A hof dual drug-loaded ultrasound-responsive targeted nebulization delivery system

Abstract

The present application relates to a kind of HOF double drug ultrasonic response targeted atomization delivery system, and its preparation method and application.The system takes amino functionalization HOF-101 nanocrystal as core carrier, nucleic acid drug is loaded on the surface and mesoporous of carrier by electrostatic adsorption, and active targeting ligand and hydrophilic polysaccharide are covalently grafted on the surface of carrier.The system realizes the spatial separation loading of double drugs with different physical and chemical properties and the sequential release of space-time controllable, has significant ultrasonic response, and has wide application prospect in the preparation of targeted inhalation drug for treating lung cancer and other solid tumors.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine delivery systems, and more particularly to an ultrasonically responsive dual-drug co-loaded targeted atomization nanodelivery system based on a hydrogen-bonded organic framework, as well as the preparation method and application of the system. Background Technology

[0002] Hydrogen-bonded organic frameworks (HOFs) are a class of ordered framework materials constructed solely from organic building blocks through intermolecular hydrogen bonds. They possess characteristics such as high specific surface area, tunable pore size, and low density, showing promising applications in gas adsorption and separation, molecular devices, fluorescent probes, proton conduction, and drug delivery. Compared to traditional metal-organic frameworks (MOFs), HOFs offer advantages such as better biocompatibility, higher degradability, and milder synthesis conditions, making them a research hotspot for novel drug carriers.

[0003] In recent years, significant progress has been made in the field of tumor immunotherapy. Tumor-associated macrophages (TAMs), as the most abundant immune cell population in the tumor microenvironment, play a crucial role in tumor growth, metastasis, and immunosuppression. Reprogramming TAMs from the pro-tumor M2 type to the anti-tumor M1 type has become an important strategy in tumor immunotherapy. However, existing technologies suffer from the following problems: First, single-drug delivery efficiency is low; current nanocarriers typically only carry a single drug, making it difficult to simultaneously achieve the synergistic effects of gene therapy (mRNA delivery) and chemotherapy (small molecule drug delivery). Second, drug release lacks spatiotemporal controllability; traditional drug carriers exhibit passive release behavior, failing to achieve precise, on-demand, targeted, and timed drug release. Furthermore, lung-targeted delivery is challenging; existing nano-formulations have poor atomization performance and uneven aerosol particle size distribution, making effective lung deposition difficult. Finally, there is a contradiction between carrier structural stability and responsiveness; overly stable carriers struggle to release drugs, while unstable carriers suffer from premature drug leakage.

[0004] HOF-101 is a dimer of 1,3,6,8-tetra(4-carboxyphenyl)pyrene (H4TBAPy) building units via carboxylic acid hydrogen bonds (R2). 2 (8) The two-dimensional layered hydrogen-bonded organic framework formed by the self-assembly of pyrene π-π stacking interactions has a rhombic one-dimensional pore structure. Studies have shown that the ultrasonic dissociation threshold of HOF-101 is about 3.94 MPa, and controllable drug release can be achieved under ultrasonic stimulation, but its application as a dual-drug co-loaded targeted atomization delivery system has not been reported. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide an ultrasound-responsive hydrogen-bonded organic framework dual-drug co-loading targeted atomization nanodelivery system. This system not only achieves spatial separation and loading of nucleic acid drugs and small molecule chemotherapeutic drugs and ultrasound-triggered "on-demand release", but also possesses excellent atomization stability and microenvironment targeting.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a nanodelivery system, specifically comprising an ultrasound-responsive hydrogen-bonded organic framework dual-drug co-loaded targeted atomization nanodelivery system, comprising: a core carrier: amino-functionalized HOF-101 nanocrystals (HOF-101-NH2); a first drug: a nucleic acid drug, such as KMT2C mRNA, loaded onto the surface and mesopores of the core carrier via electrostatic adsorption; a second drug: a small molecule chemotherapy drug, such as alectinib, loaded into the hydrophobic micropores of the core carrier via solvent diffusion; and a targeting modification layer: an active targeting ligand (such as S-adenosylmethionine, SAM) and a hydrophilic polysaccharide (such as CM-Dex), grafted onto the surface of the core carrier via covalent amide bonds.

