Preparation method of nano preparation capable of releasing hydrogen sulfide gas in acidic response manner in targeted bone resorption area

Abstract

The invention discloses a preparation method of a nano preparation for releasing hydrogen sulfide gas in an acidic response manner in a targeted bone resorption area. The preparation method comprises the following steps: S1, preparing polypeptide phospholipid DSPE-PEG-ASP8 with bone resorption area targeting property; s2, synthesizing a prodrug JK-1; s3, preparing a bone targeting liposome loaded with JK-1; the nano preparation is prepared by a method of entrapping a prodrug JK-1 capable of releasing HS under an acidic condition in a liposome with a bone resorption region targeting characteristic, so that accurate release of HS in a bone resorption microenvironment can be realized, and the problems of poor targeting property, uncontrollable release, low loading efficiency and the like in a traditional hydrogen sulfide delivery system are solved; the treatment efficiency is improved; and systemic side effects are reduced.

Description

Technical Field

[0001] This invention relates to a method for preparing a nano-formulation that releases hydrogen sulfide gas in response to acidic conditions in bone resorption regions. Background Technology

[0002] The main characteristics of osteoporosis are decreased bone mass, destruction of bone microstructure, and increased bone fragility, which often leads to fractures and movement disorders, seriously affecting patients' quality of life. Currently, clinical treatment mainly uses anti-resorption drugs such as bisphosphonates, selective estrogen receptor modulators, and calcitonin. Although these can slow bone loss, their efficacy is limited, and long-term use may lead to serious side effects (such as mandibular osteonecrosis and thrombosis), and patient compliance is also poor.

[0003] In recent years, studies have found that hydrogen sulfide (H2S), an endogenous gaseous signaling molecule, plays a crucial role in maintaining the dynamic homeostasis of bone remodeling. It can promote osteoblast activity and inhibit osteoclast formation, thus possessing potential osteoprotective effects. However, H2S gas itself suffers from drawbacks such as unstable release, poor targeting, and high systemic toxicity, severely limiting its application in the treatment of bone diseases. Especially in bone resorption areas, where osteoclast activity creates an acidic microenvironment, targeted release of H2S within this specific environment could provide a new technological pathway for the precision treatment of osteoporosis. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the present invention provides a method for preparing a nano-formulation that releases hydrogen sulfide gas in response to acidic conditions in bone resorption regions. The prepared nano-formulation can achieve precise targeted delivery of H2S gas in bone resorption regions and controllable release under acidic conditions.

[0005] This invention provides a method for preparing a nano-formulation that releases hydrogen sulfide gas in response to acidic conditions in bone resorption regions, specifically comprising the following steps: S1. Using distearate phosphatidylethanolamine-polyethylene glycol-maleimide (DSPE-PEG-MAL) and thiol-modified aspartic acid peptide (ASP8-SH) as raw materials, a peptide phospholipid (DSPE-PEG-ASP8) with bone resorption zone targeting properties was synthesized by Michael addition reaction and then purified by dialysis. The product was verified using FTIR and H-NMR to confirm its chemical structure, purity, and quality.

[0006] S2. Using phenylthiophosphoric dichloride as raw material, it was reacted sequentially with 3-hydroxypropionitrile and glycine methyl ester to obtain a yellow oily intermediate product, which was then hydrolyzed by LiOH, concentrated and freeze-dried to obtain JK-1. The product was verified by Fourier transform infrared spectroscopy (FTIR), hydrogen nuclear magnetic resonance spectroscopy (H-NMR), and H2S release curve. The chemical structure was used to verify whether the final product was JK-1, and its functionality in releasing H2S in acid response was also verified.

[0007] S3. DOTAP, DOPE, cholesterol, DSPE-PEG-MAL and DSPE-PEG-ASP8 were selected as raw materials and dissolved in chloroform at a molar ratio of 42:15:38:5. The mixture was dried into a thin film by rotary evaporation, and then JK-1 aqueous solution was added. The mixture was incubated in a water bath to form multilayer lipid vesicles. The particle size was gradually reduced by a liposome extruder to obtain a single-layer vesicle. Finally, unencapsulated JK-1 was removed by ultrasonic emulsification and dialysis to obtain JK-1-loaded liposomes. The product quality was confirmed by cryo-transmission electron microscopy (Cryo-TEM), dynamic light scattering (DLS), and zeta potential techniques.

[0008] Under different pH conditions, the H2S release curves of the product of step S3 were characterized by the methylene blue-zinc sulfide precipitation method to confirm the acid response release performance of the nano-formulation. The particle size and morphology changes of the product of step S3 under different pH conditions were compared by DLS and Cryo-TEM to evaluate the physical stability and structural integrity of the nano-formulation in different acid and alkaline environments.

