IGF1-BP coupled lipid nanoparticles as well as preparation method and application thereof

Abstract

The invention relates to IGF1-BP coupled lipid nanoparticles as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. In order to solve the problem that an endometrial nano-carrier and a corresponding targeting drug are still lacked in the existing preeclampsia prevention and treatment, the invention provides a preparation method of IGF1-BP coupled lipid nanoparticles, which comprises the following steps: respectively preparing an organic phase solution and a water phase solution, assembling the lipid nanoparticles by using the organic phase solution and the water phase solution, and preparing the IGF1-BP coupled lipid nanoparticles. And connecting IGF1-BP to the surface of the lipid nanoparticle through a coupling reaction by virtue of an EDC / NHS technology, so as to obtain the IGF1-BP coupled lipid nanoparticle. According to the IGF1-BP coupled lipid nanoparticles provided by the invention, morpholino oligonucleotide for reducing endometrial APOD expression is delivered to endometrial tissues in a targeted manner, so that not only can the symptoms of preeclampsia be relieved during pregnancy, but also prophylactic treatment can be performed on lying-in women with high risk of preeclampsia during prepregnancy.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to IGF1-BP conjugated lipid nanoparticles and their preparation methods and applications. Background Art

[0002] Preeclampsia is a serious pregnancy complication, manifested as symptoms such as hypertension and proteinuria, seriously endangering the lives of mothers and infants. Traditional treatment methods for preeclampsia include taking antihypertensive drugs to control blood pressure and using drugs such as aspirin to improve placental perfusion. However, the causes of preeclampsia are complex and diverse, and the use of non-specific drugs often makes it difficult to accurately treat specific causes, not only with limited treatment effects but also prone to unnecessary side effects.

[0003] In recent years, targeted drugs have been favored in the treatment of preeclampsia due to their characteristics of precisely hitting lesions and reducing side effects. A variety of nano-carriers targeting the placenta have been applied to the clinical treatment practice of preeclampsia. For example, siRNA is conjugated to PAMAM (polyamidoamine) nano-carriers, and the siRNA is targeted and delivered to placental cells through the nano-carriers, specifically inhibiting the synthesis of sFlt-1 (soluble fms-like tyrosine kinase 1), which can promote placental vascular development, improve uteroplacental perfusion, and thus relieve the related symptoms of preeclampsia.

[0004] Although nano-carriers targeting the placenta have achieved preliminary results in the treatment of preeclampsia, there is still a lack of nano-carriers targeting the endometrium and corresponding targeted drugs. The endometrium, as an important interface between the mother and the fetus, its abnormal function is also an important factor in the pathogenesis of preeclampsia. During normal pregnancy, trophoblast cells of the placenta will differentiate into vascular endothelial cells to form spiral arteries to ensure that the fetus obtains sufficient nutrition and oxygen. In the case of preeclampsia, patients often have abnormal lipid metabolism, such as increased lipid peroxides. Free radicals generated by lipid peroxides can damage vascular endothelial cells, resulting in insufficient remodeling of spiral arteries, thus affecting placental blood supply. Therefore, the development of nano-carriers and targeted drugs targeting the endometrium is of great significance for the prevention and treatment of preeclampsia. Summary of the Invention

[0005] To solve the problem that there is still a lack of nano-carriers targeting the endometrium and corresponding targeted drugs in the existing prevention and treatment of preeclampsia, the present invention provides IGF1-BP conjugated lipid nanoparticles and their preparation methods and applications.