[0007] Secondly, the present invention provides a method for preparing a nanodelivery system, comprising the following steps: 1. Synthesis of HOF-101 nanocrystals: The organic building block 1,3,6,8-tetra(4-carboxyphenyl)pyrene (H4TBAPy) was dissolved in a good solvent and injected into a pre-cooled antisolvent under stirring to carry out antisolvent crystallization. The solid was collected and freeze-dried to obtain rod-shaped HOF-101 nanocrystals. 2. Amino functionalization modification: The HOF-101 nanocrystals were dispersed in a buffer solution, a polyamine crosslinking agent was added and the mixture was shaken and purified to obtain HOF-101-NH2; 3. First drug loading: The nucleic acid drug aqueous solution and the HOF-101-NH2 are mixed at low temperature in a weakly acidic buffer solution. The nucleic acid drug is bound to the carrier surface and mesopores by electrostatic adsorption. The primary drug-loaded powder is obtained by freeze drying. 4. Second drug loading: The small molecule chemotherapy drug is dissolved in an organic solvent and added dropwise to the aqueous suspension of the primary drug-loaded powder containing a surfactant. The small molecule chemotherapy drug is then allowed to enter the hydrophobic micropores by solvent diffusion. The mixture is then ultrasonically dispersed and centrifuged to obtain a dual-drug-loaded nanoplatform. 5. Surface targeting modification: The dual drug-carrying nanoplatform is suspended in a buffer solution, and an active targeting ligand and a hydrophilic polysaccharide are added for pre-adsorption. Then, a condensing agent is added for amidation covalent coupling, followed by dialysis purification to obtain the nanodelivery system.

[0008] Thirdly, this invention provides an application and formulation of an ultrasound-responsive dual-drug co-loaded targeted atomization nanodelivery system based on a hydrogen-bonded organic framework. Specifically, this invention provides an atomized formulation comprising the aforementioned nanodelivery system and pharmaceutically acceptable isotonic agents, buffer solutions, and surfactants; the application of the nanodelivery system or atomized formulation in the preparation of drugs for treating tumor-associated macrophage (TAM)-related diseases, preferably lung cancer, breast cancer, or colorectal cancer.

[0009] Compared with the prior art, the beneficial effects of the present invention are as follows: First, this invention cleverly utilizes the unique hierarchical pore structure of HOF-101 (micropores of ~0.7 nm and mesopores of 5-8 nm) to encapsulate small molecule chemotherapy drugs inside hydrophobic micropores and adsorb large molecule nucleic acid drugs onto hydrophilic mesopores and surfaces, thus achieving efficient co-loading of two drugs with completely different physicochemical properties. Secondly, the system of the present invention exhibits extremely high drug-locking ability under normal physiological conditions (24-hour background leakage rate <5%). Once local ultrasound stimulation is applied in vitro, the framework of HOF-101 rapidly dissociates due to the sensitivity of the hydrogen bond network to mechanical shear force, thereby achieving precise "on-demand" release of the drug. Finally, in this invention, the modified nanoplatform exhibits good colloidal stability. After atomization by an atomizer, the aerosol particle size is precisely controlled at 1.5-3.5μm, making it easy to deposit in the deep lungs and alveolar areas, directly targeting lung TAMs. Attached Figure Description

[0010] Figure 1 This is a scanning electron microscope (SEM) characterization image of HOF-101 nanocrystals.

[0011] Figure 2 Transmission electron microscopy (TEM) images of HOF-101 nanocrystals are shown; where a is a TEM image of a single crystal (scale bar 200 nm); c is a TEM image of multiple crystals (scale bar 500 nm); and b is a macroscopic photograph of the aqueous yellow suspension. The rod-like morphology, high crystallinity, and good dispersibility provide an ideal material basis for drug loading and nebulized drug delivery.

