Polymitochondrial targeting peptide PSS31 and preparation method and application of lung targeting nucleic acid drug delivery system

Abstract

The invention provides a preparation method and application of a polymitochondrial targeting peptide PSS31 and a lung targeting nucleic acid drug delivery system, and belongs to the technical field of biochemical synthesis pharmacy. According to the invention, SS31 is polymerized through TK to form a polymer material sensitive to ROS (reactive oxygen species), namely the polymer mitochondrial targeting peptide PSS31. The PSS31 not only can efficiently load a nucleic acid drug through an electrostatic adsorption effect, but also can efficiently release the nucleic acid drug under the stimulation of ROS in cells. According to the invention, on the basis of the PSS31, a fucoidin (Fuco) modified lung-targeted nucleic acid drug delivery system siHIF-1alpha / PSS31 (at) Fuco is prepared through an electrostatic adsorption effect. The siHIF-1alpha / PSS31 (at) Fuco can be used for efficiently loading the siHIF-1alpha, improving the stability of the siHIF-1alpha, relieving pulmonary vascular remodeling, reducing pulmonary arterial pressure and efficiently treating hypoxic pulmonary hypertension in multiple ways.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biochemical synthesis pharmaceutical technology, and particularly relates to a polymeric mitochondrial targeting peptide PSS31, a preparation method and application of a lung-targeted nucleic acid drug delivery system. BACKGROUND

[0002] Gene therapy can specifically regulate the expression of target genes in target cells by delivering nucleic acid drugs (DNA or RNA) into target cells through delivery tools, so as to achieve the effect of treating related diseases. How to safely and efficiently deliver therapeutic nucleic acid drugs into target cells and efficiently edit gene expression in cells is the core step of gene therapy, which directly affects the therapeutic effect of nucleic acid drugs and the feasibility of clinical application. Therefore, developing an efficient and safe delivery material is a key link for pushing gene therapy from theory to clinic, and has important clinical value.

[0003] Current nucleic acid delivery vectors mainly include viral vectors and non-viral vectors. Traditional viral vectors have extraordinary ability in gene transfection, but have the problem of poor safety, such as carcinogenicity, immunogenicity, mutation, etc. In non-viral vectors, ionizable lipids in lipid nanoparticles (LNP) can efficiently load nucleic acid drugs into LNP and help LNP enter target cells and escape from endosomes. However, a large number of reports show that LNP can significantly stimulate the inflammatory response of target tissues after entering the body, which limits the clinical application of LNP. Cationic polymers such as polyethyleneimine, polyarginine, polylysine, etc. are one of the most studied gene delivery vectors. Cationic polymers not only can efficiently load nucleic acid drugs, but also can promote the escape of nucleic acid drugs from endosomes by using the "proton sponge effect". However, cationic polymers generally have the problems of greater toxicity and inability to respond to drug release.

[0004] HPH (hypoxic pulmonary hypertension, HPH) is a progressive malignant pulmonary vascular disease caused by high altitude or long-term hypoxic exposure, which is an abnormal increase in pulmonary arterial pressure, and belongs to the third type of pulmonary hypertension (PH) international classification. The results of epidemiological surveys show that about 140 million people in the world live in areas above 2500m, and the incidence of HPH is 10% to 15%, which develops rapidly and has poor prognosis, can induce right ventricular compensatory hypertrophy, and cause patients to usually die of right heart failure. It is reported that hypoxia-inducible factor 1-alpha (HIF-1α) activated by hypoxia is highly expressed in hypoxic damaged pulmonary arterial endothelial cells (PAECs) and pulmonary arterial smooth muscle cells (PASMCs). As a key transcription factor for oxygen homeostasis, HIF-1α binds to the hypoxic response element (HRE) of the gene in the nucleus, activates the HIF signal pathway, regulates the expression of the downstream related target gene, and causes pulmonary vascular and right heart remodeling. In addition, hypoxia can also cause mitochondrial oxidative stress, mitochondrial structure damage and membrane potential change, further promote the increase of mitochondrial ROS level, and the mutual induction and activation of the increased mitochondrial ROS and HIF-1α aggravate HPH.

[0005] In summary, as one of the key targets for treating HPH, HIF-1α can only be reduced in hypoxic damaged PAECs and PASMCs to block pulmonary vascular remodeling. SUMMARY

[0006] The purpose of the present application is to provide a preparation method and application of a poly-mitochondrial targeting peptide PSS31 and a lung-targeted nucleic acid drug delivery system. The present application polymerizes SS31 by using a ROS-sensitive dicarboxy ketone thioether (Thioketal, TK), synthesizes a poly-mitochondrial targeting peptide PSS31, then electrostatically adsorbs a specific small interfering RNA (siHIF-1α) of hypoxia-inducible factor-1α (HIF-1α) by using PSS31 to compress and form a nano-complex siHIF-1α / PSS31, and finally adsorbs a negatively charged fucoidan on the surface of siHIF-1α / PSS31 by electrostatic adsorption to prepare siHIF-1α / PSS31@Fuco.

[0007] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0008] The present application provides a preparation method of a poly-mitochondrial targeting peptide PSS31, comprising the following steps:

[0009] (1) After dissolving SS31, sodium bicarbonate is added, and after stirring, 9-fluoromethyl-N-succinimidyl carbonate is added, and then reaction, water is added, extraction is performed, separation is performed, and a compound 1 as shown in formula I is obtained;

[0010]

[0011] (2) Mix and dissolve compound 1 and ketithiolide, add benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate and diisopropylethylamine, react for 8-12 h to obtain compound 2 as shown in formula II;

[0012]

[0013] (3) Mix and dissolve compound 2 and compound 3 as shown in formula III, add benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate and diisopropylethylamine, react for 8-12 h to obtain compound 4 as shown in formula IV;

[0014]

[0015] (4) Compound 4 was added to a solution of N,N-dimethylformamide containing piperidine and reacted for 1 to 3 hours to obtain compound 5 as shown in formula V;

[0016]

[0017] (5) Mix and dissolve compound 5 and compound 2, add benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate and diisopropylethylamine, and react for 8-12 h to obtain compound 6 as shown in formula VI;

[0018]

[0019] (6) Compound 6 was dissolved in a solution of N,N-dimethylformamide containing piperidine and reacted for 1 to 3 h to obtain the polymitochondrial targeting peptide PSS31 as shown in Formula VII;

[0020]

[0021] Preferably, in step (1), the molar mass ratio of SS31, sodium bicarbonate and 9-fluorenmethyl-N-succinimide carbonate is 630-640:80-90:330-340; the reaction is carried out under nitrogen protection.

[0022] Preferably, in step (2), the molar ratio of compound 1 to ketethiocyanate is 830-840:250-260; the molar ratio of compound 1 to benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate and diisopropylethylamine is 830-840:370-380:100-110; the reaction is carried out under nitrogen protection.

[0023] Preferably, in step (3), the molar ratio of compound 2 to compound 3 is 1090-1100:860-870; the molar ratio of compound 2 to benzotriazole-NN,N′,N′-tetramethylurea hexafluorophosphate and diisopropylethylamine is 1090-1100:370-380:120-130; the reaction is carried out under nitrogen protection.

[0024] Preferably, in step (5), the molar ratio of compound 5 to compound 2 is 1490–1500:1090–1100; the molar ratio of compound 5 to benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate and diisopropylethylamine is 1490–1500:370–380:120–130; the reaction is carried out under nitrogen protection.

[0025] Preferably, the volume fraction of piperidine in the N,N-dimethylformamide solution is 15-25%; and the solvent used for dissolution is dichloromethane.

