Targeted drug-loaded microbubbles, and preparation method and application thereof

Abstract

The application provides a targeted drug-loaded microbubble as well as a preparation method and application thereof, and belongs to the technical field of methods for preparing special forms of medicines. The targeted drug-loaded microbubble comprises a PEG shell, a lipid bilayer and a DBCO anchor point, the lipid bilayer is loaded with PTX, the lipid bilayer is located in the PEG shell, and the DBCO anchor point is located on the PEG shell. The targeted drug-loaded microbubble provided by the application can regulate the shape and size of the microbubble, is applied to ultrasonic contrast diagnosis imaging, has excellent targeted killing effect on tumors, and has good biological safety.

Description

Technical Field

[0001] This application relates to a targeted drug-loaded microbubble, its preparation method and application, belonging to the technical field of methods for making drugs into special forms of administration. Background Technology

[0002] Paclitaxel (PTX) is a broad-spectrum antitumor drug, characterized by its wide biodistribution and significant adverse reactions, limiting its clinical use. Targeted drug delivery systems can improve the efficacy of chemotherapy drugs by modifying the diversity of carrier material structures. Numerous studies have reported that unmodified liposomes, nanoparticles, micelles, and other traditional drug delivery systems facilitate drug accumulation in tumors, but the drugs readily accumulate in tissues such as the liver, spleen, and kidneys. Therefore, there is an urgent need to develop delivery strategies that can improve the pharmacokinetic behavior of PTX, enhance its tumor targeting, and reduce systemic toxicity. However, a key limitation is that these traditional carriers and the drugs they carry still tend to accumulate significantly in tissues rich in the mononuclear phagocyte system (MPS), such as the liver, spleen, and kidneys, leading to off-target toxicity and reducing the amount of drug reaching the target lesion, thus affecting efficacy.

[0003] Ultrasound-targeted microbubble destruction (UTMD) is a technique that facilitates the delivery of targeted groups and drugs, offering advantages such as high efficiency, targeting, safety, and good reproducibility. Examples include the polypeptide microbubbles provided by CN113929740A for use in ultrasound imaging diagnostics, and the nanovesicles provided by CN104725645A that simultaneously enhance ultrasound imaging capabilities and sensitivity. However, regarding systemic toxicity during drug delivery, the main drawback of these microbubbles or vesicles is the lack of targeting during drug delivery. Therefore, there is an urgent need to develop drugs that combine targeted drug delivery systems with UTMD technology to improve the tumor targeting of paclitaxel and reduce systemic toxicity. Summary of the Invention

[0004] In view of this, the first objective of this application is to provide a targeted drug-loaded microbubble, which uses alkynyl DBCO modified on a PEG group as a raw material. The formed targeted drug-loaded microbubble can be irradiated with ultrasound after being inflated, and its morphology and size can be controlled.

[0005] Specifically, this application is implemented through the following scheme:

[0006] A targeted drug-loaded microbubble includes a PEG shell, a lipid bilayer, and a DBCO anchor point, wherein PTX is loaded on the lipid bilayer, the lipid bilayer is located inside the PEG shell, and the DBCO anchor point is located on the PEG shell.

[0007] The aforementioned targeted drug-loaded microvesicles not only possess a PEG shell, but also contain a lipid bilayer loaded with PTX within the shell. Furthermore, the shell is modified with alkyne-based DBCO groups, forming multiple DBCO anchor sites. These anchor DBCO groups can undergo specific bioorthogonal reactions with azide groups introduced onto the surface of tumor cells through metabolic glycoengineering, thereby achieving precise drug release. This results in a highly selective, rapid reaction kinetic, and biocompatible system, constructing an efficient and safe targeted drug delivery system that ultimately achieves targeted, imaging, and therapeutic effects.

[0008] The applicant's second objective is to provide a method for preparing the aforementioned targeted drug-loaded microbubbles, the steps of which are as follows:

[0009] Step 1, DNCO-NHS ( DMSO solution, DSPE-PEG-NH2 ( Mix the product DSPE-PEG-DBCO with a DMSO solution containing lipid tails (R is the lipid tail), stir overnight at room temperature, dialyze the resulting mixture, and freeze-dry under vacuum to obtain the product DSPE-PEG-DBCO.

