Ultrasonic responsive apoptosis bionic targeting nano-carrier delivery material and preparation method thereof

Abstract

The invention belongs to the technical field of nano-drugs, and particularly relates to an ultrasonic responsive apoptosis bionic targeting nano-carrier delivery material and a preparation method thereof. According to the invention, a beta-cyclodextrin polymer (PCD) is used as a core skeleton, and phosphorylserine (PS) is modified through a phosphate bond so as to enhance the targeting ability; pFH (perfluorohexane) is entrapped as an ultrasonic response core under a low-temperature condition by utilizing interaction of a subject and an object; liposome (soya bean lecithin and cholesterol) is used for outer layer coating, so that the in-vivo stability and the circulation half-life period of the system are remarkably improved, and the breakthrough of the PCD-PS system from local administration to systemic administration is realized for the first time; the system has excellent ultrasonic responsiveness, and can be accurately triggered at a targeted part and release contents; meanwhile, the apoptotic cell imitating design endows the system with active targeting property, and the dual-coating structure ensures the stability of in-vivo circulation, so that the synergistic interaction of stable circulation, active targeting and ultrasonic triggering is realized.

Description

Technical Field

[0001] This invention belongs to the field of nanomedicine technology, specifically relating to an ultrasound-responsive apoptosis-inspired biomimetic targeted nanocarrier delivery material and its preparation method. Background Technology

[0002] The clearance of apoptotic cells is a crucial process for maintaining homeostasis in the body. During apoptosis, phosphorylserine (PS) on the inner side of the cell membrane everts to the outer side, where it is recognized by specific receptors (such as Tim-4 and the TAM receptor family) on the surface of phagocytes like macrophages. Utilizing this biological characteristic of PS, modifying its surface onto nanocarriers allows the carriers to mimic apoptotic cells, enabling active targeting against inflammatory tissues.

[0003] Perfluorohexane (PFH) is a low-boiling-point liquid perfluorocarbon compound. When encapsulated in a closed space (such as a liposome), the input of external energy (such as ultrasound) can trigger a dramatic gas-liquid phase transition or promote its dissociation from the cavity. This characteristic makes it very suitable as a physical switch to disrupt the structure of the carrier and release its contents. However, PFH faces two major technical bottlenecks in practical applications: first, poor physical stability, due to its volatility, it is prone to escape during storage and circulation; second, insufficient targeting ability, traditional PFH formulations mainly rely on passive targeting (such as the EPR effect), and their specific enrichment efficiency in diseased tissues (especially in inflammatory areas) is limited, making it difficult to meet the needs of precision diagnosis and treatment.

[0004] Cyclodextrin polymers (PCDs) are three-dimensional network macromolecules formed by the cross-linking polymerization of β-cyclodextrin units. Each cyclodextrin unit possesses a hydrophobic cavity, enabling effective encapsulation and stabilization of PFH molecules through host-guest interactions, thus overcoming the volatility problem of PFH to some extent. Existing attempts to modify PS onto PCDs (i.e., PCD-PS systems) have significant limitations: these systems typically only allow for local injection and cannot utilize the bloodstream to treat deep lesions far from the administration site. Liposomes, on the other hand, are vesicle structures composed of a phospholipid bilayer, possessing excellent biocompatibility and encapsulation capabilities, significantly improving the in vivo stability and circulating half-life of the carrier. The internal structure is stabilized under the protection of the liposome shell, and the volatile PFH is doubly locked in through the cavity of the PCD and the bilayer membrane of the liposome.

[0005] Therefore, by encapsulating PCD-PS / PFH with liposomes, this nanocarrier system has achieved a leap from "local passive diffusion" to "systemic active targeting + ultrasound-triggered response", which has important clinical value and application prospects in the treatment of inflammation-related diseases. Summary of the Invention

[0006] The purpose of this invention is to address existing problems by providing an ultrasound-responsive apoptosis-inspired biomimetic targeted nanocarrier delivery material and its preparation method.

[0007] This invention is achieved through the following technical solution: The first objective of this invention is to provide an ultrasound-responsive apoptosis-inspired biomimetic targeted nanocarrier delivery material, comprising: A phosphorylserine-modified β-cyclodextrin polymer core, wherein phosphorylserine is modified on the β-cyclodextrin polymer backbone via a phosphate bond; Perfluorohexane encapsulated within the hydrophobic cavity of the β-cyclodextrin polymer via host-guest interactions; And liposomes that coat the outer layer of the core.