[0006] The technical solution of the present invention: A preparation method of IGF1-BP conjugated lipid nanoparticles, comprising the following steps: Step 1, prepare an organic phase solution: The cationic lipid Dlin-MC3-DMA (ionizable lipid of MC3 type), DSPC (distearoyl phosphatidylcholine), cholesterol, PEG2000-DMG (PEGylated lipid), and PEG2000-COOH (carboxylated PEG lipid) are dissolved in an alcohol solvent as mixed organic solutes to obtain an organic phase solution; Step 2: Prepare an aqueous phase solution: Using DEPC (diethyl pyrocarbonate) water as the solvent, dissolve the gene drug in DEPC water to obtain an aqueous phase solution; Step 3: Prepare lipid nanoparticles: Using the organic phase solution obtained in Step 1 as the continuous phase and the aqueous phase solution obtained in Step 2 as the dispersed phase, use a microfluidic mixing device to complete the precise assembly of lipid nanoparticles, and ultrafilter and purify the obtained mixture to obtain lipid nanoparticles; Step 4: Prepare IGF1-BP (insulin-like growth factor 1 binding protein) conjugated lipid nanoparticles: Connect IGF1-BP to the surface of the lipid nanoparticles obtained in Step 3 through a coupling reaction by EDC / NHS technology to obtain IGF1-BP conjugated lipid nanoparticles.

[0007] Furthermore, in the organic phase solution obtained in Step 1, the concentration ratio of the cationic lipid Dlin-MC3-DMA, DSPC, cholesterol, PEG2000-DMG, and PEG2000-COOH is 48.8:9.8:36.6:2.4:2.4, and the alcohol solvent is absolute ethanol.

[0008] Furthermore, the total concentration of the mixed organic solutes in the organic phase solution obtained in Step 1 is 12.5 mM, and the nitrogen-phosphorus ratio of the mixed organic solutes is 4:1.

[0009] Furthermore, the gene drug in Step 2 is a morpholino oligonucleotide, the nucleotide sequence of the morpholino oligonucleotide is as shown in SEQ ID No: 1, and the mass-volume ratio of the morpholino oligonucleotide to DEPC water is 1 mg:1 mL.

[0010] Furthermore, in the microfluidic mixing device in Step 3, the flow rate ratio of the continuous phase to the dispersed phase is 3:1, and the flow rate is 12 mL / min.

[0011] Furthermore, the ultrafiltration in Step 3 is carried out by centrifuging at 2000 g for 20 min at room temperature using an ultrafiltration tube, and the purification treatment is to add a PBS buffer solution with a pH of 7.4 to the ultrafiltration tube and centrifuge again to displace the original solvent.

[0012] Furthermore, the EDC / NHS technique described in Step 4 involves suspending 1 mg of purified lipid nanoparticles in 3 mL of MES (2-(N-morpholino)ethanesulfonic acid) buffer with a concentration of 0.1 mol / L. Then, 28.3 μg of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and 17 μg of NHS (N-hydroxysuccinimide) are added to the resulting suspension. After mixing, the reaction mixture is shaken for 20 min to obtain a reaction mixture. First, 0.5 mg of IGF1-BP is added to 500 μL of deionized water, and then the resulting solution is added to the reaction mixture. Subsequently, 250 μL of 20-fold concentrated phosphate buffer is added to the reaction mixture, and the pH of the system is adjusted to 7.0 - 8.0. The resulting reaction system is stirred overnight, and impurities are removed by ultrafiltration and centrifugation to obtain IGF1-BP conjugated lipid nanoparticles.

[0013] The IGF1-BP conjugated lipid nanoparticles prepared by the preparation method provided by the present invention have an average particle size of 62.3 nm, a number concentration of 5.5×10 6 particles / mL, and a potential of -23 ± 1.89 mV.

[0014] Application of the IGF1-BP conjugated lipid nanoparticles prepared by the present invention in the clinical prevention and treatment of preeclampsia.

[0015] Furthermore, the IGF1-BP conjugated lipid nanoparticles are directly delivered to the endometrial tissue by intravenous injection.

[0016] Advantages of the present invention: IGF1R is a highly expressed receptor in endometrial epithelial cells, especially highly expressed on the surface of glandular epithelial and luminal epithelial cells. The present invention uses IGF1 as a ligand on the synthesized lipid nanoparticles to construct IGF1-BP conjugated lipid nanoparticles targeting the endometrium. The IGF1-BP conjugated lipid nanoparticles targetedly deliver gene drugs that reduce the expression of endometrial APOD (apolipoprotein D) to the endometrial tissue. Through multiple mechanisms such as reducing the production of lipid peroxides, alleviating endothelial damage, promoting spiral artery remodeling, and regulating lipid balance by reducing the expression of endometrial APOD, the symptoms of preeclampsia are jointly alleviated.