[0026] The present invention also provides the application of the aforementioned polymitochondrial targeting peptide PSS31 in the delivery of nucleic acid drugs.

[0027] This invention also provides a method for preparing the lung-targeted nucleic acid drug delivery system siHIF-1α / PSS31@Fuco, comprising the following steps:

[0028] Hypoxia-inducible factor-1α-specific small interfering RNA was mixed with the polymitochondrial targeting peptide PSS31, and fucoidan was added. After incubation, the lung-targeting nucleic acid drug delivery system siHIF-1α / PSS31@Fuco was obtained.

[0029] The present invention also provides the application of the lung-targeted nucleic acid drug delivery system siHIF-1α / PSS31@Fuco in the preparation of drugs for treating hypoxic pulmonary hypertension.

[0030] The beneficial effects of this invention compared to the prior art are as follows:

[0031] The polymitochondrial targeting peptide PSS31 provided by this invention is composed of SS31 and TK. SS31 is polymerized using TK to form a ROS-sensitive polymer material. The PSS31 can efficiently load nucleic acid drugs via electrostatic adsorption and also efficiently release nucleic acid drugs under intracellular ROS stimulation. Based on PSS31, this invention also prepares a Fuco-modified lung-targeting nucleic acid drug delivery system siHIF-1α / PSS31@Fuco via electrostatic adsorption. The siHIF-1α / PSS31@Fuco can efficiently load siHIF-1α and improve its stability. It can be recognized by hypoxia-damaged PMVECs and PASMCs, enter cells via caveolin-1-mediated endocytosis, and PSS31 cleaves under high intracellular ROS conditions, efficiently releasing siHIF-1α and SS31. By inhibiting HIF-1α expression, it simultaneously alleviates oxidative stress in mitochondria of hypoxic PMVECs and PASMCs, restores mitochondrial membrane potential, reduces mitochondrial ROS release, and blocks the mutual induction and activation of HIF-1α and mitochondrial ROS. Furthermore, the siHIF-1α / PSS31@Fuco can alleviate pulmonary vascular remodeling, reduce pulmonary artery pressure, and provide multi-pathway, highly effective treatment for hypoxic pulmonary hypertension.

[0032] This invention verified the composition of siHIF-1α / PSS31@Fuco, the stability of siHIF-1α, and its targeting of hypoxic-damaged lung tissue through gel retardation assays, flow cytometry, and in vivo imaging techniques. In vivo and in vitro experiments were conducted to investigate the silencing effect of siHIF-1α / PSS31@Fuco on HIF-1α hyperexpression in patients with HPH, elucidating the therapeutic effects and mechanisms of siHIF-1α / PSS31@Fuco on HPH through multiple pathways.

[0033] The cationic polymer PSS31 synthesized in this invention can efficiently load the nucleic acid drug siHIF-1α. The prepared siHIF-1α / PSS31@Fuco can target the lung tissue of HPH rats and efficiently release siHIF-1α in the high ROS environment of hypoxic-damaged PMVECs and PASMCs, thereby inhibiting the high expression of HIF-1α in the lung tissue of HPH rats, restoring pulmonary artery endothelial function, inhibiting the excessive proliferation and contraction of pulmonary artery smooth muscle, and significantly improving the therapeutic effect of siHIF-1α on hypoxic pulmonary hypertension, which has good application prospects.

[0034] The polymer material PSS31 provided by this invention has the characteristics of high efficiency in loading nucleic acid drugs and low toxicity. Furthermore, a drug delivery system siHIF-1α / PSS31@Fuco is constructed using PSS31, capable of targeting and accumulating in hypoxic-damaged lung tissue, and efficiently loading siHIF-1α. By inhibiting HIF-1α expression and reducing ROS generated by mitochondrial oxidative stress, the mutual activation between HIF-1α and ROS is blocked, thereby achieving a highly effective therapeutic effect on hypoxic pulmonary hypertension.

[0035] The inventors discovered through research that using Fuco as a carrier for modified drugs allows them to bind to p-selectin on the cell surface and be taken up by target cells via caveolin-1-mediated endocytosis, thus avoiding the destruction of nucleic acid drugs by endosomes and lysosomes and improving the transfection efficiency of nucleic acid drugs. For the highly expressed HIF-1α, this invention employs specific small interfering RNA targeting HIF-1α to reduce its expression.

[0036] Furthermore, this invention achieves highly efficient transfection of nucleic acid drugs into target cells by loading nucleic acid drugs onto PSS31 and encapsulating Fuco on the outer layer. Using HPH as a disease model and siRNA drugs as model drugs, this invention verifies the delivery efficiency of the nucleic acid delivery vector. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0038] Figure 1 The synthetic route for the polymitochondrial targeting peptide PSS31;

[0039] Figure 2 High-resolution mass spectrometry image of polymitochondrial targeting peptide PSS31;

[0040] Figure 3 Performance test results for PSS31 compressing siHIF-1α:

[0041] Figure 4The results of characterization measurements of siHIF-1α / PSS31@Fuco are shown below. In this table, A represents the particle size distribution of siHIF-1α / PSS31, B represents the particle size distribution of siHIF-1α / PSS31@Fuco, C represents the potential distribution of siHIF-1α / PSS31, and D represents the potential distribution of siHIF-1α / PSS31@Fuco.

[0042] Figure 5 The results of Experiment 3 are shown in Figure A, where A represents the protective effect of siHIF-1α / PSS31@Fuco on siHIF-1α, and B represents the agarose gel electrophoresis image of siHIF-1α / PSS31@Fuco releasing siHIF-1α in response to ROS.

[0043] Figure 6 To detect the uptake of siHIF-1α / PSS31 and siHIF-1α / PSS31@Fuco by hypoxic-damaged PMVECs and PASMCs by flow cytometry, where A represents the uptake of siHIF-1α / PSS31 by hypoxic-damaged PMVECs, B represents the uptake of siHIF-1α / PSS31@Fuco by hypoxic-damaged PMVECs, C represents the uptake of siHIF-1α / PSS31 by hypoxic-damaged PASMCs, and D represents the uptake of siHIF-1α / PSS31@Fuco by hypoxic-damaged PASMCs.

[0044] Figure 7 The results are for Experiment 5. In Experiment 5, A is the Western Blot detection of the silencing effect of siHIF-1α / PSS31@Fuco on HIF-1α protein in hypoxic-exposed PMVECs, and B is the Western Blot detection of the silencing effect of siHIF-1α / PSS31@Fuco on HIF-1α protein in hypoxic-exposed PASMCs (n=3, Mean±SD, *P<0.05, **P<0.01, ns: no significance).

[0045] Figure 8 The results of the targeting experiment of siHIF-1α / PSS31@Fuco on lung tissue of HPH rats are shown. Among them, A is the distribution of siHIF-1α / PSS31@Fuco in major organs of HPH rats, B is the distribution of siHIF-1α / PSS31@Fuco in lung tissue of HPH rats, and C is the distribution of siHIF-1α / PSS31@Fuco in lung tissue sections.

[0046] Figure 9To investigate the expression of HIF-1α in the pulmonary artery of HPH rats using immunofluorescence staining;

[0047] Figure 10 To detect the effect of siHIF-1α / PSS31@Fuco on HIF-1α protein in lung tissue of HPH rats using Western Blot. Detailed Implementation

[0048] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0049] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0050] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0051] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0052] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0053] This invention provides a method for preparing the polymitochondrial targeting peptide PSS31, comprising the following steps:

[0054] (1) After dissolving SS31, sodium bicarbonate was added, and after stirring, 9-fluorenylmethyl-N-succinimide carbonate was added. The reaction was carried out, water was added, extraction was performed, and separation was carried out to obtain compound 1 as shown in Formula I.