[0012] Figure 3 The nitrogen adsorption-desorption isotherm of HOF-101 is shown; HOF-101 exhibits typical type IV adsorption behavior.

[0013] Figure 4The image shows the BJH pore size distribution curve of HOF-101. The BJH pore size distribution exhibits a multi-peak distribution in the range of 2-20 nm, with the main peak located at 5-8 nm. HK / SF micropore analysis reveals ultrafine pores of 0.66-0.75 nm. The synergistic effect of micropores and mesopores enables the partitioned loading of small molecule drugs and large molecule mRNAs, and achieves time-sequential release through the differentiated diffusion resistance of different pore sizes.

[0014] Figure 5 Scanning electron microscopy (SEM) characterization and macroscopic morphology of the compound formulation (CPP) are shown in the following images: (a) is a low-magnification SEM image (50,000×, scale bar 1 μm), showing that the CPP retains its rod-like morphology after dual-drug loading and surface-targeting modification; (b) is a high-magnification SEM image (100,000×, scale bar 500 nm), further revealing details of surface roughening of the rod-like particles and slight irregularities at the edges, confirming the presence of the surface modification layer; the crystal framework remains intact, demonstrating the good structural stability of the HOF-101 framework during drug loading and functionalization modification; (c) is the lyophilized CPP powder, which is a uniform yellow powder with a fine texture and no clumping, demonstrating the feasibility of the freeze-drying process and the good powder flowability of the product, facilitating the preparation and storage of subsequent atomized formulations.

[0015] Figure 6 Transmission electron microscopy (TEM) characterization of the compound formulation (CPP), where a is a high-magnification TEM image (scale bar 200 nm) showing the fine structure of a single CPP particle; b is a low-magnification TEM image (scale bar 500 nm) showing the aggregation state of multiple CPP particles.

[0016] Figure 7EDS elemental mapping and quantitative analysis of the compound formulation (CPP) were performed. Image a shows a multi-element overlay mapping image, displaying the spatial distribution of six elements (P (cyan), S (purple), F (blue), O (yellow), N (green), and C (red)) on the CPP surface. Image b shows a SEM image, displaying the morphology of the rod-shaped particles, serving as the spatial localization reference for elemental mapping. Image c shows the C element mapping (red), exhibiting the strongest and most uniform distribution, with dense signals covering the entire rod-shaped particle region, at a content of 55.25 wt%, derived from the HOF-101 organic ligand backbone, carboxymethyl dextran, mRNA ribose and bases, alectinib, and SAM. Image d shows the N element mapping (green), showing a dense and uniform distribution, at a content of 20.28 wt%, significantly higher than pure HOF-101, providing direct evidence of successful loading of nitrogen-rich drugs (mRNA nucleobases, alectinib piperazine ring and amino group, and SAM adenosine moiety). Image e shows the P element mapping (cyan), at a content of 2.31 wt%, a specific marker of successful mRNA loading. Since HOF-101, dextran, alectinib, and SAM do not contain P, the P element only comes from the mRNA ribose-phosphate backbone and is evenly distributed, indicating that mRNA is widely adsorbed on the CPP surface without local aggregation; f is the S element mapping (purple), present in trace amounts (0.34 wt%), possibly from the methionine moiety of SAM; g is the O element mapping (yellow), with a content of 21.55 wt%, showing a uniform distribution, originating from the carboxyl group of HOF-101 ligand, the hydroxyl and carboxyl groups of carboxymethyl dextran, the mRNA phosphate backbone, and the ribose hydroxyl groups; h is the F element mapping (blue), present in trace amounts (0.28 wt%), possibly from the fluorine substituent of alectinib or residual reagents used in preparation.