[0010] The structure of the product DSPE-PEG-DBCO is represented as follows:

[0011] ,or Mainly includes the lipid tail ( ), anion head part ( ), PEG shell portion ( ) and click the anchor point part ( ).

[0012] Step two, the DSPE-PEG-DBCO and DSPE-PEG2000 prepared in step one above ( ), DSPC ( Paclitaxel (PTX) was mixed in chloroform, and then the chloroform was evaporated and dried under a nitrogen stream to remove the chloroform, resulting in a dried lipid film.

[0013] Step 3: After adding the dried lipid membrane to the degassing buffer, heat it at 60~65℃ to obtain a clear liposome suspension.

[0014] Step four: The liposome suspension is placed in a sealed container, and the air inside the bottle is replaced with C3F8 gas. The mixture is then mechanically shaken to obtain the finished targeted drug-loaded microbubbles, denoted as P@MB-DBCO.

[0015] Preferred:

[0016] In step one,

[0017] The equivalent ratio of DBCO-NHS to DSPE-PEG-NH2 is 1:5.

[0018] The dialysis process involves first dialyzing the mixture in DMSO, and then dialyzing it in distilled water.

[0019] In step two,

[0020] The molar ratio of DSPC, DSPE-PEG-DBCO, and DSPE-PEG2000 is 5:5:90.

[0021] The evaporation and drying process further includes rotary evaporator treatment or overnight incubation to remove all chloroform. Preferably, the rotary evaporator rotates at 60-80 rpm, and the treatment or overnight incubation temperature is 45-50°C.

[0022] In step three,

[0023] The degassing buffer is a mixture of NaCl, isopropanol, and glycerol in a volume ratio of 8:1:1.

[0024] The heat treatment includes: first heating and shaking in a water bath, and then heating and shaking in an ultrasonic cleaner. More preferably, the water bath temperature is 62~65℃ and the treatment time is 10~15min; the ultrasonic cleaner temperature is 60~62℃ and the treatment time is 20~30min.

[0025] In step four, mechanical vibration is performed in a dental amalgam mixer.

[0026] The applicant's third objective is to provide the application of the aforementioned targeted drug-loaded microbubbles in tumor diagnosis, wherein the targeted drug-loaded microbubbles are used as ultrasound contrast imaging reagents. As a specific application example, the ultrasound contrast imaging instrument parameters for this targeted drug-loaded microbubble application are set to MI 0.122, probe frequency 18MHz, and the ultrasound contrast imaging reagent dose is 100uL, administered via peripheral intravenous bolus injection.

[0027] The aforementioned targeted drug-loaded microbubbles can also be applied to tumor treatment, specifically as an ultrasound-targeted therapy agent for tumors. As a specific application example, the application environment for this ultrasound-targeted therapy agent is: ultrasound power of 1.5 W / cm². 2 The frequency is 1 MHz and the duration is 2 min.

[0028] This study provides targeted drug-loaded microbubbles that utilize the azido-acetylene cycloaddition reaction (SPAAC) in click chemistry to modify the outer shell material of ultrasound microbubbles. This constructs a highly efficient and safe targeted drug delivery system, building upon ultrasound contrast imaging for diagnostic purposes. When applied to ultrasound contrast imaging or ultrasound-targeted therapy, the ultrasound waves shatter the microbubbles within the target tissue, releasing the carried genes and drugs. The instantaneous rupture of the microbubbles generates a "cavitation effect" and an "acoustic aperture effect," achieving targeted and efficient drug release and precise targeting, thereby improving the efficiency of tumor treatment.

[0029] Compared with the prior art, the present invention has the following advantages:

[0030] 1) The novel targeted drug-loaded microbubbles of this invention are prepared using a thin-film hydration method. The above-mentioned special microbubble design endows the novel targeted microbubbles with high drug loading and encapsulation efficiency, uniform microbubble size, good stability, and changes in morphology and size after ultrasonic irradiation.

[0031] 2) The novel targeted drug-loaded microbubbles of this invention use DBCO-modified liposome shells, which are simple to prepare, have appropriate raw material ratios, and are stable after inflation. They have the same ultrasound imaging effect as SonoVe and a longer duration of action within the tumor.