[0055]

[0056] (2) Mix and dissolve compound 1 and ketethiocyanate, add benzotriazole-N,N,N,N′-tetramethylurea hexafluorophosphate and diisopropylethylamine, and react for 8-12 h to obtain compound 2 as shown in formula II;

[0057]

[0058] (3) Mix and dissolve compound 2 and compound 3 as shown in formula III, add benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate and diisopropylethylamine, react for 8-12 h to obtain compound 4 as shown in formula IV;

[0059]

[0060] (4) Compound 4 was added to a solution of N,N-dimethylformamide containing piperidine and reacted for 1 to 3 hours to obtain compound 5 as shown in formula V;

[0061]

[0062] (5) Mix and dissolve compound 5 and compound 2, add benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate and diisopropylethylamine, and react for 8-12 h to obtain compound 6 as shown in formula VI;

[0063]

[0064] (6) Compound 6 was dissolved in a solution of N,N-dimethylformamide containing piperidine and reacted for 1 to 3 h to obtain the polymitochondrial targeting peptide PSS31 as shown in Formula VII;

[0065]

[0066] In this invention, the molar ratio of SS31, sodium bicarbonate, and 9-fluorenylmethyl-N-succinimide carbonate in step (1) is 630-640:80-90:330-340, more preferably 634-638:84-88:334-338, and more preferably 635:85:337; the dissolution is preferably performed using a 1,4-dioxane solution, which is prepared by mixing 1,4-dioxane with water at a volume ratio of 1:1; the stirring... The stirring is preferably carried out under ice bath conditions, and the stirring time is preferably 14-16 min, more preferably 15 min; the addition of fmoc-osu is preferably done in batches, and the number of additions is preferably 3 times; the reaction temperature is preferably 20-30°C, more preferably 24-28°C, and even more preferably 25°C; the reaction time is preferably 8-12 h, more preferably 10 h; the extraction solvent used is preferably ethyl acetate; the separation method is preferably silica gel column chromatography. Compound 1 is (9H-fluorene-9-yl)methyl((R)-6-(((R)-1-amino-1-oxo-3-phenylpropane-2-yl)amino)-5-((R)-2-((S)-2-amino-5-guanidinepentanamino)-3-(4-hydroxy-2,6-dimethylphenyl)propionamide)-6-oxohexyl)carbamate.

[0067] In this invention, the structural formula of SS31 is shown in Formula VIII:

[0068]

[0069] In this invention, the molar ratio of compound 1 to TK (ketothioglycol) in step (2) is preferably 830-840:250-260, more preferably 834-838:254-258, and even more preferably 835:255; the solvent used for dissolution is preferably dichloromethane; the molar ratio of compound 1 to benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate and diisopropylethylamine is preferably 830-840:370-380:100-110, more preferably 834-838:374-378:104-108, and even more preferably 835:375. : 105; the reaction time is preferably 10 h; the reaction is preferably carried out under nitrogen protection; the reaction temperature is preferably 20-30℃, more preferably 24-28℃, and even more preferably 25℃; the reaction is preferably accompanied by stirring; after the reaction, thin-layer chromatography analysis is preferably performed, resuspended, deionized water with an equal volume of dichloromethane (DCM) is added, filtered, and purified by adding DCM containing petroleum ether to obtain compound 2; the purpose of the thin-layer chromatography analysis is to detect whether the reaction is complete; the purpose of the resuspension is to remove a large amount of solvent; the purpose of adding deionized water with an equal volume of DCM is to precipitate.

[0070] In this invention, compound 2 is (9R,12R,15S)-9-(((R)-1-amino-1-oxo-3-phenylpropane-2-yl)carbamoyl)-1-(9H-fluorene-9-yl)-15-(3-guanidinopropyl)-12-(4-hydroxy-2,6-di-methylbenzyl)-21,21-dimethyl-3,11,14,17-tetraoxy-2-oxa-20,22-dithia-4,10,13,16-tetraazapentane-25-acid.

[0071] In this invention, SS31 (D-Arg-2'6'-dimethylTyr-Lys-Phe-NH2) is a cationic polypeptide, and multiple clinical trials have shown that SS31 has good safety. SS31 has an isoelectric point of 10.3 and is positively charged under physiological conditions; however, its molecular weight is relatively small (639.8 g) and its positive charge is weak, limiting its ability to load nucleic acid drugs. The amino and carboxyl groups of the dicarboxylic ketone thioglycolate on SS31 exhibit strong reactivity. Therefore, by polymerizing SS31 using a ROS-sensitive dicarboxylic acid ketone (TK), ROS-responsive polystyrene (PSS31) is formed. This not only enables efficient loading of nucleic acid drugs but also allows for the release of nucleic acid drugs in the high ROS environment of diseased cells, effectively correcting the expression of target genes.

[0072] In this invention, the molar ratio of compound 2 to compound 3 in step (3) is preferably 1090–1100:860–870, more preferably 1094–1098:864–868, and even more preferably 1095:865; the molar ratio of compound 2 to benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate and diisopropylethylamine is preferably 1090–1100:370–380:120–130, more preferably 1094–1098:374–378:124–128, and even more preferably 1095:375:125; the dissolution process uses… The solvent is preferably dichloromethane; the reaction time is preferably 10 h; the reaction temperature is preferably 20–30 °C, more preferably 24–28 °C, and even more preferably 25 °C; the reaction is preferably carried out under nitrogen protection; the reaction is preferably accompanied by stirring; after the reaction, thin-layer chromatography analysis is preferably performed, followed by resuspension, addition of an equal volume of deionized water to DCM, filtration, and purification with DCM containing petroleum ether to obtain compound 4; the purpose of the thin-layer chromatography analysis is to detect whether the reaction is complete; the purpose of resuspension is to remove a large amount of solvent; the purpose of adding an equal volume of deionized water to DCM is to precipitate a precipitate.

[0073] In this invention, compound 3 is (9H-fluorene-9-yl)methyl((6S,9R,12R,15S)-1,16-diamino-12-(4-aminobutyl)-15-benzyl-9-(4-hydroxy-2,6-di-methylbenzyl)-1-imino-7,10,13,16-tetraoxo-2,8,11,14-tetraazahexanosaccharide-6-yl)carbamate; compound 4 is bis((9H-fluoro-9-yl)methyl)((6S,9R,12R,28S,31)methyl ...28S,28S,28S,28S,28S,28S,28S,28S,28S,28S,28S,28S,28S,28S,28S,28S,28S,28S,28S,28S,28S,28S,28S,28S,28S,28S,28S,28S,28S,28S,28S,28S,28S,28S,28S,28S,28S,28S,28S, R,34R)-1-amino-12,34-bis(((S)-1-amino-1-oxo-3-phenylpropane-2-yl)carbamoyl)-28-(3-guanidinopropyl)-9,31-bis(4-hydroxy-2,6-di-methylbenzyl)-1-imino-22,22-dimethyl-7,10,18,26,29,32-hexyloxy-21,23-dithia-2,8,11,17,27,30,33-heptazaoctazotricontan-6,38-di-yl)dicarbamate.