[0009] In step S3, the JK-1 aqueous solution was replaced with an aqueous solution containing ICG to prepare ICG-loaded liposomes. The liposomes were injected into the tail vein of mice, and imaging analysis was performed using an in vivo imaging system (IVIS) 12 hours after injection to evaluate the in vivo distribution and bone tissue targeting of the liposomes.

[0010] The beneficial effects of this invention are as follows: By synthesizing polypeptide phospholipids with targeting properties to bone resorption regions and constructing stable liposomes, this invention efficiently encapsulates the prodrug JK-1, which releases hydrogen sulfide in response to acidity, thereby achieving precise release of H2S in the acidic microenvironment of bone resorption regions. This significantly improves the targeting and controllability of the nano-formulation, overcoming the problems of poor targeting, uncontrolled release, and low loading efficiency of hydrogen sulfide delivery in existing technologies. It can effectively improve the treatment effect of osteoporosis and other bone diseases, reduce systemic side effects, and has good clinical application prospects. Attached Figure Description

[0011] Figure 1 Flowchart for the preparation and performance verification of Lipo-ASP8@JK-1 nanoformulation; Figure 2The relevant spectra of the polypeptide phospholipid (DSPE-PEG-ASP8) with bone resorption zone targeting properties are shown in Part A (FTIR spectrum of DSPE-PEG-ASP8; H-NMR spectrum of DSPE-PEG-ASP8). Figure 3 The relevant spectra and release curves of JK-1 are shown in Part A (FTIR spectrum of JK-1; H-NMR spectrum of JK-1; H2S release kinetic curves of JK-1 under different pH conditions). Figure 4 The images show the particle size distribution, zeta potential, and cryo-electron microscopy (Cryo-TEM) images of Liposome and Liposome-ASP8 (Part A shows the particle size distribution of Liposome; Part B shows the particle size distribution of Liposome-ASP8; Part C shows the zeta potential of Liposome and Liposome-ASP8; Part D shows the Cryo-TEM images of Liposome and Liposome-ASP8). Figure 5 The images show the release curves, particle size distribution, and cryo-electron microscopy (Cryo-TEM) images of Lip-ASP8@JK-1 under different pH conditions (Part A shows the H2S release kinetic curves of Lip-ASP8@JK-1 under different pH conditions; Part B shows the particle size distribution of Lip-ASP8@JK-1 under different pH conditions; Part C shows the particle size distribution of Lip-ASP8@iJK-1 under different pH conditions; Parts D and E show the Cryo-TEM images of Lip-ASP8@JK-1 under different pH conditions). Figure 6 The distribution and statistical analysis of Lip@ICG and Lip-ASP8@ICG in mice are shown in Figure A (the distribution of Lip@ICG and Lip-ASP8@ICG in mice; and the statistical analysis of the distribution of Lip@ICG and Lip-ASP8@ICG in the femur and tibia of mice). Detailed Implementation

[0012] The preparation method of the nano-formulation that releases hydrogen sulfide gas in response to acidic conditions in the bone resorption region according to an embodiment of the present invention includes the following steps: S1. Using distearate phosphatidylethanolamine-polyethylene glycol-maleimide (DSPE-PEG-MAL) and thiol-modified aspartic acid peptide (ASP8-SH) as raw materials, a peptide phospholipid (DSPE-PEG-ASP8) with bone resorption zone targeting properties was synthesized by Michael addition reaction and then purified by dialysis. The product was verified using FTIR and H-NMR to confirm its chemical structure, purity, and quality.

[0013] S2. Using phenylthiophosphoric dichloride as raw material, it was reacted sequentially with 3-hydroxypropionitrile and glycine methyl ester to obtain a yellow oily intermediate product, which was then hydrolyzed by LiOH, concentrated and freeze-dried to obtain JK-1. The product was verified by Fourier transform infrared spectroscopy (FTIR), hydrogen nuclear magnetic resonance spectroscopy (H-NMR), and H2S release curve. The chemical structure was used to verify whether the final product was JK-1, and its function of releasing H2S in acid response was also verified.

[0014] S3. DOTAP, DOPE, cholesterol, DSPE-PEG-MAL and DSPE-PEG-ASP8 were selected as raw materials and dissolved in chloroform at a molar ratio of 42:15:38:5. The mixture was dried into a thin film by rotary evaporation. Then, JK-1 (50mM) aqueous solution was added and incubated in a 50℃ water bath to form multilayer lipid vesicles. The particle size was gradually reduced by a liposome extruder to obtain monolayer vesicles. Finally, unencapsulated JK-1 was removed by ultrasonic emulsification and dialysis to obtain JK-1-loaded liposomes. The product quality was confirmed by cryo-transmission electron microscopy (Cryo-TEM), dynamic light scattering (DLS), and zeta potential techniques.