[0008] Furthermore, the liposomes are composed of soybean lecithin and cholesterol.

[0009] Furthermore, the mass ratio of soybean lecithin to cholesterol is 5:4.

[0010] A second objective of this invention is to provide a method for preparing the ultrasound-responsive apoptosis-inspired biomimetic targeted nanocarrier delivery material according to any one of claims 1 to 3, comprising the following steps: (1) Preparation of phosphorylserine-modified β-cyclodextrin polymer; (2) Under conditions of 0-4°C, the phosphorylserine-modified β-cyclodextrin polymer and perfluorohexane were mixed in phosphate buffer at a mass ratio of 5:2 to form a complex suspension; (3) Dissolve soybean lecithin and cholesterol in an organic solvent, remove the solvent to form a lipid film; (4) Add the complex suspension obtained in step (2) to the lipid film, and perform hydration, ultrasonic treatment and membrane extrusion to obtain the ultrasonic-responsive apoptosis biomimetic targeted nanocarrier delivery material.

[0011] Further, the synthesis method of the β-cyclodextrin polymer in step (1) is as follows: β-Cyclodextrin was dissolved in an aqueous sodium hydroxide solution, epichlorohydrin was added, and the mixture was reacted at 30 ± 2°C for 24-48 h. After the reaction was completed, acetone was added to precipitate the polymer, which was then washed, dialyzed, and freeze-dried to obtain the final product.

[0012] Furthermore, the synthesis method of the phosphorylserine-modified β-cyclodextrin polymer in step (1) is as follows: The product was obtained by reacting FMOC-L-serine methyl ester, a phosphorus-containing crosslinking agent, and a β-cyclodextrin polymer sequentially in an organic solvent, followed by oxidation, deprotection, filtration, dialysis, and freeze-drying.

[0013] Furthermore, the phosphorus-containing crosslinking agent is bis(diisopropylamino)(2-cyanoethoxy)phosphine; The organic solvent is N,N-dimethylformamide.

[0014] Further, the organic solvent mentioned in step (3) is dichloromethane.

[0015] Furthermore, in step (4), the hydration is carried out at 37°C and 400 rpm for 30 min.

[0016] Further, in step (4), the ultrasonic treatment uses an ultrasonic cell disruptor with a working frequency of 20 kHz and a power of 100 W. It adopts an intermittent operation mode, pausing for 1 minute after every 2 minutes of ultrasonic treatment, with a total treatment time of 10 minutes.

[0017] A third objective of this invention is to provide the application of the aforementioned ultrasound-responsive apoptosis-inspired biomimetic targeted nanocarrier delivery material in the preparation of drugs for treating inflammation-related diseases.

[0018] The present invention has the following advantages over the prior art: This invention proposes an ultrasound-activated liposome-encapsulated cyclodextrin polymer delivery system with phosphorylserine modification and perfluorohexane encapsulation. The system comprises: a β-cyclodextrin polymer (PCD) as the core framework, phosphorylserine (PS) modified with phosphate bonds to enhance targeting ability; perfluorohexane (PFH) encapsulated at low temperatures using host-guest interactions as the ultrasound-responsive core; and an outer coating of liposomes (soy lecithin and cholesterol), significantly improving the system's in vivo stability and circulating half-life. This represents a breakthrough in PCD-PS system delivery, moving from local to systemic administration. The system exhibits excellent ultrasound responsiveness, allowing for precise triggering and release of contents at the target site. Simultaneously, its apoptotic cell-mimicking design endows the system with active targeting, while the dual-encapsulation structure ensures stable in vivo circulation, thus achieving a synergistic effect of stable circulation, active targeting, and ultrasound triggering. Attached Figure Description

[0019] Figure 1 A schematic diagram of the synthetic route for PCD-PS / PFH@Lip; Figure 2Fourier transform infrared (FT-IR) spectrum of PCD-PS Figure 3 The phosphorus NMR spectrum of PCD-PS / PFH@Lip ( 31 P NMR); Figure 4 Particle size distribution of the PCD-PS / PFH@Lip delivery system; Figure 5 Potential diagram of the PCD-PS / PFH@Lip delivery system; Figure 6 Transmission electron microscopy (TEM) images of the PCD-PS / PFH@Lip delivery system; Figure 7 Transmission electron microscopy (TEM) images of the PCD-PS / PFH@Lip delivery system after ultrasonic treatment; Figure 8 The fluorescence intensity of macrophages taking up DiL-labeled PCD-PS / PFH@Lip delivery system; Figure 9 The effects of ultrasound response on anti-inflammatory cytokines in lipopolysaccharide-induced macrophages before and after the PCD-PS / PFH@Lip delivery system. Detailed Implementation

[0020] To further explain the present invention, the following specific embodiments are described.