[0074] In this invention, the volume fraction of piperidine in the N,N-dimethylformamide solution in step (4) is preferably 15-25%, more preferably 18-22%, and even more preferably 20%; the reaction time is preferably 1.5-2.5 h, more preferably 2 h; the reaction temperature is preferably 20-30 °C, more preferably 24-28 °C, and even more preferably 25 °C; the reaction is preferably accompanied by stirring; after the reaction, thin-layer chromatography analysis is preferably performed, resuspended, DCM is added, and DCM containing petroleum ether is added for purification to obtain compound 5; the purpose of the thin-layer chromatography analysis is to detect whether the reaction is complete; the purpose of the resuspension is to remove a large amount of solvent; after adding DCM, stirring and rotary evaporation are preferably performed, and the number of stirring and rotary evaporation treatments is preferably 3-5 times, more preferably 4 times.

[0075] In this invention, compound 5 is (R)-6-amino-N-((S)-1-amino-1-oxo-3-phenylpropane-2-yl)-2-((6S,9R,12R,28S,31R)-1,6-diamino-12-(((S)-1-amino-1-oxo-3-phenylpropane-2-yl)carbamoyl)-28-(3-guanidinopropyl)-9,31-bis(4-hydroxy-2,6-dimethylbenzyl)-1-imino-22,22-dimethyl-7,10,18,26,29-pentoxy-21,23-dithia-2,8,11,17,27,30-hexazinetricontan-32-amide)hexanoamide.

[0076] In this invention, the molar ratio of compound 5 and compound 2 in step (5) is preferably 1490–1500:1090–1100, more preferably 1494–1498:1094–1098, and even more preferably 1495:1095; the solvent used for dissolution is preferably dichloromethane; the molar ratio of compound 5 to benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate and diisopropylethylamine is preferably 1490–1500:370–380:120–130, more preferably 1494–1498:374–378:124–128, and even more preferably... 1495:375:125; the reaction time is preferably 10 h; the reaction temperature is preferably 20-30℃, more preferably 24-28℃, and even more preferably 25℃; the reaction is preferably accompanied by stirring; the reaction is preferably carried out under nitrogen protection; after the reaction, thin-layer chromatography analysis is preferably performed, resuspended, deionized water of equal volume to DCM is added, filtered, and purified by adding DCM containing petroleum ether to obtain compound 6; the purpose of the thin-layer chromatography analysis is to detect whether the reaction is complete; the purpose of the resuspension is to remove a large amount of solvent; the purpose of adding deionized water of equal volume to DCM is to precipitate.

[0077] In this invention, compound 6 is bis((9H-fluorene-9-yl)methyl)((5S,8R,11R,23S,26R,29R,45S,48R,51R,67S,70R,73R)-5,73-bis(((R)-1-amino-1-oxo-3-phenylpropane-2-yl)carbamoyl)-29,51-bis((S)-1-amino-1-oxo-3-phenylpropane-2-yl)carbamoyl)-11,23,45,67-tetrayl(3-guanidinopropyl)-8, 26,48,70-Tetramethyl(4-hydroxy-2,6-dimethylbenzyl)-17,17,39,39,61,61-hexamethyl-7,10,13,21,24,27,35,43,46,49,57,65,68,71-tetracarboxoxy-16,18,38,40,60,62-hexaoxy-6,9,12,22,25,28,34,44,47,50,56,66,69,72-tetracarbonylnitrogen heptaheptylcontan-1,77-di-yl)dicarbamate.

[0078] In this invention, the volume fraction of piperidine in the N,N-di-methylformamide solution in step (6) is preferably 15-25%, more preferably 18-22%, and even more preferably 20%; the reaction time is preferably 1.5-2.5 h, more preferably 2 h; the reaction temperature is preferably 20-30 °C, more preferably 24-28 °C, and even more preferably 25 °C; the reaction is preferably accompanied by stirring; after the reaction, thin-layer chromatography analysis is preferably performed, resuspended, DCM is added, and DCM containing petroleum ether is added for purification to obtain the polymitochondrial targeting peptide PSS31; the purpose of the thin-layer chromatography analysis is to detect whether the reaction is complete; the purpose of the resuspension is to remove a large amount of solvent; after adding DCM, stirring and rotary evaporation are preferably performed, and the number of stirring and rotary evaporation treatments is preferably 3-5 times, more preferably 4 times.

[0079] In this invention, the polymitochondrial targeting peptide PSS31 is (S)-6-amino-N-((R)-1-amino-1-oxo-3-phenylpropane-2-yl)-2-((2R, 5R, 8R, 11S, 27R, 30R, 33S, 49R, 52R, 55S, 67R, 70R)-1-amino-27,49-bis((S)-1-amino-1-oxo-3-phenylpropane-2-yl)carbamoyl)-5-(4-aminobutyl)-2-benzyl-11,33,55,67-tetramethyl (3-Guidinylpropyl)-8,30,52,70-Tetramethyl(4-hydroxy-2,6-dimethylbenzyl)-17,17,39,39,61,61-Hexamethyl-1,4,7,10,13,21,29,32,35,43,51,54,57,65,68-Pentoxy-16,18,38,40,60,62-Hexamethyl-3,6,9,12,22,28,31,34,44,50,53,56,66,69-Tetracarboxamide-71-amide)hexamethylenetetramine.

[0080] In this invention, the preparation method of the polymitochondrial targeting peptide PSS31 is applicable to the preparation of polymitochondrial targeting peptide PSS31 with ketothiols as linkers of any degree of polymerization.

[0081] The present invention also provides the application of the aforementioned polymitochondrial targeting peptide PSS31 in the delivery of nucleic acid drugs.

[0082] This invention also provides a method for preparing the lung-targeted nucleic acid drug delivery system siHIF-1α / PSS31@Fuco, comprising the following steps:

[0083] Hypoxia-inducible factor-1α-specific small interfering RNA was mixed with the polymitochondrial targeting peptide PSS31, and fucoidan was added. After incubation, the lung-targeting nucleic acid drug delivery system siHIF-1α / PSS31@Fuco was obtained.

[0084] In this invention, the mass ratio of hypoxia-inducible factor-1α-specific small interfering RNA (siHIF-1α) to PSS31 is preferably 1:4 to 6, more preferably 1:5; the mixing is preferably allowed to stand for 20 to 40 minutes, more preferably 25 to 35 minutes, and even more preferably 30 minutes; the incubation temperature is preferably 35 to 40°C, more preferably 36 to 38°C, and even more preferably 37°C; the incubation time is preferably 0.5 to 1.5 hours, more preferably 0.8 to 1.2 hours, and even more preferably 1.0 hour.

[0085] The present invention also provides the application of the lung-targeted nucleic acid drug delivery system siHIF-1α / PSS31@Fuco in the preparation of drugs for treating hypoxic pulmonary hypertension.