[0015] Under different pH conditions, the H2S release curves of the product of step S3 were characterized by the methylene blue-zinc sulfide precipitation method to confirm the acid response release performance of the nano-formulation. The particle size and morphology changes of the product of step S3 under different pH conditions were compared by DLS and Cryo-TEM to evaluate the physical stability and structural integrity of the nano-formulation in different acid and alkaline environments.

[0016] In step S3, the JK-1 aqueous solution was replaced with an aqueous solution containing ICG to prepare ICG-loaded liposomes. The liposomes were injected into the tail vein of mice, and imaging analysis was performed using an in vivo imaging system (IVIS) 12 hours after injection to evaluate the in vivo distribution and bone tissue targeting of the liposomes.

[0017] In addition, other types of aqueous solution contents can be used to prepare non-targeted or bone-targeted liposomes loaded with PBS and iJK-1, which can be used as a control group in experimental design to compare the targeting and release performance of the targeted liposomes and verify the specificity and effectiveness of the nano-formulation of the present invention.

[0018] Experimental verification: In the initial stage, bone-targeting liposomes were constructed, and JK-1 was synthesized. First, DSPE-PEG-MAL and ASP8-SH were mixed in HEPES buffer at pH 7.2, and bone-targeting DSPE-PEG-ASP8 was synthesized via Michael addition reaction. After dialysis purification, it was identified by FTIR and H-NMR. Figure 2 As shown in Part A, DSPE-PEG-MAL exhibits weak NH and CO-NH peaks (3552, 3475 cm⁻¹) in its FTIR spectrum; however, the signal intensity of the NH and CO-NH peaks is significantly enhanced in the DSPE-PEG-ASP8 spectrum, which is attributed to the large number of NH and CO-NH structures contained in ASP8. The weakening of the maleimide C=C peak at 1465 cm⁻¹ in DSPE-PEG-ASP8 further indicates that ASP8 has partially substituted maleimide. Additionally, the NMR spectrum of DSPE-PEG-MAL shows a characteristic peak of maleimide at 6.7 ppm, while this peak disappears in the spectrum of DSPE-PEG-ASP8; this indicates that the maleimide group in DSPE-PEG-MAL has reacted with ASP-SH (…). Figure 2 Part B). Based on the above, DSPE-PEG-MAL or DSPE-PEG-ASP8 was mixed with DOTAP, DOPE, and Chol in a certain proportion to construct liposomes for particle size and morphology characterization. Cyro-TEM and DLS results showed that the particle size of pure Liposome and Liposome-ASP8 both exhibited a normal distribution trend of about 60-200 nm. Figure 4 Parts A and B), through Zeta potential analysis, can be used to observe the charge characteristics of both ( Figure 4 The morphological characteristics of both (part C) can be observed through Cryo-TEM images. Figure 4 (Part D). On the other hand, JK-1 was synthesized using phenylthiophosphoric dichloride as a starting material, and FTIR, H-NMR, and H2S release behavior tests were performed to verify the compound. Figure 3 As shown in Part A, the NH and C=O peaks at 3400 and 1605 cm⁻¹ suggest the presence of an amide structure; the CH bonds at 1400 and 1350 cm⁻¹, the C(=O)-O bonds at 1150 and 1100 cm⁻¹, and the =CH bonds at 750 and 625 cm⁻¹ correspond to the CH₂, ester group, and benzene ring in the JK-1 structural formula, respectively. The H₂NMR spectrum shows characteristic peaks of NH and CH at 7.72 and 7.21 ppm, respectively, representing hydrogen atoms in the benzene ring, while 3.15 and 2.9 ppm are characteristic peaks for NH and CH. Figure 3Part B). Finally, the release characteristics of JK-1 at different pH values ​​were detected by the methylene blue-zinc sulfide precipitation method. The results showed that acidic pH accelerated the release of H2S from JK-1, while almost no H2S release was detected at neutral pH. Figure 3 Part C).

[0019] After constructing Liposome-ASP8 loaded with JK-1, its H2S release performance under different pH conditions was further characterized. The H2S release curve of Lip-ASP8@JK-1 was determined by the methylene blue-zinc sulfide precipitation method. The results showed that it promoted the release of H2S from liposomes in an acidic pH environment. Figure 5 In Part A of the study, comparing the acidic environment (pH=4.0) simulating bone resorption region with the weakly alkaline environment (pH=7.4) simulating normal physiological conditions, the H2S release was significantly higher in the acidic environment than in the weakly alkaline environment. This demonstrates the formulation's high loading efficiency and good release responsiveness during H2S delivery. Simultaneously, DLS analysis of the particle size changes of Lip-ASP8@JK-1 under different pH conditions revealed a significant decrease in liposome particle size at acidic pH. Figure 5 (Part B); however, for Lip-ASP8@iJK-1, its particle size does not change significantly under acidic pH ( Figure 5 (Part C). Furthermore, the morphological characteristics of Lip-ASP8@JK-1 under different pH conditions were observed using Cryo-TEM, and corresponding electron microscopic images were obtained: at acidic pH, shrinkage and discoloration of the liposome morphology were observed (…). Figure 5 Part D); liposome morphology was almost normal at neutral pH (part D); Figure 5 (Part E of the equation). These results collectively reveal the release behavior, particle size variation, and morphological characteristics of Lip-ASP8@JK-1 under different pH conditions, providing strong experimental evidence for its pH response characteristics.