[0032] 3) The novel targeted drug-loaded microbubbles of this invention improve the uptake and accumulation of drugs in tumors through rapid and efficient click reactions, while ultrasound-targeted destruction of microbubbles (UTMD) enables precise drug release, enhances drug transcellular transport, and further improves the tumor treatment rate. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 This is a synthetic route diagram for targeted drug-loaded microbubbles in this application;

[0035] Figure 2 For the compound DBCO-NHS involved in this application 1 HNMR spectrum;

[0036] Figure 3 The compound DSPE-PEG-NH2 involved in this application 1 HNMR spectrum;

[0037] Figure 4 The polymer DSPE-PEG-DBCO involved in this application1 HNMR spectrum;

[0038] Figure 5 Fourier transform infrared chromatograms of DBCO-NHS, DSPE-PEG-NH2, and DSPE-PEG-DBCO;

[0039] Figure 6 The images show the DLS spectra of the targeted drug-loaded microbubbles and the microbubbles after ultrasounding involved in this application.

[0040] Figure 7 This is a transmission electron microscope image of the targeted drug-loaded microbubbles involved in this application.

[0041] a - Unstained state, b - Stained state;

[0042] Figure 8 The fluorescence image is of the targeted drug-loaded microbubbles involved in this application;

[0043] Figure 9 This is an image showing the contrast-enhanced imaging effect of the targeted drug-loaded microbubble ultrasound contrast imaging mode involved in this application;

[0044] Figure 10 This is a toxicity test of different drug concentrations involved in this application on 4T1 tumor cells;

[0045] Figure 11 This application describes the killing effect of the acoustic sensitizer on 4T1 tumor cells under ultrasound stimulation. Detailed Implementation

[0046] To make the technical problems, technical solutions, and beneficial effects to be solved by this application clearer, the technical solutions in the embodiments of this application will be further described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain this application and are not intended to limit the technical solutions of this application. All other embodiments obtained by those skilled in the art based on the embodiments in this application without creative effort are within the scope of protection of this application.

[0047] Example 1

[0048] This embodiment provides a targeted drug-loaded microbubble. The embodiment of this application is described below with reference to the accompanying drawings.

[0049] See Figure 1 , Figure 1 The synthetic route of the targeted drug-loaded microbubbles in this embodiment is shown.

[0050] This embodiment employs a method combining thin-film hydration and mechanical vibration to prepare targeted drug-loaded microbubbles. First, appropriate amounts of DSPC, DSPE-PEG2000, DSPE-PEG-DBCO, and PTX are mixed in chloroform in a test tube. The chloroform is then removed under a gentle nitrogen flow, followed by evaporation using a rotary evaporator for 1 hour or overnight to remove all chloroform (rotating at 60 rpm at 50°C). Next, the dried lipid film is added to an appropriate amount of degassing buffer. The test tube is then heated in water at 62°C for 15 minutes, followed by sonication for 30 minutes. Then, 1 mL of the aqueous liposome suspension is added to a sealed vial, and the air in the vial is replaced with C3F8 gas. Finally, the vial is mechanically vibrated in a dental amalgam mixer for 45 seconds. Successfully prepared drug-loaded microbubbles are stored at 4°C.

[0051] The specific steps are as follows:

[0052] Step 1: DBCO-NHS was dissolved in 2 mL of DMSO, while DSPE-PEG-NH2 was dissolved separately in 10 mL of DMSO. The two solutions were then mixed and stirred overnight at room temperature. Afterward, the reaction mixture was dialyzed against DMSO for one day, and then against distilled water for one day. Finally, the product was freeze-dried under vacuum for one day to obtain the product DSPE-PEG-DBCO.

[0053] The equivalent ratio of DBCO-NHS to DSPE-PEG-NH2 is 1:5.

[0054] Step 2: Mix DSPE-PEG-DBCO, DSPE-PEG2000, DSPC, and PTX in chloroform in a flask. Then, evaporate the chloroform to dryness under a gentle nitrogen flow, followed by treatment with a rotary evaporator for 1 hour or overnight (60 rpm, 50°C) to remove all chloroform and obtain a dried lipid film.

[0055] The molar ratio of DSPC / DSPE-PEG-DBCO / DSPE-PEG2000 is 5:5:90.

[0056] Step 3: Add an appropriate amount of degassing buffer to the dried lipid film, place the flask in water at 62°C and heat for 15 minutes, shaking once every 5 minutes, then perform ultrasonic cleaning at 60°C for 30 minutes, shaking once every 5 minutes and moving the flask to obtain a clear liposome suspension.