[0086] Example 1

[0087] A method for preparing the polymitochondrial targeting peptide PSS31, comprising the following steps:

[0088] (1) Accurately weigh 768 mg (1.2 mol) of SS31 and dissolve it in 25 ml of a 1,4-dioxane-water mixed solvent (volume ratio 1:1). Then accurately weigh 202 mg (2.4 mol) of sodium bicarbonate into the reaction system and stir in an ice bath for 15 min. Subsequently, add 472 mg (1.4 mol) of 9-fluorenemethyl-N-succinimide carbonate (fmoc-osu) in three portions into the reaction system and stir overnight at room temperature. After monitoring the completion of the reaction by thin-layer chromatography, add 30 ml of deionized water to quench the reaction. Extract the product with ethyl acetate and concentrate by rotary evaporation. Separate and purify the target product by silica gel column chromatography to obtain compound 1 as shown in Formula I:

[0089] (2) Accurately weigh 1000 mg and 1.2 mmol of compound 1, 328 mg and 1.3 mmol of TK, place them in a round-bottom flask, add 5 ml of DCM, stir and dissolve at 25 °C, add 531 mg and 1.4 mmol of benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate (HBTU) and 129 mg and 1.2 mmol of DIPEA, stir and react at 25 °C for 10 h under nitrogen protection, monitor the reaction by thin-layer chromatography, resuspend to remove a large amount of solvent, add an equal volume of deionized water to precipitate the precipitate, filter, add DCM containing petroleum ether to recrystallize and purify, to obtain compound 2 as shown in Formula II;

[0090] (3) Accurately weigh 1096 mg and 1.0 mmol of compound 2 and 948 mg and 1.1 mmol of compound 3 as shown in Formula III, place them in a round-bottom flask, add 5 ml of DCM, stir and dissolve at 25 °C, add 455 mg and 1.2 mmol of benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate and 129 mg and 1.0 mmol of diisopropylethylamine, stir and react at 25 °C for 10 h under nitrogen protection, monitor the reaction by thin-layer chromatography, resuspend to remove a large amount of solvent, add an equal volume of deionized water to precipitate the precipitate, filter, add DCM containing petroleum ether to recrystallize and purify, to obtain compound 4 as shown in Formula IV;

[0091] (4) Accurately weigh 2328 mg and 1.2 mmol of compound 4, add 10 ml of N,N-dimethylformamide solution containing 20% ​​piperidine to dissolve, stir at 25 °C for 2 h, monitor the reaction by thin-layer chromatography to ensure complete reaction, resuspend to remove a large amount of solvent, add 20 ml of DCM and stir thoroughly, then evaporate to dryness (repeat this step 3 times), add DCM containing petroleum ether for recrystallization and purification to obtain compound 5 as shown in formula V;

[0092] (5) Accurately weigh 1496 mg and 1.0 mmol of compound 5 and 1206 mg and 1.1 mmol of compound 2, place them in a round-bottom flask, add 10 ml of DCM, stir and dissolve at 25 °C, add 455 mg and 1.2 mmol of benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate and 129 mg and 1.0 mmol of diisopropylethylamine, stir and react at 25 °C for 10 h under nitrogen protection, monitor the reaction by thin-layer chromatography, resuspend to remove a large amount of solvent, add an equal volume of deionized water to precipitate the precipitate, filter, add DCM containing petroleum ether to recrystallize and purify, to obtain compound 6 as shown in formula VI;

[0093] (6) Accurately weigh 4383 mg and 1.2 mmol of compound 6, add 10 ml of N,N-dimethylformamide solution containing 20% ​​piperidine to dissolve, stir at 25 °C for 2 h, monitor the reaction for completeness by thin-layer chromatography, resuspend to remove a large amount of solvent, add 20 ml of DCM and stir thoroughly, then evaporate to dryness (repeat this step 3 times), add DCM containing petroleum ether for recrystallization and purification to obtain the polymitochondrial targeting peptide PSS31 as shown in Formula VII.

[0094] Example 2

[0095] A method for preparing the polymitochondrial targeting peptide PSS31, comprising the following steps:

[0096] (1) Accurately weigh 768 mg, 1.2 mol of SS31 and dissolve it in 25 ml of a 1,4-dioxane-water mixed solvent (volume ratio 1:1). Then accurately weigh 202 mg, 2.4 mol of sodium bicarbonate into the reaction system and stir in an ice bath for 15 min. Subsequently, add 472 mg, 1.4 mol of 9-fluorenylmethyl-N-succinimide carbonate (fmoc-osu) in three portions into the reaction system and stir overnight at room temperature. After monitoring the completeness of the reaction by thin-layer chromatography, add 30 ml of deionized water to quench the reaction. Extract the product with ethyl acetate and concentrate by rotary evaporation. Separate and purify the target product by silica gel column chromatography to obtain compound 1 as shown in Formula I.

[0097] (2) Accurately weigh 1000 mg and 1.2 mmol of compound 1, 328 mg and 1.3 mmol of TK, place them in a round-bottom flask, add 5 ml of DCM, stir and dissolve at 20 °C, add 531 mg and 1.4 mmol of benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate (HBTU) and 129 mg and 1.2 mmol of diisopropylethylamine (DIPEA), stir and react at 20 °C for 12 h under nitrogen protection, monitor the reaction for completeness by thin-layer chromatography, resuspend to remove a large amount of solvent, add an equal volume of deionized water to precipitate the precipitate, filter, add DCM containing petroleum ether for recrystallization and purification to obtain compound 2 as shown in Formula II;

[0098] (3) Accurately weigh 1096 mg and 1.0 mmol of compound 2 and 948 mg and 1.1 mmol of compound 3 as shown in Formula III, place them in a round-bottom flask, add 5 ml of DCM, stir and dissolve at 20 °C, add 455 mg and 1.2 mmol of benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate and 129 mg and 1.0 mmol of diisopropylethylamine, stir and react at 20 °C for 12 h under nitrogen protection, monitor the reaction by thin-layer chromatography, resuspend to remove a large amount of solvent, add an equal volume of deionized water to precipitate the precipitate, filter, add DCM containing petroleum ether to recrystallize and purify, to obtain compound 4 as shown in Formula IV;

[0099] (4) Accurately weigh 2328 mg and 1.2 mmol of compound 4, add 10 ml of N,N-di-methylformamide solution containing 15% piperidine to dissolve, stir at 20 °C for 3 h, monitor the reaction by thin-layer chromatography to ensure complete reaction, resuspend to remove a large amount of solvent, add 20 ml of DCM and stir thoroughly, then evaporate to dryness (repeat this step 4 times), add DCM containing petroleum ether for recrystallization and purification to obtain compound 5 as shown in formula V;

[0100] (5) Accurately weigh 1496 mg and 1.0 mmol of compound 5 and 1206 mg and 1.1 mmol of compound 2, place them in a round-bottom flask, add 10 ml of DCM, stir and dissolve at 20 °C, add 455 mg and 1.2 mmol of benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate and 129 mg and 1.0 mmol of diisopropylethylamine, stir and react at 20 °C for 12 h under nitrogen protection, monitor the reaction by thin-layer chromatography, resuspend to remove a large amount of solvent, add an equal volume of deionized water to precipitate the precipitate, filter, add DCM containing petroleum ether to recrystallize and purify, to obtain compound 6 as shown in formula VI;

[0101] (6) Accurately weigh 4383 mg and 1.2 mmol of compound 6, add 10 ml of N,N-dimethylformamide solution containing 15% piperidine to dissolve, stir at 20 °C for 3 h, monitor the reaction by thin-layer chromatography to ensure complete reaction, resuspend to remove a large amount of solvent, add 20 ml of DCM and stir thoroughly, then evaporate to dryness (repeat this step 4 times), add DCM containing petroleum ether for recrystallization and purification to obtain the polymitochondrial targeting peptide PSS31 as shown in Formula VII.

[0102] Example 3

[0103] A method for preparing the polymitochondrial targeting peptide PSS31, comprising the following steps:

[0104] (1) Accurately weigh 768 mg, 1.2 mol of SS31 and dissolve it in 25 ml of a 1,4-dioxane-water mixed solvent (volume ratio 1:1). Then accurately weigh 202 mg, 2.4 mol of sodium bicarbonate into the reaction system and stir in an ice bath for 15 min. Subsequently, add 472 mg, 1.4 mol of 9-fluorenylmethyl-N-succinimide carbonate (fmoc-osu) in three portions into the reaction system and stir overnight at room temperature. After monitoring the completeness of the reaction by thin-layer chromatography, add 30 ml of deionized water to quench the reaction. Extract the product with ethyl acetate and concentrate by rotary evaporation. Separate and purify the target product by silica gel column chromatography to obtain compound 1 as shown in Formula I.