[0017] Figure 8Comparison of SEM / TEM morphology of CPP before and after ultrasonic treatment. Image a shows the SEM image before ultrasonic treatment (100,000×, scale bar 500 nm), showing that the CPP maintains a typical rod-shaped crystal morphology with a smooth and regular surface, a length of approximately 500 nm-1 μm, a diameter of approximately 100-200 nm, good dispersion, and no obvious structural defects, indicating a complete and stable carrier structure. Image b shows the TEM image before ultrasonic treatment (scale bar 200 nm), further confirming the clear outline and uniform electron density distribution of individual crystals, sharp crystal edges, no obvious internal pores or cracks, and a dense and complete framework structure. Image c shows the SEM image after ultrasonic treatment (80,000×, scale bar 500 nm), showing significant morphological changes in the rod-shaped particles, with obvious surface roughening, edge breakage, and size inhomogeneity. Some particles exhibit cracks or delamination, and the crystal integrity is significantly compromised. Image d shows the TEM image after ultrasonic treatment (scale bar 200 nm). The image (nm) shows that the edges of individual crystals become blurred, and there are obvious contrast changes inside. The electron density in some areas is reduced, reflecting the destructive effect of ultrasonic cavitation on the hydrogen bond network of HOF-101. The crystal skeleton undergoes local dissociation and fragmentation.

[0018] Figure 9 Alectinib release curves. The control group without ultrasound (gray curve) released only about 35% within 72 h, exhibiting typical slow diffusion release behavior; the ultrasound-treated group (red curve) achieved a release rate of about 75% within the same time period, with significant jumps in release rate at the ultrasound application time points (arrow marks, approximately 12, 24, 36, 48, 60, and 72 h), indicating that ultrasound cavitation instantaneously opened hydrophobic channels to promote the diffusion of small molecule drugs.

[0019] Figure 10 KMT2C mRNA release curves. The control group without ultrasound (gray curve) released approximately 45% at 72 h; the ultrasound-treated group (red curve) reached approximately 62%. As a macromolecule, mRNA release in mesoporous structures is relatively slow, but it still exhibits significant ultrasound responsiveness. The accelerated release at the ultrasound treatment time point demonstrates the rapid desorption of electrostatically adsorbed mRNA under the perturbation of the carrier structure. Detailed Implementation

[0020] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0021] Example 1: Preparation and Characterization of HOF-101 Nanocrystals 30.0 mg of 1,3,6,8-tetra(4-carboxyphenyl)pyrene (H4TBAPy) was completely dissolved in 3.0 mL of N,N-dimethylamide (DMF). The solution was then added dropwise at a rate of 0.5 mL / min to 12.0 mL of ultrapure water pre-cooled to 4 °C while stirring at a constant speed of 1000 rpm. After the addition was complete, the mixture was stirred for another 10 min to induce sufficient antisolvent crystallization. After the reaction was complete, the suspension was centrifuged at 12000 g for 5 min, the supernatant was discarded, and the precipitate was washed three times each with acetone, anhydrous ethanol, and ultrapure water. The precipitate was then dried in a freeze dryer (-50 °C) for 48 h to obtain white solid HOF-101 nanocrystals.

[0022] Crystal characterization: SEM and TEM analyses showed that the HOF-101 prepared in this invention has a highly uniform rod-like structure with a smooth and regular surface, and a length distribution between 500 nm and 1 μm. Individual crystals exhibit a clear rod-like outline and a uniform electron density distribution. It forms a stable yellow suspension in aqueous phase, demonstrating good dispersibility and colloidal stability. The nitrogen adsorption-desorption isotherm curves show typical type IV adsorption behavior, and its BET surface area is calculated to be 497.73 m². 2 / g, total pore volume is 0.401cm³ 3 / g, with a micropore volume of 0.154 and a mesopore volume of 0.267; BJH pore size analysis showed a multi-peak mesopore distribution in the range of 2-20 nm (the main peak is located at 5-8 nm), and HK model analysis confirmed the existence of micropores of 0.66-0.75 nm, indicating the successful construction of a micropore-mesopore hierarchical structure. XRD patterns showed multiple sharp diffraction peaks in the low-angle region (5-15°), with the strongest peaks located at approximately 8° and 12°, matching the theoretical crystal structure of HOF-101. See Figure 1-4 .

