δ: 173.51, 64.12, 34.10, 28.33, 25.51, 24.56.

Under Ar, dry DCM (50 ml) PEG-COOH (2.00 g, 0.20 mmol, 1.0 eq) and DIEA (78.4 mg, 0.99 mmol, 3.0 eq) were stirred at 0 degrees Celsius for 10 minutes. Then NHS (46.0 mg, 0.40 mmol, 2.0 eq), EDC (76.6 mg, 0.40 mmol, 2.0 eq) and compound j (3.00 g, 0.20 mmol, 1.0 eq) were added to the reactor at 0 degrees Celsius. Stir at room temperature for 48 hours and rinse with water (3 x 20ml). The organic layer was dried over anhydrous Na ₂ SO ₄ under reduced pressure to give terminal white solid (t) (3.90 g, 78%). ¹ H NMR (500MHz, CDCl ₃ )δ: 4.02(t), 3.60(s), 2.26(t), 1.60(m), 1.34(m). ¹³ CNMR(126MHz, CDCl ₃ )δ: 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 nano-drug-loaded microspheres (PssL-NACNPs)

N-acetylcysteine (NAC, A7250, Sigma-Aldrich, China) was embedded with the PssL polymer carrier obtained according to the above preparation method. That is, PssL (50 mg) was dissolved in deionized water (50 ml). To the above suspension was added NAC (10 mg) in dichloromethane suspension (5 ml) and sonicated for 5 min. After sonication for 30 min, centrifugation, washing with deionized water 5 times to remove residues, and collection of 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. Particle size and particle size distribution of PEG ss PCL and PssL NAC NPs in complete cell culture medium were assessed by dynamic light scattering (Brookhaven Instruments, USA). The morphology of PEG ssPCL and PssL NAC NPs was examined using transmission electron microscopy (TEM, JEOL TEM-100). The molecular weight and polydispersity of PEG-ss-PCL were determined by gel permeation chromatography (GPC). Using polystyrene as a reference, the number average molecular weight (Mn), weight average molecular weight (Mw) and polydispersity index (PDI, Mw/Mn) were determined.

The cleavage of thioketal bonds in PEG-ss-PCL after hydrogen peroxide treatment was systematically analyzed by Malvern gel permeation chromatography (Viscotek GPC/SEC, Malvern, USA). Pure tetrahydrofuran (THF) was used as the mobile phase. The temperature was fixed at 35°C and the flow rate was set at 1.0 ml/min. PssL was injected through a Malvern autosampler and the results were measured using polystyrene (PSt) as a standard sample. Relative molecular weights were analyzed with OmniSEC.

The size and morphology of the nanoparticles were observed by transmission electron microscopy (TEM), the molecular weight of the nanoparticles was observed by gel permeation chromatography (GPC), and the hydrated particle size of the nanoparticles was observed by dynamic light diffraction (DLS).

Figure 1 shows the characterization 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 image of PssL NPs. (D) TEM image of PssL-NAC NPs. (E) TEM image of PssL-NAC NPs after ROS treatment. (F) GPC image of PssL NPs; (G) GPC image of PssL NPs treated with ROS. (H) DLS image.

After the successful synthesis of PssL NPs, as an amphiphilic polymer, PssL can self-assemble into nanoparticles, which can then be used as a powerful drug carrier for NAC. The morphologies of PssL-NPs and PssL-NAC-NPs were observed by transmission electron microscopy. As shown in Figure 1C, the PssL NPs displayed a uniformly dispersed spherical morphology with a size of about 80 nm. After encapsulating the NAC, the size of the PssL NAC NPs was slightly larger, about 120 nm, as shown in Fig. 1D. However, upon exposure to ROS, the spherical morphology of PssL NAC NPs was disrupted (Fig. 1E). As shown in Figure 1F-G, after ROS treatment, the molecular weight decreased and the retention time on the gel column became longer, indicating thioketal bond cleavage and polymer disruption. In the dynamic light scattering (DLS) results (PEG-PCL-NAC i.e. PssL NACNPs, PEG-PCL i.e. PssL NPs in Figure 1H), PssL NAC NPs (~155 nm) showed slightly larger fluidity than PssL NPs (~102 nm) kinetic diameter, which showed similar results to TEM. There were three peaks in PssL-NAC-NPs after PssL-NAC-NPs participated in the ROS reaction, indicating the cleavage of the ROS-responsive thioketal bond and the cleavage of the amphiphilic polymer.

Example 3 Biocompatibility of PssL-NAC NPs

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

Figure 2 is a schematic diagram of the results of CCK-8. The results showed that compared with the control group, NAC and PssL-NAC had no significant effect on cell proliferation, and both had better biocompatibility.

Example 4 ROS scavenging ability of PssL-NAC NPs

The intracellular ROS were labeled with DCFH-DA fluorescent probe, and the intracellular ROS in each group was observed by confocal microscope and flow cytometry. The apoptosis of cells was detected by Annexin V-FITC/PI apoptosis detection kit and observed by flow cytometry.

Figure 3: Regulation of ROS and apoptosis rate determination by PssL-NAC treatment. (A) CLSM images of hPDLSCs incubated with three groups (Control, NAC, PssL-NAC NPs) and different LPS concentrations (0, 1, 5, 10 and 20 μg/ml), the scale bar is 100 μm. The excitation wavelength was 488 nm. (B) The mean fluorescence intensity (MFI) of the three groups was detected by flow cytometry. . (C) Apoptotic rates of hPDLSCs treated with different concentrations of LPS (0, 5, 10 μg/ml) in the presence or absence of NAC and PssL-NAC.