[0105] (2) Accurately weigh 1000 mg and 1.2 mmol of compound 1, 328 mg and 1.3 mmol of TK, place them in a round-bottom flask, add 5 ml of DCM, stir and dissolve at 30 °C, add 531 mg and 1.4 mmol of benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate (HBTU) and 129 mg and 1.2 mmol of DIPEA, stir and react at 30 °C for 8 h under nitrogen protection, monitor the reaction by thin-layer chromatography, resuspend to remove a large amount of solvent, add an equal volume of deionized water to precipitate the precipitate, filter, add DCM containing petroleum ether to recrystallize and purify, to obtain compound 2 as shown in Formula II;

[0106] (3) Accurately weigh 1096 mg and 1.0 mmol of compound 2 and 948 mg and 1.1 mmol of compound 3 as shown in Formula III, place them in a round-bottom flask, add 5 ml of DCM, stir and dissolve at 30 °C, add 455 mg and 1.2 mmol of benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate and 129 mg and 1.0 mmol of diisopropylethylamine, stir and react at 30 °C for 8 h under nitrogen protection, monitor the reaction by thin-layer chromatography, resuspend to remove a large amount of solvent, add an equal volume of deionized water to precipitate the precipitate, filter, add DCM containing petroleum ether for recrystallization and purification to obtain compound 4 as shown in Formula IV;

[0107] (4) Accurately weigh 2328 mg and 1.2 mmol of compound 4, add 10 ml of N,N-dimethylformamide solution containing 25% piperidine to dissolve, stir at 30 °C for 1 h, monitor the reaction by thin-layer chromatography to ensure complete reaction, resuspend to remove a large amount of solvent, add 20 ml of DCM and stir thoroughly, then evaporate to dryness (repeat this step 5 times), add DCM containing petroleum ether for recrystallization and purification to obtain compound 5 as shown in formula V;

[0108] (5) Accurately weigh 1496 mg and 1.0 mmol of compound 5 and 1206 mg and 1.1 mmol of compound 2, place them in a round-bottom flask, add 10 ml of DCM, stir and dissolve at 30 °C, add 455 mg and 1.2 mmol of benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate and 129 mg and 1.0 mmol of diisopropylethylamine, stir and react at 30 °C for 8 h under nitrogen protection, monitor the reaction by thin-layer chromatography, resuspend to remove a large amount of solvent, add an equal volume of deionized water to precipitate the precipitate, filter, add DCM containing petroleum ether to recrystallize and purify, to obtain compound 6 as shown in Formula VI;

[0109] (6) Accurately weigh 4383 mg and 1.2 mmol of compound 6, add 10 ml of N,N-di-methylformamide solution containing 25% piperidine to dissolve, stir at 30 °C for 1 h, monitor the reaction by thin-layer chromatography to ensure complete reaction, resuspend to remove a large amount of solvent, add 20 ml of DCM and stir thoroughly, then evaporate to dryness (repeat this step 5 times), add DCM containing petroleum ether for recrystallization and purification to obtain the polymitochondrial targeting peptide PSS31 as shown in Formula VII.

[0110] Example 4

[0111] A method for preparing a lung-targeted nucleic acid drug delivery system siHIF-1α / PSS31@Fuco, comprising the following steps:

[0112] Weigh out siHIF-1α and the polymitochondrial targeting peptide PSS31 described in Example 1, and place them in ultrapure water (DEPC water) to prepare a 20 μM siHIF-1α solution and a 1 mg / mL polymitochondrial targeting peptide PSS31 solution. Slowly add 5 μL of siHIF-1α solution dropwise to the PSS31 solution, vortex for 30 s to mix, and let stand for 30 min to obtain a siHIF-1α / PSS31 complex solution. Slowly add 10 μL of fucoidan (Fuco) solution (1 mg / mL) diluted with DEPC water dropwise, vortex for 30 s to mix, and incubate at 37 °C for 1 h to obtain the lung-targeting nucleic acid drug delivery system siHIF-1α / PSS31@Fuco.

[0113] Example 5

[0114] A method for preparing a lung-targeted nucleic acid drug delivery system siHIF-1α / PSS31@Fuco, comprising the following steps:

[0115] Weigh out siHIF-1α and the polymitochondrial targeting peptide PSS31 described in Example 2, and place them in ultrapure water (DEPC water) to prepare a 20 μM siHIF-1α solution and a 1 mg / mL polymitochondrial targeting peptide PSS31 solution. Slowly add 5 μL of siHIF-1α solution dropwise to the PSS31 solution, vortex for 30 s to mix, and let stand for 20 min to obtain a siHIF-1α / PSS31 complex solution. Slowly add fucoidan solution diluted with DEPC water dropwise, vortex for 30 s to mix, and incubate at 35 °C for 1.5 h to obtain the lung-targeting nucleic acid drug delivery system siHIF-1α / PSS31@Fuco.

[0116] Example 6

[0117] A method for preparing a lung-targeted nucleic acid drug delivery system siHIF-1α / PSS31@Fuco, comprising the following steps:

[0118] Weigh out siHIF-1α and the polymitochondrial targeting peptide PSS31 described in Example 3, and place them in ultrapure water (DEPC water) to prepare a 20 μM siHIF-1α solution and a 1 mg / mL polymitochondrial targeting peptide PSS31 solution. Slowly add 5 μL of siHIF-1α solution dropwise to the PSS31 solution, vortex for 30 s to mix, and let stand for 40 min to obtain a siHIF-1α / PSS31 complex solution. Slowly add fucoidan solution diluted with DEPC water dropwise, vortex for 30 s to mix, and incubate at 40 °C for 0.5 h to obtain the lung-targeting nucleic acid drug delivery system siHIF-1α / PSS31@Fuco.

[0119] Experiment 1: Screening for the optimal mass ratio of the siHIF-1α / PSS31 complex

[0120] The structure of the polymitochondrial targeting peptide PSS31 prepared in Example 1 was identified by high-resolution mass spectrometry. Simultaneously, siHIF-1α / PSS31 complex solutions with mass ratios of 1:1, 1:3, 1:5, 1:7, and 1:10 were prepared according to the method in Example 4, and the efficiency of PSS31 in compressing siHIF-1α was investigated using gel retardation experiments. The results are as follows... Figure 2 and Figure 3 As shown.

[0121] The results showed that the theoretical molecular weight of the polymitochondrial targeting peptide PSS31 was 3208.15; the [M+3H]-value was 3211.174; the [M+4H]-value was 3212.182; the [M+5H]-value was 3213.190; and the [M+6H]-value was 3214.198. The TOF-MS results for PSS31 were: [M+3H]-value 3211.647; [M+4H]-value 3212.624; [M+5H]-value 3213.645; and [M+6H]-value 3213.648, indicating that PSS31 was successfully synthesized.

[0122] Depend on Figure 3 It can be seen that the optimal feeding ratio of PSS31 to siHIF-1α is 1:5.

[0123] Example 2 Characterization Determination

[0124] The siHIF-1α / PSS31 complex solution and siHIF-1α / PSS31@Fuco prepared in Example 4 were diluted to 1.5 mL with DEPC water, respectively. The particle size and zeta potential of the siHIF-1α / PSS31 complex solution and siHIF-1α / PSS31@Fuco were measured using a laser particle size analyzer. The results are as follows: Figure 4As shown.