[0021] Example 1: Synthesis of Cyclodextrin Polymer (PCD) Main synthesis steps: Approximately 8.8 mmol of β-cyclodextrin was added to a 250 mL three-necked flask, followed by a slow dropwise addition of a total of 16 mL of NaOH aqueous solution. The mixture was mechanically stirred at room temperature until the solid was completely dissolved, forming a homogeneous, transparent, alkaline viscous liquid. After the β-cyclodextrin was fully dispersed, the stirring speed was increased to 500 rpm, and approximately 85 mmol of epichlorohydrin was added rapidly in one go (controlling the molar ratio of β-cyclodextrin to epichlorohydrin to approximately 1:10). The reaction was continued at 30 ± 2 °C for 24–48 h. The endpoint of the polymerization reaction was determined by a significant and continuous increase in the viscosity of the system (manifested as an increase in stirring resistance). After the reaction was completed, approximately 200 mL of pre-cooled acetone was added to the flask, and the mixture was stirred vigorously for 30 min to allow the polymer to fully precipitate. After standing for 10 min, the supernatant was discarded. The precipitate was then washed twice with anhydrous ethanol (100 mL each time, stirred for 30 min each time) to thoroughly remove small molecule impurities. 100 mmol of acetone was added to the precipitate. The solution was redispersed with mL of deionized water, and the resulting clear, light yellow solution was transferred to a dialysis bag with a molecular weight cutoff of 8-14 kDa. Ultrapure water was used as the external solution, and dialysis was performed for 5 days (the external solution was changed 3 times a day). Finally, the dialysis internal solution was freeze-dried (-50℃, 0.1 mbar, 48 h) to obtain a white, fluffy, spongy PCD solid powder.

[0022] Product characterization: (1) Particle size analysis: Dynamic light scattering (DLS) was used to measure the size of the synthesized PCD aqueous solution. The results showed that the hydrated particle size was concentrated around 10 nm (see Figure 4 The PCD control curves show that the nanoscale polymer was successfully synthesized.

[0023] (2) Confirmation of chemical structure: Fourier transform infrared spectroscopy (FT-IR) analysis showed that PCD was visible at 1022 cm⁻¹. -1 A significant C–O–C stretching vibration absorption peak is observed at the wavenumber (see [link]). Figure 2 This is a characteristic signal of successful crosslinking of epichlorohydrin to form ether bonds, confirming the construction of the target polymer backbone.

[0024] Example 2: Synthesis of phosphorylserine-modified cyclodextrin polymer (PCD-PS) Main synthesis steps: 1 mol of FMOC-L-serine methyl ester was dissolved in the organic solvent N,N-dimethylformamide (DMF), followed by the addition of 0.9 mol of bis(diisopropylamino)(2-cyanoethoxy)phosphine (as a crosslinking agent with phosphorus-containing functional groups), and the reaction was stirred at room temperature for 6 h; 0.9 mol of PCD was added to the above reaction solution, and the mixture was stirred for another 24 h; then 30 wt% H2O2 solution (corresponding to 5 mol) was added to carry out the oxidation step (lasting 2 h); then, pure water was added to dissolve the PCD-PO, and sodium hydroxide was added according to the molar ratio of n(NaOH):n(PCD-PO) = 3.5:1 to carry out the deprotection reaction (lasting 1 h), accompanied by the formation of a white precipitate; the resulting mixture was filtered through a 0.22 μm microporous membrane, dialyzed for 3 days, and then freeze-dried to finally obtain the PCD-PS product.

[0025] Characterization results: (1) Nuclear magnetic resonance phosphorus spectroscopy analysis: the collected products 31 P NMR spectrum. The results show a characteristic resonance peak attributable to phosphorus in phosphorylserine around a chemical shift δ ≈ 7 ppm (see P NMR spectrum). Figure 3 This directly proves the success of the Photoshop retouching.

[0026] (2) Surface charge characteristics: The zeta potential of PCD–PS was measured by dynamic light scattering technique. The potential value of PCD–PS was -32 mV (while the potential of unmodified PCD was close to 0 mV, see [reference]). Figure 5 This significant change in electronegativity strongly confirms that negatively charged PS molecules have been successfully modified into the PCD backbone via covalent bonds.