[0057] Step 4: Add 1 ml of liposome suspension to a sealed vial and replace the air in the vial with 10 ml of C3F8 gas. Finally, mechanically vibrate the vial for 45 seconds using a dental amalgam mixer. The successfully prepared targeted drug-loaded microbubbles P@MB-DBCO are stored at 4°C.

[0058] Among them: the compounds involved are DBCO-NHS, DSPE-PEG-NH2, and DSPE-PEG-DBCO. 1 HNMR spectrum as follows Figures 2 to 4 As shown.

[0059] DBCO-NHS 1 HNMR data can be found Figure 2 : 1 H NMR (400MHz, DMSO-d6), δ ppm: 8.10 (d,2H), 7.80-7.40 (m, 6H), 3.00 (s, 4H), 2.60-1.80 (m, 8H).

[0060] DSPE-PEG-NH2 1 HNMR data can be found Figure 3 : 1 H NMR (400MHz, DMSO-d6), δ ppm: 8-7 (br,1-2H), 4.10 (m, 2H), 3.90-3.40 (m, ~180H), 3.10 (t, 2H), 2.25 (t, 4H), 1.70-1.50 (m, 4H), 1.40-1.20 (m, ~60H), 0.85 (t, 6H).

[0061] DSPE-PEG-DBCO 1 HNMR data can be found Figure 4 : 1 H NMR (400MHz, DMSO-d6), δ ppm: 8.10-7.20 (m, 8H), 3.90-3.40 (m, ~180H), 3.10 (t, 2H), 2.80-2.50 (m, 8H), 2.25 (t,4H), 1.70-1.50 (m, 4H), 1.40-1.20 (m, ~60H), 0.85 (t, 6H).

[0062] Fourier transform infrared chromatograms of DBCO-NHS, SPDE-PEG-NH2, and SPDE-PEG-DBCO are shown below. Figure 5 The reaction between NHS and NH2 proves that the DBCO functional group was successfully coupled.

[0063] The morphology, size, and stability of the prepared targeted drug-loaded microbubbles were characterized by DLS. The results showed that the polymers all self-assembled into nanoparticles with a particle size of approximately 3 μm. Figure 6 This matches the spherical shape and size observed in transmission electron microscopy (TEM) images. Figure 7 (a) shows that it has a bilayer structure ( Figure 7 (b) in the middle, and Figure 1 The reaction end products have a consistent structure. After ultrasound-targeted destruction of microbubbles, the size of the microbubbles can promote their aggregation in solid tumors through enhanced permeation and retention (EPR).

[0064] The results of co-incubation of P@MB-DBCO with azide-Cy5.5 fluorescent dye for 1 hour, observed using an inverted fluorescence microscope, are as follows: Figure 8 As shown: the microbubble surface exhibits fluorescence after the DBCO group reacts with azide-Cy5.5 containing azide groups, proving the presence of DBCO groups on the microbubble surface.

[0065] Application Example 1

[0066] This embodiment is an intratumoral imaging experiment of targeted drug-loaded microbubbles in ultrasound contrast imaging mode.

[0067] To investigate the imaging effect and duration of microbubbles in contrast-enhanced ultrasound, 4T1 tumor cells were injected subcutaneously into mice. Tumor-bearing mice with tumors approximately 7 mm in size were selected and injected with 100 μL of microbubbles via the tail vein. Intratumoral imaging was observed and recorded. Results are shown below. Figure 9 As shown: In in vivo contrast imaging mode, targeted drug-loaded contrast imaging has the same contrast intensity and effect as SonoVe within the tumor, but lasts longer.

[0068] Application Example 2

[0069] This example illustrates the effect of targeted drug-loaded microbubbles on the survival rate of 4T1 cells.

[0070] 4T1 cells were loaded at 5 × 10 3 Cells were seeded at a concentration of 10 cells / mL in 96-well plates and cultured for 24 h. Then, cells were seeded with different concentrations of PTX (0–625 μg / mL). -1 The original cell culture medium was replaced with P@MB-DBCO medium. After incubation for 6 h, the cells were washed with PBS, and cell viability was assessed using the standard CCK-8 assay. Figure 10 It can be seen that even at 625 μg mL -1Even at RFGN concentrations, cell survival rates remained high, indicating that our microvesicles are relatively safe for cells.

[0071] Application Example 3

[0072] This embodiment demonstrates the killing effect of targeted drug-loaded microbubbles on tumor cells under ultrasound.