[0125] Depend on Figure 4 It can be seen that the average particle size of siHIF-1α / PSS31 is 161.5 nm and the zeta potential is 8.05 mV; the average particle size of siHIF-1α / PSS31@Fuco is 184.8 nm and the zeta potential is -23.8 mV.

[0126] Experimental Example 3: Investigation of the protective effect of siHIF-1α / PSS31@Fuco on siHIF-1α and its ROS-responsive drug release characteristics.

[0127] 100 μL of 37.5 nmol / L siHIF-1α / PSS31@Fuco solution (based on siHIF-1α) was prepared according to the method in Example 4. RNase enzyme solution (10 mg / ml) was added, and the solution was incubated at 37°C for 2 h. Residual RNase enzyme was removed by ultrafiltration and centrifugation. 150 μL and 1 mM H2O2 solutions were added to the siHIF-1α / PSS31 and siHIF-1α / PSS31@Fuco groups, respectively, and the solutions were incubated at 37°C for another 8 h. The protective effect of siHIF-1α / PSS31@Fuco on siHIF-1α was investigated using gel electrophoresis retardation assays.

[0128] A 100 μL solution of 37.5 nmol / L siHIF-1α / PSS31@Fuco (calculated as siHIF-1α) was prepared according to the method in Example 4. 150 μL of 1 mM H2O2 solution was added, and the solution was incubated at 37℃ for 0, 2, 4, 6, and 8 h. The release of siHIF-1α from siHIF-1α / PSS31@Fuco in H2O2 was investigated using gel electrophoresis retardation experiments. The results are as follows: Figure 5 As shown.

[0129] Depend on Figure 5 The agarose gel electrophoresis results after 2 hours of incubation of siHIF-1α / PSS31@Fuco with RNase enzyme showed that naked siHIF-1α was completely degraded after 2 hours of incubation with RNase enzyme; however, siHIF-1α in siHIF-1α / PSS31@Fuco was almost completely degraded, indicating that siHIF-1α / PSS31@Fuco can protect siHIF-1α from RNase enzyme degradation to a certain extent.

[0130] Depend on Figure 5It can be seen that as the incubation time of siHIF-1α / PSS31@Fuco with H2O2 increases, the amount of siHIF-1α released also gradually increases. After 8 hours, the release of siHIF-1α reaches its maximum, indicating that siHIF-1α / PSS31@Fuco has H2O2-responsive drug release characteristics.

[0131] Experiment 4: Effects of hypoxia-induced PMVECs and PASMCs on the uptake of siHIF-1α / PSS31@Fuco

[0132] Second-generation adherent PMVECs and PASMCs were directly extracted and isolated from SD rats (purchased from the Animal Center of Air Force Medical University, Xi'an, Shaanxi, China), and seeded into 12-well plates. The plates were then cultured in a normoxic cell incubator until cell adhesion was achieved. After 24 hours of culture, the old culture medium was discarded, and serum-free DMEM high-glucose medium (purchased from Wuhan Saiwei Biotechnology Co., Ltd.) was added. The plates were then cultured in a hypoxic incubator (5% O2) for another 24 hours. The culture medium was then replaced with 1 mL of siHIF-1α / PSS31 and siHIF-1α / PSS31@Fuco (siHIF-1α was FAM-labeled at a concentration of 37.5 nmol / L) diluted in serum-free DMEM high-glucose medium, and subsequently incubated in a hypoxic incubator (5% O2) for 1, 3, and 6 hours. Cells were collected and resuspended in pre-chilled 400 μL PBS buffer. Flow cytometry was used to investigate the uptake of siHIF-1α / PSS31 and siHIF-1α / PSS31@Fuco prepared in Example 4 by hypoxic-induced PMVECs and PASMCs. Results are as follows: Figure 6 As shown.

[0133] Flow cytometry results showed that siHIF-1α / PSS31@Fuco was taken up by hypoxic-damaged PMVECs or PASMCs in a time-dependent manner. Furthermore, at the same uptake time point, compared to siHIF-1α / PSS31, siHIF-1α / PSS31@Fuco accumulated significantly more in hypoxic-damaged PMVECs or PASMCs. These results indicate that siHIF-1α / PSS31@Fuco can better target hypoxic-damaged PMVECs or PASMCs.

[0134] Experimental Example 5: Silent Effect of siHIF-1α / PSS31@Fuc on HIF-1α Protein in Hypoxic-Induced PMVECs and PASMCs.

[0135] Second-generation adherent PMVECs and PASMCs were directly extracted and isolated from SD rats (purchased from the Animal Center of Air Force Medical University, Xi'an, Shaanxi, China), and seeded into 6-well plates. The plates were then cultured in a normoxic cell incubator until cell adhesion was achieved. After 24 hours, the old culture medium was discarded and replaced with 2 mL of serum-free DMEM high-glucose medium diluted with siHIF-1α / PSS31@Fuco or siNC / PSS31@Fuco (siNC and siHIF-1α were purchased from Gemma Gene in Shanghai, China; the preparation method of siNC / PSS31@Fuco was the same as that of siHIF-1α / PSS31@Fuco). Solutions of siHIF-1α / PSS31@Fnco, siHIF-1α / PSS31, and Lipofectamine3000@siHIF-1α (Lipofectamine3000@siHIF-1α was prepared according to the instructions for commercially available Lipofectamine3000 transfection reagent) were used. All solutions were prepared at a concentration of 75 nmol / L for siNC and siHIF-1α, respectively. A blank control group was added to 1 mL of fresh serum-free DMEM high-glucose culture medium. Transfection was performed in a hypoxic cell incubator (5% O2) for 48 h. After transfection, PMVECs or PASMCs were collected, and 85 μL of a mixture of RIPA lysis buffer (purchased from Beyotime Biotechnology, Shanghai) and a protease inhibitor (purchased from Servicebio, Wuhan) was added. The cells were lysed on ice for 30 min. Total protein was then extracted using a scraper (the extraction method was as follows: protein lysis buffer was collected in an EP tube, centrifuged at 12000 rpm for 15 min at 4℃, and the supernatant was collected). Protein concentration was quantified using a BCA protein assay kit (purchased from Xinsaimei, Suzhou), and the loading volume for each group was calculated to ensure that 25 μg of protein was loaded per group. The remaining protein sample was added to 1 / 5 of the sample volume of SDS-PAGE protein loading buffer, mixed thoroughly, and boiled at 100℃ for 8 min to denature the protein. After cooling to room temperature, it was stored at -20℃ for later use. Western blot experiments were performed to detect the expression level of HIF-1α protein in PMVECs or PASMCs cells. The results are as follows: Figure 7 As shown.

[0136] The results showed that siNC / PSS31@Fuco could not reduce the expression of HIF-1α protein in hypoxic-damaged PMVECs or PASMCs. The silencing effect of siHIF-1α / PSS31@Fuco on HIF-1α protein in hypoxic-damaged PMVECs or PASMCs was significantly better than that of siHIF-1α / PSS31, and essentially comparable to that of siHIF-1α@Lipo3000. These results indicate that siHIF-1α / PSS31@Fuco can effectively improve the transfection efficiency of siHIF-1α and its ability to silence HIF-1α protein in hypoxic-damaged PMVECs or PASMCs.