[0017] The present invention delivers a morpholino oligonucleotide that reduces endometrial APOD expression to endometrial tissue through IGF1-BP conjugated lipid nanoparticles, which can not only relieve the symptoms of preeclampsia during pregnancy, but also provide preventive treatment for pregnant women at high risk of preeclampsia in the pre-pregnancy period. The IGF1-BP conjugated lipid nanoparticles provided by the present invention are administered by intravenous injection, which can safely and effectively improve the clinical symptoms, maternal and child outcomes, and placental pathological manifestations of preeclamptic mice, thereby providing new experimental evidence and strategies for the clinical prevention and treatment of preeclampsia and drug research and development. Description of the Drawings

[0018] Figure 1 Transmission electron microscopy image of the IGF1-BP conjugated lipid nanoparticles prepared in Example 1, with the scale bar in the figure being 200 nm; Figure 2 Transmission electron microscopy image of the IGF1-BP conjugated lipid nanoparticles prepared in Example 1, with the scale bar in the figure being 50 nm; Figure 3 Comparison chart of the changes in systolic blood pressure during pregnancy of three groups of pregnant mice in Example 3, where E in the figure represents the number of days of pregnancy; Figure 4 Comparison chart of the weights of fetuses obtained by sacrificing three groups of pregnant mice at 17.5 days of pregnancy in Example 3; Figure 5 Comparison chart of the weights of placentas obtained by sacrificing three groups of pregnant mice at 17.5 days of pregnancy in Example 3; Figure 6 Comparison chart of stained sections of placentas obtained by sacrificing three groups of pregnant mice at 17.5 days of pregnancy in Example 3; Figure 7 Schematic diagram of the construction method and administration method of the IGF1-BP conjugated lipid nanoparticles carrying morpholino oligonucleotide of the present invention. Detailed Embodiments

[0019] The technical solutions of the present invention will be further described below in conjunction with the embodiments, but are not limited thereto. Any modification or equivalent replacement of the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention shall be covered within the protection scope of the present invention. For the process equipment or devices not specifically noted in the following embodiments, conventional equipment or devices in the art are used. Unless otherwise specified, the raw materials used in the embodiments of the present invention can be obtained commercially; unless otherwise specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0020] Example 1 This example provides a preparation method of IGF1-BP conjugated lipid nanoparticles, including the following steps: Step 1. Prepare an organic phase solution: Dissolve cationic lipid Dlin-MC3-DMA, DSPC, cholesterol, PEG2000-DMG and PEG2000-COOH as mixed organic solutes in absolute ethanol to obtain an organic phase solution; In the obtained organic phase solution, the concentration ratio of cationic lipid Dlin-MC3-DMA, DSPC, cholesterol, PEG2000-DMG and PEG2000-COOH is 48.8:9.8:36.6:2.4:2.4, the total concentration of the mixed organic solutes is 12.5 mM, and the nitrogen-phosphorus ratio of the mixed organic solutes is 4:1.

[0021] Step two: Prepare an aqueous phase solution: Use DEPC water as the solvent. DEPC water is ultrapure water treated with diethyl pyrocarbonate and sterilized by high temperature and high pressure; dissolve 1 mg of the gene drug morpholino oligonucleotide that inhibits the expression of endometrial APOD in 1 mL of DEPC water to obtain an aqueous phase solution; The gene drug used in this example to inhibit the expression of endometrial APOD is morpholino oligonucleotide, denoted as APOD morpholino oligonucleotide, and its nucleotide sequence is as shown in SEQ ID No:1, and the specific sequence is: 5'-ACGTAGGTACGTAGGTACGCAT-3'.