The results showed that: (1) After LPS stimulation, intracellular ROS was significantly increased, NAC could regulate cellular ROS to the level of the control group, while PssL-NAC NPs regulated intracellular ROS to twice the level of the control group. (2) Both NAC and PssL-NAC could reduce LPS-induced apoptosis, and the ROS retained by PssL-NAC that was twice the level of the control group did not promote apoptosis.

Example 5 The effect of PssL-NAC NPs on the osteogenic differentiation of stem cells

Cellular RNA was extracted by Trizol method, and the gene expression of intracellular osteogenic markers was analyzed by PCR. The intracellular protein was extracted, and the protein expression of osteogenic markers was detected by Western Blot. After 7 days of cell culture, the expression of alkaline phosphatase was detected, and alizarin red staining was performed to observe the number of mineralized nodules, and semi-quantitative analysis was performed.

Figure 4: Different concentrations of LPS (0, 5, 10 μg/ml) induce osteogenic differentiation of hPDLSCs in the absence of NAC and PssL-NAC. (A) ALP activity. (B) BMP-2 mRNA expression. (C) Runx2 mRNA expression. (D) Expression of PKA mRNA. (E) BMP-2, Runx2, PKA protein expression. (F) Alizarin red S staining image, scale bar: 100 μm. (G) Quantitative mineralization analysis of group F. NAC had no significant effect on the number of mineralized nodules, while PssL-NAC promoted the formation of mineralized nodules.

The results showed that: (1) PssL-NAC promoted the activity of ALP and the expression of osteogenic markers better than NAC. (2) NAC had no significant effect on the number of mineralized nodules, while PssL-NAC promoted the formation of mineralized nodules.

Example 6 In vivo experiment

Thirty-two male SD rats, weighing about 240 g, were divided into 4 groups with 8 rats in each group. Rats were anesthetized with 7% chloral hydrate (0.4 ml/100 g), and 0.2 mm ligature was used to pass through the mesial and distal spaces of the maxillary second molars and ligated on the buccal side. A periodontitis model was established. After 1 week of ligation, 10 μl of 2 mg/ml NAC or 2 mg/ml PssL-NAC was injected into the center of the submucosal periosteum of the palatine with a microsyringe every 3 days, and the ligation silk was retained during the treatment. Each group is control group (unligated healthy rats), periodontitis group (ligation not treated), NAC group (NAC treated group) and PssL-NAC group (psl-NAC treated group). One week later, the 4-week-treated (two-week, five-week post-ligation) rats were sacrificed and intact maxillae containing maxillary dentition were collected for micro-CT and histological analysis. Collect heart, liver, spleen, lung and kidney for drug toxicity test.

Figure 5: Micro-CT images of rats after 1 and 4 weeks of NAC and PssL-NAC treatment. ((A-H)-1) The buccal-palatal section image of the maxillary second molar. ((A-H)-2) Three-dimensional reconstruction of digitized images. Figure 6: Application of Micro-CT to analyze the distance between CEJ-ABC in 6 parts of maxillary second molars in different groups. Figure 7: Histological analysis of maxillary second molars 1 week after treatment. (A-D)-1 and (A-D)-2 correspond to the red and black boxes in A-D. ((A-D)-3) Representative TRAP images. A-D scale bar: 500 μm, A-D (1-3): 100 μm. Figure 8: Histological analysis 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. A-D scale bar: 500 μm, A-D (1-3): 100 μm. Figure 9: Masson's trichrome staining of maxillary second molars after 4 weeks of NAC and PssL-NAC treatment. A-D is 500 μm, E-H is 100 μm.

Figure 10: Histopathological images of heart, liver, spleen, lung, kidney. Scale bar: 50 μm.

the result shows:

(1) With the increase of ligation time, the damage around the alveolar bone after the ligation of the maxillary second molar worsened. Ligation significantly increased the loss of attachment (periodontitis group), indicating that the periodontitis model was successfully established.

(2) After injection of NAC and PssL-NAC, PssL-NAC inhibited the bone destruction caused by ligation, and it could be observed that NAC aggravated the destruction of periodontal bone at 4 weeks.

(3) After injection of NAC and PssL-NAC, the loss of attachment was shortened, while it was shorter in the PssL-NAC group, indicating less bone loss. The above results suggest that PssL-NAC can improve bone loss in ligation-induced periodontitis.

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

(5) NAC and PssL-NAC alleviated tissue damage compared with untreated ligation. One week after injection, the NAC group had better tissue repair effect than the PssL-NAC group, and the osteoclast activity decreased. Over time, the tissue destruction worsened, forming an abscess (marked by a green square) around the alveolar ridge. The tissue structure of the PssL-NAC group was relatively restored, and the osteoclast activity decreased. Inflammatory cell infiltration effect of NAC group was obvious.

(6) Masson staining at 4 weeks showed that the collagen fibers were degenerated, arranged disorderly and sparsely after ligation, while the periodontal ligament fibers in the PssL-NAC group were densely arranged and neatly arranged.

(7) PssL-NAC showed no obvious drug toxicity.

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

The above descriptions are only the embodiments of the present application, and are not intended to limit the scope of the patent of the present application. Any equivalent structure or equivalent process transformation made by using the contents of the description and drawings of the present application, or directly or indirectly applied to other related technologies Fields are similarly included within the scope of patent protection of this application.

Claims (10)

1. A sulfur-containing polymer having a 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.

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)

(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 10-200.

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)

(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)

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.

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