[0020] Reference Figure 6 As shown, the distribution of ICG in different organs of mice 12 hours after Lip@ICG and Lip-ASP8@ICG injection into the tail vein was observed using an IVIS in vivo imaging system. Figure 6 Part A of the study showed that the fluorescence intensity of Lip-ASP8@ICG in bone tissue was significantly higher than that of non-targeted Lip@ICG. Furthermore, statistical analysis of fluorescence signals in the mouse femur and tibia regions revealed… Figure 6 Part B further confirms that the liposomes prepared in this invention have excellent bone tissue targeting ability.

[0021] The above embodiments are merely one preferred embodiment of the present invention. Ordinary variations and substitutions made by those skilled in the art within the scope of the technical solution of the present invention are all included within the protection scope of the present invention.

Claims

1. A method for preparing a nano-formulation that releases hydrogen sulfide gas in response to acidic conditions in bone resorption regions, characterized in that, Includes the following steps: S1. Using distearate phosphatidylethanolamine-polyethylene glycol-maleimide (DSPE-PEG-MAL) and thiol-modified aspartic acid peptide (ASP8-SH) as raw materials, a peptide phospholipid (DSPE-PEG-ASP8) with bone resorption zone targeting properties was synthesized by Michael addition reaction and then purified by dialysis. S2. Using phenylthiophosphoric dichloride as raw material, it was reacted sequentially with 3-hydroxypropionitrile and glycine methyl ester to obtain a yellow oily intermediate product, which was then hydrolyzed by LiOH, concentrated and freeze-dried to obtain JK-1. S3. DOTAP, DOPE, cholesterol, DSPE-PEG-MAL and DSPE-PEG-ASP8 were selected as raw materials, dissolved in chloroform in proportion, dried into a thin film by rotary evaporation, and then JK-1 aqueous solution was added. Multilayer lipid vesicles were formed by water bath incubation, and the particle size was gradually reduced by a liposome extruder to obtain a single layer of vesicles. Finally, unencapsulated JK-1 was removed by ultrasonic emulsification and dialysis to obtain JK-1-loaded liposomes.

2. The method for preparing a nano-formulation that releases hydrogen sulfide gas in response to acidic conditions in bone resorption regions according to claim 1, characterized in that: The product of step S1 was verified by Fourier transform infrared spectroscopy (FTIR) and hydrogen nuclear magnetic resonance spectroscopy (H-NMR).

3. The method for preparing a nano-formulation that releases hydrogen sulfide gas in response to acidic conditions in bone resorption regions according to claim 1, characterized in that: The product of step S2 was verified by FTIR, H-NMR and H2S release curves.

4. The method for preparing a nano-formulation that releases hydrogen sulfide gas in response to acidic conditions in bone resorption regions according to claim 1, characterized in that: In step S3, the molar ratio of DOTAP, DOPE, cholesterol, and DSPE-PEG-ASP8 is 42:15:38:

5.

5. The method for preparing a nano-formulation that releases hydrogen sulfide gas in response to acidic conditions in bone resorption regions according to claim 1, characterized in that: The product of step S3 was verified by cryo-transmission electron microscopy (Cryo-TEM), dynamic light scattering (DLS), and zeta potential techniques.

6. The method for preparing a nano-formulation that releases hydrogen sulfide gas in response to acidic conditions in bone resorption regions according to claim 1, characterized in that: The H2S release curves of the product in step S3 were characterized by methylene blue-zinc sulfide precipitation under different pH conditions. The particle size and morphology changes of the product in step S3 under different pH conditions were compared by DLS and Cryo-TEM.

7. The method for preparing a nano-formulation that releases hydrogen sulfide gas in response to acidic conditions in bone resorption regions according to claim 1, characterized in that: In step S3, the JK-1 aqueous solution was replaced with an aqueous solution containing ICG to prepare ICG-loaded liposomes. The liposomes were injected into the tail vein of mice, and imaging analysis was performed using an in vivo imaging system (IVIS) 12 hours after injection to evaluate the in vivo distribution and bone tissue targeting of the liposomes.

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