[0023] Figure 1 The image shows the scanning electron microscope (SEM) characterization of HOF-101 nanocrystals (accelerating voltage 20.00 kV, working distance 9.1 mm). HOF-101 exhibits a typical rod-shaped crystal morphology, with a length of approximately 500 nm-1 μm. The surface is smooth and the edges are regular, with good dispersion, and some crystals are aggregated in bundles. Under high magnification (80,000×), the fine striation structure on the crystal surface can be clearly observed, reflecting the high crystallinity and long-range order of the HOF-101 framework.

[0024] Figure 2Images show transmission electron microscopy (TEM) characterization of HOF-101 nanocrystals. Image a shows a single HOF-101 crystal (scale bar: 200 nm), displaying a clear rod-like outline and uniform electron density distribution, sharp edges, and no obvious internal defects, confirming the high crystallinity and structural integrity of the HOF-101 framework. Image b shows a macroscopic photograph of a stable yellow suspension formed by HOF-101 in aqueous solution. No precipitation occurred after 48 h of standing, demonstrating the material's excellent dispersibility and colloidal stability. This unique rod-like morphology, combined with nano- to micron-scale dimensions and porous structure, makes HOF-101 ideal as a drug carrier for efficient loading. Its regular morphology also facilitates the formation of a uniform aerosol particle size distribution during nebulization, ensuring consistent and controllable lung deposition. Image c shows TEM images of multiple HOF-101 crystals (scale bar: 500 nm). The nm) showed that the rod-shaped particles were dispersed or slightly aggregated, with a uniform size distribution and no obvious agglomeration or morphological distortion, indicating that HOF-101 prepared by the antisolvent crystallization method has good dispersibility and morphological controllability.

[0025] Figure 3 To determine the nitrogen adsorption-desorption isotherm curves, a fully automated specific surface area and pore size analyzer was used to measure the nitrogen adsorption-desorption isotherms of HOF-101 at 77 K liquid nitrogen temperature. The samples were pretreated by vacuum degassing at 150°C for 12 h to remove adsorbed moisture and impurities from the surface. The relative pressure (P / P0) range was 0–1.0, and the adsorption capacity was determined using the static volumetric method. Figure 4 The results show the calculation results of the pore structure parameters. The specific surface area was calculated using the BET multi-point method, the mesopore size distribution was calculated using the BJH method, and the micropore size distribution was calculated using the HK / SF method.

[0026] Example 2: Amino-functionalization modification of the core carrier (HOF-101-NH2) 100.0 mg of the HOF-101 nanocrystals prepared in Example 1 were weighed and uniformly dispersed in 20.0 mL of PBS buffer (pH 8.0) using ultrasound. Then, 1,4-butanediamine was added to the system to bring the final concentration to 5 mM. The mixture was placed in a constant-temperature shaker at 25°C and reacted in the dark for 48 h. During this period, the supernatant was discarded by centrifugation every 12 h, and the solution was replaced with fresh PBS containing the same concentration of 1,4-butanediamine. After the reaction was completed, the product was collected by centrifugation at 15000 g for 20 min, washed five times with ultrapure water to completely remove the free amine, and then freeze-dried to obtain HOF-101-NH2.

[0027] Dynamic light scattering (DLS) measured its hydrated particle size to be approximately 259.6 nm; the Zeta potential changed from the negative potential of the original HOF-101 to +14.5 mV (pH 7.4), indicating that the surface carboxyl groups successfully underwent hydrogen bond self-assembly with 1,4-butanediamine, introducing protonable amino groups.

[0028] Example 3: Sequential loading of the first drug (mRNA) and the second drug (alectinib) mRNA loading: Take 500 μL of KMT2C mRNA aqueous solution with a concentration of 0.5 mg / mL and add it directly to PBS buffer (1.0 mL, pH 6.5) containing 10 mg HOF-101-NH2. Gently mix at 4°C for 60 min. After pre-freezing the mixture at -20°C for 2 h, transfer it to a freeze dryer and dry at -50°C for 24 h, and then dry at -10°C for 12 h to obtain mRNA@HOF-101 lyophilized powder.