[0022] Step three: Prepare lipid nanoparticles: Inject the organic phase solution and the aqueous phase solution into the microfluidic mixing device NanoAssemblr Ignite. Use the organic phase solution obtained in step one as the continuous phase and the aqueous phase solution obtained in step two as the dispersed phase. Set the flow rate ratio of the continuous phase to the dispersed phase to 3:1, the flow rate to 12 mL / min, and the total volume to 11.20 mL. Use the microfluidic mixing device to complete the precise assembly of lipid nanoparticles. Centrifuge the obtained mixture at 2000 g for 20 min at room temperature using an ultrafiltration tube (Amicon Ultra-4 centrifugal filtration unit), discard the filtrate, add PBS buffer with a pH of 7.4 to the ultrafiltration tube, and centrifuge again to replace the original solvent to obtain purified lipid nanoparticles; Step four: Prepare IGF1-BP conjugated lipid nanoparticles: The IGF1-BP was connected to the surface of the lipid nanoparticles obtained in Step 3 through an EDC / NHS technology coupling reaction. 1 mg of the purified lipid nanoparticles was suspended in 3 mL of MES buffer with a concentration of 0.1 mol / L and a pH of 5.5. To pre-activate the carboxyl groups, 28.3 μg of EDC and 17 μg of NHS were added to the obtained suspension. After mixing, the reaction was carried out on an oscillator for 20 min to obtain a reaction mixture. Subsequently, first, 0.5 mg of IGF1-BP (purchased from Shanghai Borsen Biotechnology Co., Ltd., numbered BES20003AP) was added to 500 μL of deionized water, and then the obtained solution was added to the reaction mixture. Then, 250 μL of 20-fold concentrated phosphate buffer was added to the reaction mixture, and the pH of the system was adjusted to 7.0 - 8.0. The obtained reaction system was stirred overnight. Finally, the obtained reaction system was filtered three times using an Amicon Ultra-4 centrifugal filter with a molecular mass of 10 kDa to remove excess peptides and other impurities (such as EDC and NHS), and IGF1-BP-conjugated lipid nanoparticles were obtained.

[0023] Example 2 In this example, the particle size, number, and zeta potential of the IGF1-BP-conjugated lipid nanoparticles prepared in Example 1 were measured.

[0024] Analysis was carried out using ZetaView software (version 8.05.11). The IGF1-BP-conjugated lipid nanoparticles obtained in the example were diluted 1:1000 with ultrapure water to obtain a sample. 500 μL of the sample was injected into the measurement cell of a laser particle size analyzer to ensure there were no bubbles, and data at 11 positions were recorded and analyzed. In the ZetaView system, 100 nm polystyrene particles were used as the alignment control. The temperature was maintained at approximately 27 °C during the measurement. The zeta potential of the IGF1-BP-conjugated lipid nanoparticles was measured by detecting the electrophoretic mobility of the lipid nanoparticles in an electric field.

[0025] The IGF1-BP-conjugated lipid nanoparticles were diluted 100-fold with ultrapure water and then placed on an electron microscope grid and incubated for 10 min. Subsequently, the grid was stained with 100 μL of 2% (mass / volume) phosphotungstic acid solution for 1 min. The TEM grid was washed with ultrapure water and semi-dried. Finally, images of the nanoparticles were taken, and the diameter of the IGF1-BP-conjugated lipid nanoparticles was quantitatively analyzed under a transmission electron microscope.

[0026] Figure 1 and Figure 2 are transmission electron micrographs of the IGF1-BP-conjugated lipid nanoparticles prepared in Example 1 at different magnifications; Figure 1 and Figure 2The results showed that the IGF1-BP conjugated lipid nanoparticles were spherical in shape, with a complete shape, clear outline, and smooth edges. After measurement, the average particle size of the IGF1-BP conjugated lipid nanoparticles obtained in Example 1 was 62.3 nm, the number concentration was 5.5×10 6 particles / mL, and the potential was -23±1.89 mV.

[0027] Example 3 In this example, the efficacy of the IGF1-BP conjugated lipid nanoparticles prepared in Example 1 in alleviating preeclampsia symptoms was investigated through animal model experiments.