[0027] (3) Functional group analysis: Infrared spectrum ( Figure 2 It is clearly shown in the image that it is located at 1737 cm. -1 The characteristic absorption peak of the C=O stretching vibration further confirms the formation of phosphate bonds, which corroborates the NMR results.

[0028] Example 3: Construction of the delivery system (PCD–PS / PFH@Lip) Step-by-step preparation process: (1) Effective encapsulation of PFH: 0.5 g of PCD-PS was dispersed in 5 mL of PBS buffer and stirred at 500 rpm for 10 min to promote complete dissolution. Then 0.2 g of PFH was added and the reaction was continued for 30 min under the same stirring conditions. The stable encapsulation of PFH into the hydrophobic cavity of PCD was achieved by relying on the host-guest molecular recognition mechanism, thereby obtaining the PCD–PS / PFH complex suspension.

[0029] (2) Formation of liposome film: Weigh 5 g of soybean lecithin and 4 g of cholesterol respectively, dissolve them together in 20 mL of dichloromethane, transfer this solution to a round bottom flask of a suitable rotary evaporator, set the transfer parameters to 30 ℃ and 100 rpm, evaporate the solvent, and finally form a uniform liposome film on the flask wall (visual inspection confirms no trace of organic solvent residue).

[0030] (3) Hydration and ultrasonic homogenization of the membrane: The entire PCD–PS / PFH complex suspension (total volume approximately 50 mL) prepared in the previous step was added to the liposome membrane. The membrane was stirred at 400 rpm for 30 min in a constant temperature water bath at 37 ℃ to drive the membrane to fully hydrate and transform it into a crude liposome morphology. Subsequently, an ultrasonic cell disruptor (working frequency 20 kHz, power 100 W) was used for treatment, with a total treatment time of 10 min. The temperature rise during the treatment process was strictly controlled to avoid damage to the liposome membrane due to overheating. An intermittent operation mode was adopted: after every 2 min of ultrasonic treatment, the process was paused for 1 min for system cooling, thereby obtaining a uniform final product delivery system (named PCD–PS / PFH@Lip).

[0031] System characterization: (1) Particle size and distribution: Based on the analysis of DLS test data, the statistical average particle size of this delivery system is 122 nm (see Figure 4 The dispersion is ideal and meets the size requirements of nano-drug delivery systems.

[0032] (2) Zeta potential: The measured Zeta potential of the system is -35 mV (see Figure 5 This negative charge property stems from the inherent negative charge of the liposome membrane material used, which helps to inhibit particle aggregation through electrostatic repulsion, thereby improving the physical stability of the formulation.

[0033] (3) Microscopic morphology: Observed with the aid of transmission electron microscopy (TEM), the delivery system is observed to have a regular spherical or near-spherical structure with clear and complete vesicle boundaries (see [reference]). Figure 6 This directly confirms that liposomes successfully encapsulated and formed structurally complete nanoparticles.

[0034] Example 4: Verification of the ultrasonic response performance of the delivery system To verify the ultrasound-responsive release and targeted therapy function of the PCD–PS / PFH@Lip delivery system, the following experiments were conducted: (1) Verification of in vitro ultrasound-triggered structural destruction: Take 1 mL of the PCD–PS / PFH@Lip suspension (concentration 1 mg / mL) prepared in Example 3 and place it in an EP tube. Use an ultrasound therapy device (frequency 1 MHz, intensity 2 W / cm²) to perform the treatment. 2 The samples were irradiated for 3 minutes. Immediately after irradiation, samples were negatively stained with phosphotungstic acid, and morphological changes were observed using transmission electron microscopy (TEM). The results are as follows: Figure 7 As shown, the delivery system without sonication exhibits a complete spherical vesicle structure; after sonication, the liposome shell is clearly ruptured and the structure disintegrates, indicating that ultrasound can effectively trigger the destruction of the liposome membrane and release the internally loaded PCD–PS / PFH complex.

[0035] (2) Evaluation of cell-targeted uptake: RAW264.7 macrophages were seeded in confocal culture dishes and divided into two groups: the experimental group was treated with DiL-labeled PCD–PS / PFH@Lip, and the control group was treated with PCD / PFH@Lip without PS modification. After incubation for 4 h, the cells were washed, and the fluorescence intensity was observed and quantified using laser confocal microscopy. The results showed that ( Figure 8 The fluorescence signal of the experimental group cells was significantly stronger than that of the control group, confirming that PS modification significantly enhanced the macrophages' active uptake of the carrier and verifying its targeting performance in mimicking apoptotic cells.