[0029] Alectinib loading: 2.0 mg of alectinib was completely dissolved in 200 μL of anhydrous ethanol. Under low-speed stirring, this ethanol solution was added dropwise to a 2.0 mL aqueous system (containing 0.5 wt% Tween-80, pH 7.2) containing all the above-mentioned mRNA@HOF-101 lyophilized powder, and the mixture was placed in a shaker at 25°C in the dark for 4 h. Intermittent sonication (100 W, 3 s on, 3 s off, total 5 min) was then performed to promote uniform drug diffusion. Finally, the mixture was centrifuged at 13000 rpm for 10 min, the solid was washed three times with cold PBS, and freeze-dried to obtain the dual-drug-loading platform K / A@HOF-101.

[0030] Characterization and drug loading determination: The hydrated particle size of K / A@HOF-101 was 266.5 nm, only slightly increased by 2.7% compared to before modification; the zeta potential decreased to +3.3 mV, confirming that the negatively charged mRNA adsorbed and neutralized part of the positive charge of the amino groups. The standard curve of alectinib at 340 nm was determined by UV-Vis spectrophotometry using the washing supernatant, and the result was y = 0.015x + 0.2408 (R²). 2 =0.9854); the standard curve for KMT2C mRNA is y = 0.03432x + 1.11022 × 10⁻⁶. -16 (R) 2 =0.9999), and calculations showed that the encapsulation efficiency of the mRNA was greater than 85%, with the drug loading of alectinib remaining stable at around 12.5 wt%. Agarose gel electrophoresis showed that the mRNA bands in K / A@HOF-101 were intact, without degradation or diffusion.

[0031] Example 4: Surface-targeting modification of a dual-drug delivery platform 20.0 mg of K / A@HOF-101 prepared in Example 3 was resuspended in 5.0 mL of PBS buffer (pH 7.4), and 15.0 mg of S-adenosylmethionine (SAM) and 12.0 mg of carboxymethyl dextran (CM-Dex) were added sequentially. The mixture was incubated at room temperature for 2 h for electrostatic pre-adsorption. Then, 10.0 mg of EDC·HCl and 6.0 mg of NHS were added to the system, and the pH was adjusted to 6.5 with dilute hydrochloric acid. The reaction was carried out under nitrogen protection at 25°C in the dark for 6 h. After the reaction, the reaction solution was transferred to a dialysis bag (MWCO 100 kDa) and dialyzed with ultrapure water for 48 h, with the water changed every 6 h. Finally, the solution was freeze-dried to obtain the final targeted nanodelivery system (named CPP nanoplatform).

[0032] Measurements revealed that the hydrated particle size of CPP was 270.4 nm, and the PDI was 0.18. The Zeta potential completely flipped to -4.6 mV, confirming successful surface coating with the negatively charged hydrophilic polysaccharide CM-Dex. EDS elemental analysis showed a P content of 2.31 wt%, a S content of 0.34 wt%, and a F content of 0.28 wt%. XPS full-spectrum and P 2p high-resolution spectra exhibited a strong single characteristic peak at 133 eV (corresponding to the phosphate group of mRNA), and a significant F 1s peak at 688 eV (corresponding to the fluorine atom of alectinib). FTIR spectra showed a broad hydroxyl absorption peak introduced by CM-Dex in the 3000-3500 cm⁻¹ region. XRD patterns showed that the characteristic diffraction peaks of the modified HOF-101 skeleton remained intact. Results are shown below. Figure 5-6 .