[0028] I. Model construction method: In this example, a preeclampsia mouse model with endometrium-specific overexpression of APOD was constructed using the prior invention patent "Method for constructing an APOD conditional knockout mouse model" with the authorization announcement number CN117467704B.

[0029] II. Grouping method: Preliminary pre-experiments verified that although morpholino oligonucleotides with mismatches encapsulated in lipid nanoparticles could target the endometrium, the mismatched morpholino oligonucleotides did not have the function of degrading APOD after entering target cells, so they did not have the therapeutic effect on preeclampsia.

[0030] In this example, the verification experiments were respectively set up with a normal pregnant mouse control group 1, a preeclampsia pregnant mouse control group 2, and a preeclampsia pregnant mouse treatment group injected with IGF1-BP conjugated lipid nanoparticles.

[0031] The administration method and dosage of the preeclampsia mouse treatment group injected with IGF1-BP conjugated lipid nanoparticles were as follows: at 5.5 days, 7.5 days, and 9.5 days of pregnancy, 100 μL of 10 μM IGF1-BP conjugated lipid nanoparticles were injected through the tail vein respectively.

[0032] The normal pregnant mouse control group 1 and the preeclampsia pregnant mouse control group 2 were respectively injected with 100 μL of normal saline through the tail vein at 5.5 days, 7.5 days, and 9.5 days of pregnancy.

[0033] III. Blood pressure monitoring of pregnant mice: The BP-2000 blood pressure analysis system was used to non-invasively measure the blood pressure of the three groups of pregnant mice at 7.5 days, 8.5 days, 9.5 days, 10.5 days, 11.5 days, 12.5 days, 14.5 days, 15.5 days, and 17.5 days of pregnancy. This measurement included placing a detector around the tail of the mouse and using a transmission light plethysmograph to measure the blood pressure.

[0034] Figure 3It is a comparison chart of the changes in systolic blood pressure during pregnancy of three groups of pregnant mice. In the chart, E represents the number of days of pregnancy; Figure 3 It shows that intravenous injection of IGF1-BP conjugated lipid nanoparticles can reduce the systolic blood pressure during pregnancy of preeclamptic pregnant mice, N = 5.

[0035] IV. Detection of the weights of the placenta and fetus of pregnant mice: At 17.5 days of pregnancy, three groups of pregnant mice were sacrificed, the placenta and fetus were collected, and the fetus and placenta were recorded and weighed. Figure 4 and Figure 5 It is a comparison chart of the weights of the fetuses and placentas obtained from sacrificing three groups of pregnant mice at 17.5 days of pregnancy; Figure 4 and Figure 5 It shows that intravenous injection of IGF1-BP conjugated lipid nanoparticles can significantly increase the fetal weight and placental weight of preeclamptic pregnant mice, N = 5 litters.

[0036] V. Pathological manifestations of the placenta of pregnant mice: At 17.5 days of pregnancy, three groups of pregnant mice were sacrificed. The placenta sections (5 µm) of the three groups of pregnant mice were dewaxed, rehydrated, incubated in hematoxylin for 8 min, then incubated in the differentiating solution for 1 - 5 s, incubated in the blue returning solution for 1 min, incubated in eosin for 1 min and dehydrated. Placenta sections passing through the sagittal midline were selected for light microscopy imaging.

[0037] Figure 6 It is a comparison chart of the stained sections of the placentas obtained from sacrificing three groups of pregnant mice at 17.5 days of pregnancy; According to Figure 6 Histopathological analysis showed that intravenous injection of IGF1-BP conjugated lipid nanoparticles alleviated the small vessel congestion and necrosis caused by arteriolar spasm in the cross-section of the placenta obtained from preeclamptic pregnant mice at 17.5 days of pregnancy, N = 5.