[0073] The in vitro antitumor effect of targeted drug-loaded microvesicles was evaluated using a cell CCK-8 assay. 4T1 cells (1×10⁻⁶) were used. 4 Cells (100 cells / well) were seeded in 96-well plates and incubated for 24 hours. After observing good cell growth under a light microscope, the cells were divided into 5 groups: Group 1 was the US group; Groups 2 to 5 were P@MB-DBCO+US groups containing different concentrations of PTX, respectively. Different concentrations of drug-loaded microbubbles were added according to the groups, and after 6 hours, the cells were treated with US (1 MHz, 1.5 W / cm²) for 2 minutes. 2 (50% empty space). Continue incubation in a cell culture incubator for 24 h, aspirate the liquid from the 96-well plate, add 200 μL of a solution containing 10% CK-8 prepared from serum-free medium to each well, and incubate again in a cell culture incubator for 2 h. Use a multi-mode microplate reader to detect the absorbance of each well at 450 nm, and calculate the survival rate of tumor cells in different treatment groups, i.e., cell viability, using a formula.

[0074] .

[0075] like Figure 11 As shown, the P@MB-DBCO +US treatment method exhibited a very good cell-killing ability. The survival rate of 4T1 cells in the P@MB-DBCO +US group was only about 13.8%, and the killing effect became more and more obvious with the increase of PTX concentration, indicating that the targeted imaging microbubbles have a good anti-tumor effect.

[0076] The above-described embodiments are merely illustrative of several feasible implementations of the present invention, and their descriptions are relatively specific and detailed. However, they should not be construed as limiting the scope of the present invention, nor are the embodiments intended to limit the scope of protection in the claims of the present invention. For those skilled in the art, various modifications and improvements can be made without departing from the concept of the present invention. All equivalent implementations or changes that do not depart from the present invention should be included within the scope of the present invention.

Claims

1. A targeted drug-loaded microbubble, characterized in that: It comprises a PEG shell, a lipid bilayer, and DBCO anchors. PTX is loaded onto the lipid bilayer, which is located within the PEG shell. The DBCO anchors are located on the PEG shell. The method for preparing the targeted drug-loaded microbubbles is as follows: Step 1: Mix the DMSO solution of DBCO-NHS and the DMSO solution of DSPE-PEG-NH2, stir overnight at room temperature, dialyze the resulting mixture, and freeze dry under vacuum to obtain the product DSPE-PEG-DBCO, with an equivalent ratio of DBCO-NHS to DSPE-PEG-NH2 of 1:

5. Step 2: DSPE-PEG-DBCO, DSPE-PEG2000, DSPC, and PTX are mixed in chloroform, and then the chloroform is evaporated and dried under a nitrogen stream to remove the chloroform, resulting in a dry lipid film. The molar ratio of DSPC, DSPE-PEG-DBCO, and DSPE-PEG2000 is 5:5:

90. Step 3: After adding the dried lipid membrane to the degassing buffer, heat it at 60~65℃ to obtain a clear liposome suspension. Step four: The liposome suspension is placed in a sealed container, and the air inside the bottle is replaced with C3F8 gas and mechanically shaken to obtain the finished targeted drug-loaded microbubbles.

2. The targeted drug-loaded microbubble according to claim 1, characterized in that: In step three, the degassing buffer solution is a mixture of NaCl, isopropanol, and glycerol.

3. The targeted drug-loaded microbubble according to claim 2, characterized in that: The volume ratio of NaCl, isopropanol, and glycerol is 8:1:

1.

4. The application of the targeted drug-loaded microbubbles as described in claim 1 in the preparation of tumor ultrasound contrast imaging reagents, wherein the tumor is breast cancer.

5. The application according to claim 4, characterized in that: The parameters of the ultrasound contrast imaging instrument were set to MI 0.122, probe frequency 18MHz, and ultrasound contrast imaging reagent dosage 100uL, which was injected via peripheral intravenous bolus injection.

6. The application of the targeted drug-loaded microbubbles as described in claim 1 in the preparation of ultrasound-targeted therapy reagents for tumors, wherein the tumor is breast cancer.

7. The application according to claim 6, characterized in that: Ultrasonic power is 1~2 W / cm 2 The frequency is 1~2 MHz, and the action time is 1~5 min.

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