[0033] EDS elemental mapping indicates successful dual-drug loading and targeted modification of CPP. The three main elements, C, N, and O (totaling 96.08%), showed a high degree of co-localization, consistent with the chemical fact that C, N, and O coexist in organic molecules. Although P content was low, its uniform distribution indicated widespread rather than localized mRNA adsorption. The C / N ratio of approximately 2.7:1 reflects the comprehensive composition of the organic framework, polysaccharide, nucleic acid, and drug; the N / P ratio of approximately 8.8:1 verifies the rationality of mRNA loading. EDS, as a bulk analysis technique (detection depth 1-2 μm), has a wider detection range than XPS (5-10 nm), yet still detected a high N content of 20.28% and a P content of 2.31%, indicating that drug and mRNA loading is not limited to a thin surface layer but is distributed throughout a certain depth range. This is attributed to the porous structure of HOF-101, which allows small molecule drugs to permeate into the pores, and mRNA forms a coating layer of a certain thickness at the pore openings and surface through electrostatic adsorption. Results are shown below. Figure 7 .

[0034] In summary, EDS data, XPS (surface elemental changes), FTIR (chemical bond formation), XRD (framework structure retention), and SEM / TEM (morphological stability and surface coating) corroborate each other at different levels, providing the most direct chemical evidence from the perspective of bulk elemental composition and spatial distribution. Together, they construct a complete chain of evidence to confirm the successful preparation of the TAM-targeted atomization HOF-101 dual-drug platform.

[0035] Example 5: Formulation and Performance Evaluation of Nebulized Formulation Weigh 20.0 mg of the CPP nanoplatform prepared in Example 4 and add it to 10.0 mL of an aqueous solution containing 90.0 mg sodium chloride, 10 mM phosphate buffer (pH 7.4), and 0.02 wt% polysorbate 80. Disperse the solution ultrasonically for 5 min (80 W) in an ice-water bath, then filter through a 0.45 μm syringe filter to obtain the final atomized solution formulation (concentration 2 mg / mL).

[0036] Laser diffraction particle size analyzer determined that the aerodynamic median particle size (MMAD) of the aerosol droplets formed after atomization by a clinical medical nebulizer was 2.45 μm (distributed between 1.5 and 3.5 μm). After being stored at 4°C for 7 days, the hydrated particle size change rate of the formulation was less than 5%, demonstrating excellent atomization stability and resistance to shear diffusion.

[0037] Example 6 Evaluation of Ultrasonic Response Release Performance The nebulized formulation prepared in Example 5 was placed under an ultrasonic generator and subjected to ultrasonic stimulation at a frequency of 1.0 MHz and a sound pressure level of 4.0 MPa for 3 min. SEM observation showed that the surface was smooth and regular before ultrasound, with a length of approximately 500 nm-1 μm. TEM images further confirmed the clear outline and uniform electron density distribution of individual crystals, indicating that the carrier structure was intact and stable. However, after ultrasonic treatment, the regular rod-shaped crystals underwent severe surface roughening, edge breakage, and delamination. TEM images showed that the edges of individual crystals became blurred, and there were obvious contrast changes inside, confirming that the hydrogen bond framework underwent structural dissociation under ultrasonic mechanical waves. This reflects the destructive effect of ultrasonic cavitation on the HOF-101 hydrogen bond network. This reversible structural response provides a physical basis for the triggered release of the drug.

[0038] Drug release was measured using in vitro dialysis with PBS (pH 7.4) containing 0.1% Tween-80 as the release medium. In the control group without ultrasound, the cumulative release rates of alectinib and KMT2C mRNA within 72 h were only 32.1% and 38.4%, respectively, with the background leakage rate strictly controlled below 5% every 24 h. In the experimental group with ultrasound stimulation, the cumulative release rate of alectinib reached 76.8% and the cumulative release rate of mRNA reached 65.2% within 72 h, successfully achieving spatiotemporally controllable ultrasound-responsive release. This provides an ideal nanoplatform for a time-sequential combined therapy strategy of mRNA-initiated gene therapy followed by alectinib synergistic chemotherapy, and for spatiotemporally controllable drug therapy in precision medicine. Results are shown below. Figure 8-10 .