Claims

1. A method for preparing IGF1-BP coupled lipid nanoparticles, characterized in that: The steps include: Step 1: Prepare organic phase solution: Cationic lipid Dlin-MC3-DMA, DSPC, cholesterol, PEG2000-DMG and PEG2000-COOH were dissolved as mixed organic solutes in an alcohol solvent to obtain an organic phase solution; Step 2: Prepare aqueous solution: Using DEPC water as solvent, dissolve the gene drug in DEPC water to obtain an aqueous solution; Step 3: Preparation of lipid nanoparticles: The organic phase solution obtained in step 1 is used as a continuous phase, and the aqueous phase solution obtained in step 2 is used as a dispersed phase, a microfluidic mixing device is used to precisely assemble lipid nanoparticles, and the obtained mixture is ultrafiltered and purified to obtain lipid nanoparticles; Step 4: Preparation of IGF1-BP coupled lipid nanoparticles: The IGF1-BP is connected to the surface of the lipid nanoparticles obtained in step three through a coupling reaction using the EDC / NHS technology to obtain IGF1-BP-coupled lipid nanoparticles.

2. The method for preparing an IGF1-BP coupled lipid nanoparticle according to claim 1, characterized in that: The concentration ratio of the cationic lipids Dlin-MC3-DMA, DSPC, cholesterol, PEG2000-DMG and PEG2000-COOH in the organic phase solution obtained in step 1 is 48.8:9.8:36.6:2.4:2.4, and the alcohol solvent is anhydrous ethanol.

3. A method for preparing an IGF1-BP coupled lipid nanoparticle according to claim 1 or 2, characterized in that: The total concentration of the mixed organic solute in the organic phase solution obtained in step 1 is 12.5 mM, and the nitrogen-phosphorus ratio of the mixed organic solute is 4:

1.

4. The method for preparing an IGF1-BP coupled lipid nanoparticle according to claim 3, characterized in that: Step 2: The gene drug is a morpholino oligonucleotide, the nucleotide sequence of the morpholino oligonucleotide is shown in SEQ ID No: 1, and the mass volume ratio of the morpholino oligonucleotide to DEPC water is 1 mg: 1 mL.

5. The method for preparing an IGF1-BP coupled lipid nanoparticle according to claim 4, characterized in that: In step 3, the flow ratio of the continuous phase to the dispersed phase in the microfluidic mixing device is 3:1, and the flow rate is 12 mL / min.

6. The method for preparing an IGF1-BP coupled lipid nanoparticle according to claim 5, characterized in that: The ultrafiltration in step 3 is performed by centrifugation at 2000 g for 20 min at room temperature using an ultrafiltration tube. The purification treatment is performed by adding PBS buffer with a pH of 7.4 to the ultrafiltration tube and centrifuging again to replace the original solvent.

7. The method for preparing an IGF1-BP coupled lipid nanoparticle according to claim 6, characterized in that: Step 4: The EDC / NHS technology is to suspend 1 mg of purified lipid nanoparticles in 3 mL of 0.1 mol / L MES buffer, add 28.3 μg of EDC and 17 μg of NHS to the resulting suspension, mix and oscillate for 20 minutes to obtain a reaction mixture; first add 0.5 mg of IGF1-BP to 500 μL of deionized water, then add the resulting solution to the reaction mixture, and then add 250 μL of 20-fold concentrated phosphate buffer to the reaction mixture, and adjust the pH of the system to 7.0-8.0; the resulting reaction system is stirred overnight, ultrafiltered and centrifuged to remove impurities, and IGF1-BP-coupled lipid nanoparticles are obtained.

8. An IGF1-BP coupled lipid nanoparticle prepared by the preparation method according to any one of claims 1 to 7, characterized in that: The average particle size of the IGF1-BP coupled lipid nanoparticles is 62.3 nm, and the number concentration is 5.5×10 6 particles / mL, and the potential was -23±1.89mV.

9. Use of the IGF1-BP coupled lipid nanoparticles as claimed in claim 8 in the clinical prevention and treatment of preeclampsia.

10. The use of IGF1-BP coupled lipid nanoparticles in clinical prevention and treatment of preeclampsia according to claim 9, characterized in that: The IGF1-BP coupled lipid nanoparticles are directly delivered to the endometrial tissue via intravenous injection.

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