[0036] (3) Verification of anti-inflammatory efficacy: An inflammation model was established using lipopolysaccharide (LPS) to induce RAW264.7 macrophages. Three groups were set up: a blank control group (cells only), an LPS model group, and an LPS + delivery system + ultrasound group. The latter group was treated with PCD–PS / PFH@Lip (containing an equal amount of dexamethasone as the model drug), and after 2 h of incubation, ultrasound (1 MHz, 1.5 W / cm²) was applied to the cell region. 2 (2 min). After culturing for another 24 h, the cell supernatant was collected, and the expression level of the key anti-inflammatory cytokine IL-10 was detected using an ELISA kit. The results are as follows: Figure 9 As shown, compared with the LPS model group, the delivery system + ultrasound group showed a significant increase in IL-10 secretion, demonstrating that the system can effectively release drugs and enhance anti-inflammatory effects under ultrasound triggering.

[0037] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An ultrasound-responsive apoptosis-inspired biomimetic targeted nanocarrier delivery material, characterized in that, include: A phosphorylserine-modified β-cyclodextrin polymer core, wherein phosphorylserine is modified on the β-cyclodextrin polymer backbone via a phosphate bond; Perfluorohexane encapsulated within the hydrophobic cavity of the β-cyclodextrin polymer via host-guest interactions; And liposomes that coat the outer layer of the core.

2. The ultrasound-responsive apoptosis-inspired biomimetic targeted nanocarrier delivery material according to claim 1, characterized in that, The liposomes are composed of soybean lecithin and cholesterol.

3. The ultrasound-responsive apoptosis-inspired biomimetic targeted nanocarrier delivery material according to claim 2, characterized in that, The mass ratio of soybean lecithin to cholesterol is 5:

4.

4. A method for preparing an ultrasound-responsive apoptosis-inspired biomimetic targeted nanocarrier delivery material according to any one of claims 1 to 3, characterized in that, Includes the following steps: (1) Preparation of phosphorylserine-modified β-cyclodextrin polymer; (2) Under conditions of 0-4°C, the phosphorylserine-modified β-cyclodextrin polymer and perfluorohexane were mixed in phosphate buffer at a mass ratio of 5:2 to form a complex suspension; (3) Dissolve soybean lecithin and cholesterol in an organic solvent, remove the solvent to form a lipid film; (4) Add the complex suspension obtained in step (2) to the lipid film, and perform hydration, ultrasonic treatment and membrane extrusion to obtain the ultrasonic-responsive apoptosis biomimetic targeted nanocarrier delivery material.

5. The preparation method according to claim 4, characterized in that, The synthesis method of the β-cyclodextrin polymer in step (1) is as follows: β-Cyclodextrin was dissolved in an aqueous sodium hydroxide solution, epichlorohydrin was added, and the mixture was reacted at 30 ± 2°C for 24-48 h. After the reaction was completed, acetone was added to precipitate the polymer, which was then washed, dialyzed, and freeze-dried to obtain the final product.

6. The preparation method according to claim 5, characterized in that, The method for synthesizing the phosphorylserine-modified β-cyclodextrin polymer in step (1) is as follows: The product was obtained by reacting FMOC-L-serine methyl ester, a phosphorus-containing crosslinking agent, and a β-cyclodextrin polymer sequentially in an organic solvent, followed by oxidation, deprotection, filtration, dialysis, and freeze-drying.

7. The preparation method according to claim 6, characterized in that, The phosphorus-containing crosslinking agent is bis(diisopropylamino)(2-cyanoethoxy)phosphine; The organic solvent is N,N-dimethylformamide.

8. The preparation method according to claim 4, characterized in that, The organic solvent mentioned in step (3) is dichloromethane.

9. The preparation method according to claim 4, characterized in that, In step (4), the hydration is carried out at 37°C and 400 rpm for 30 min.

10. The preparation method according to claim 4, characterized in that, In step (4), the ultrasonic treatment is performed using an ultrasonic cell disruptor with a working frequency of 20 kHz and a power of 100 W. The intermittent operation mode is adopted, with a 1-minute pause after every 2 minutes of ultrasonic treatment, and a total treatment time of 10 minutes.

Images