[0039] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A HOF dual-drug delivery system with ultrasonic response and targeted atomization, characterized in that, include: Core carrier: The core carrier is amino-functionalized HOF-101 nanocrystals; First drug: The first drug is a nucleic acid drug, which is loaded onto the surface and mesopores of the core carrier by electrostatic adsorption; Second drug: The second drug is a small molecule chemotherapy drug, which is loaded in the hydrophobic micropores of the core carrier; Targeted modification layer: The targeted modification layer includes an active targeting ligand and a hydrophilic polysaccharide, which are grafted onto the surface of the core carrier via covalent amide bonds.

2. The delivery system as claimed in claim 1, characterized in that, The nucleic acid drug is KMT2C mRNA; the small molecule chemotherapy drug is alectinib; the active targeting ligand is S-adenosylmethionine; and the hydrophilic polysaccharide is carboxymethyl glucan.

3. The delivery system as described in claim 1 or 2, characterized in that, The amino-functionalized HOF-101 nanocrystals are rod-shaped crystals with a microporous-mesoporous hierarchical pore structure formed by the self-assembly of 1,3,6,8-tetra(4-carboxyphenyl)pyrene building units through intermolecular hydrogen bonds, and surface amino groups are introduced through the self-assembly of surface carboxyl groups using polyamine crosslinking agents.

4. The delivery system as claimed in claim 3, characterized in that, The polyamine crosslinking agent is 1,4-butanediamine; the zeta potential of the amino-functionalized HOF-101 nanocrystals at pH 7.4 is +10 mV to +20 mV.

5. The delivery system as claimed in claim 1, characterized in that, The zeta potential of the first drug after loading is +1 mV to +5 mV; the overall hydrated particle size of the delivery system is 250-350 nm, the overall zeta potential is -5 mV to -20 mV, and the polymer dispersibility index (PDI) is < 0.

3.

6. The delivery system as claimed in claim 1, characterized in that, The delivery system is ultrasonically responsive, exhibiting frame disintegration under ultrasonic stimulation at frequencies of 0.5-2.0 MHz and sound pressure levels greater than 3.94 MPa; the background drug leakage rate is < 5% under no ultrasonic conditions and the total drug release rate is > 70% under ultrasonic triggering.

7. The preparation method of the HOF dual-drug-carrying ultrasonic-responsive targeted atomization delivery system as described in claim 1, characterized in that, Includes the following steps: S1. Dissolve 1,3,6,8-tetra(4-carboxyphenyl)pyrene in a good solvent, and inject it into a pre-cooled antisolvent under stirring to carry out antisolvent crystallization. After washing and freeze-drying, rod-shaped HOF-101 nanocrystals are obtained. S2. The HOF-101 nanocrystals were dispersed in an alkaline buffer solution, a polyamine crosslinking agent was added and the mixture was shaken to react. After purification, amino-functionalized HOF-101 nanocrystals were obtained. S3. The nucleic acid drug aqueous solution and the amino-functionalized HOF-101 nanocrystals are mixed at low temperature in a weakly acidic buffer solution for electrostatic adsorption, and then freeze-dried to obtain a primary drug-loaded powder. S4. Dissolve the small molecule chemotherapy drug in an organic solvent, add it dropwise to the aqueous suspension of the primary drug-loaded powder containing a surfactant for solvent diffusion loading, disperse by ultrasonication and collect by centrifugation to obtain a dual-drug-loaded nanoplatform. S3. The dual-drug-carrying nanoplatform is suspended in a buffer solution, and an active targeting ligand and a hydrophilic polysaccharide are added sequentially for pre-adsorption. Then, a condensing agent is added for amidation covalent coupling, followed by dialysis purification and freeze-drying to obtain the delivery system.

8. The preparation method according to claim 7, characterized in that, The good solvent is N,N-dimethylformamide, and the antisolvent is ultrapure water.

9. An atomizing formulation, characterized in that, The delivery system comprising any one of claims 1 to 6, and pharmaceutically acceptable isotonic agents, buffers, and surfactants.

10. The use of the delivery system according to any one of claims 1 to 6, or the atomized formulation according to claim 9, in the preparation of a medicament for treating tumor-associated macrophage-related diseases, wherein the disease is lung cancer, breast cancer, or colorectal cancer.

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