[0137] Experiment 6: Establishment of the HPH rat model and in vivo targeting study of siHIF-1α / PSS31@Fuco

[0138] The experiments were conducted using siHIF-1α / PSS31 and Cy5-siHIF-1α / PSS31@Fuco, prepared in Example 4. A hypoxic-hypoxic model was established in Sprague-Dawley rats (SD rats). Specifically, 27 male SD rats (approximately 200g each, purchased from the Animal Center of Air Force Medical University, Xi'an, Shaanxi, China) were acclimatized to standard conditions (noroxic environment) for one week. Nine rats were randomly selected and kept in a normoxic environment for another two weeks as the wild-type rat control (WT-C group). The remaining 18 rats were placed in a hypoxic-hypoxic chamber at a simulated altitude of 5500m (pressure 380mmHg, oxygen concentration 10%) for two weeks, with 8 hours of hypoxic-hypoxic conditions daily, serving as the wild-type rat hypoxia group (WT-H group). The rearing temperature was room temperature; relative humidity was 50%-60%; the lighting was 12 hours / darkness, with free access to water and food during this period.

[0139] Cy5-labeled Cy5-siHIF-1α / PSS31 and Cy5-siHIF-1α / PSS31@Fuco were prepared using siHIF-1α (GenePharma, Shanghai) labeled with Cy5 fluorescent dye. The preparation methods for Cy5-siHIF-1α / PSS31 and Cy5-siHIF-1α / PSS31@Fuco were the same as for siHIF-1α / PSS31@Fuco; Cy5-siHIF-1α was a fluorescently labeled siRNA purchased from GenePharma in Shanghai, China. Rats in the WT-H group were injected intravenously with either Cy5-siHIF-1α / PSS31 or Cy5-siHIF-1α / PSS31@Fuco solution (Cy5-siHIF-1α dosage was 0.5 mg / kg); rats in the WT-C group were injected intravenously with Cy5-siHIF-1α / PSS31@Fuco nanoparticle solution. Rats were euthanized 3, 12, and 24 hours after administration via tail vein. Major organs and tissues, including the brain, heart, liver, spleen, lungs, and kidneys, were removed. The dynamic distribution of Cy5-siHIF-1α / PSS31@Fuco in the rats' organs and lungs was observed using in vivo imaging at these times. Results are as follows: Figure 8 As shown.

[0140] In vivo imaging results showed that siHIF-1α / PSS31@Fuco was distributed in the lung tissue of HPH rats in a time-dependent manner. At the same time point after administration, siHIF-1α / PSS31@Fuco was significantly more distributed in the lung tissue of HPH rats than siHIF-1α / PSS31. Furthermore, compared to normoxic rats, siHIF-1α / PSS31@Fuco accumulated more readily in the lung tissue of HPH rats. This indicates that siHIF-1α / PSS31@Fuco has better targeting specificity in HPH rats.

[0141] Experiment 7: Co-localization of siHIF-1α / PSS31@Fuco with pulmonary artery endothelium and pulmonary artery smooth muscle in HPH rat lung tissue

[0142] Rapidly frozen sections of lung tissue isolated 12 hours after drug administration in Example 6 were used to investigate the co-localization of Cy5-labeled siHIF-1α / PSS31@Fuco with pulmonary artery endothelium (CD31 antibody labeling) and pulmonary artery smooth muscle cells (α-SMA antibody labeling) in lung tissue using immunohistochemistry. The specific steps are as follows:

[0143] First, the lung tissue samples from each group were briefly rinsed in pre-cooled physiological saline and immediately stored at -80°C. The sections were then rapidly frozen and placed at room temperature for 30 minutes. Acetone fixative (from Servicebio, Wuhan) was added and fixed for 10 minutes. The fixative was discarded, and the sections were washed three times in PBS buffer, 5 minutes each time. After antigen retrieval, the sections were placed in PBS buffer and washed three times on a shaker, 5 minutes each time. After drying, a circle was drawn around the tissue, and a biological tissue autofluorescence quencher (from Servicebio, Wuhan) was added. After washing with PBS, 3% BSA bovine serum albumin (from Servicebio, Wuhan) was added and blocked at room temperature for 30 minutes, followed by washing with pure water for 5 minutes. Primary antibody incubation: Appropriate amounts of CD31 antibody (1:100 dilution, from Affnity, Suzhou) and αt-SMA antibody (1:100 dilution, from Affnity, Suzhou) were added to the sections, and incubated overnight at 4°C. Secondary antibody incubation: Discard the primary antibody, immerse the slides in PBS buffer, and wash three times on a shaker for 5 min each time. After drying the slides, add 500 μl of FITC-labeled secondary antibody (1:200 dilution) and Alexa Fluor 488-labeled secondary antibody (1:100 dilution, both purchased from Abcam, Cambridge, England), and incubate at room temperature in the dark for 50 min.

[0144] After removing the secondary antibody, the slides were immersed in PBS buffer and washed three times (5 min each time) on a shaker. After drying, DAPI staining solution was added to the slide rings, and the slides were incubated at room temperature in the dark for 10 min. After mounting, the co-localization of Cy5-siHIF-1α / PSS31@Fuco with pulmonary artery endothelium and pulmonary artery smooth muscle in HPH rat lung tissue was observed using a fluorescence microscope. Results are as follows: Figure 9 As shown.

[0145] The results showed that CD31 (green), α-SMA (green), and Cy5 (red) were significantly co-localized in the pulmonary artery endothelium and pulmonary artery smooth muscle cells of HPH rats only after administration of siHIF-1α / PSS31@Fuco. These results indicate that siHIF-1α / PSS31@Fuco can accumulate in the pulmonary artery endothelium and pulmonary artery smooth muscle of HPH rats, demonstrating its targeting ability for hypoxic pulmonary artery endothelium and smooth muscle.

[0146] Experiment 8: Effect of siHIF-1α / PSS31@Fuco on HIF-1α protein expression in lung tissue of HPH rats

[0147] The HPH model SD rats constructed in Experiment 6 were divided into 5 groups of 6 rats each. Each group received a tail vein injection of saline, high-dose siHIF-1α / PSS31@Fuco (1.5 mg / kg, calculated based on siHIF-1α concentration), low-dose siHIF-1α / PSS31@Fuco (0.5 mg / kg, calculated based on siHIF-1α concentration), siNC / PSS31@Fuco (1.5 mg / kg, calculated based on siNC concentration), siHIF-1α / PSS31 (1.5 mg / kg, calculated based on siHIF-1α concentration), and PSS31 (PSS31 dosage was 9 mg / kg). Administration began on day 2 after entering the hypobaric chamber and continued every 2 days for a total of 4 times. During the administration period, the WT-H group continued to be placed in the hypobaric chamber for 8 hours daily. After treatment, the expression level of HIF-1α in the lung tissue of HPH rats in each group was detected by immunofluorescence staining.

[0148] After treatment, lung tissue was obtained from rats in each group, weighed, and rinsed with PBS. The lung tissue was then homogenized in a glass homogenizer, with 200 μL of RIPA lysis buffer added per 100 mg of tissue. The homogenate was thoroughly homogenized and lysed at 4°C on ice for 30 min. Total protein was extracted using a scraper, and the protein lysate was collected in EP tubes. After centrifugation at 12000 rpm for 15 min at 4°C, the supernatant was collected. Western blot analysis was used to detect the expression level of HIF-1α protein in the lung tissue of HPH rats. Results are as follows: Figure 10 As shown.

[0149] Western blot results showed that hypoxia significantly increased HIF-1α protein expression in lung tissue of HPH rats compared to normoxic rats. PSS31, siNC / PSS31@Fuco, and siHIF-1α / PSS31 all reduced HIF-1α protein expression in lung tissue of HPH rats to varying degrees. Among them, siHIF-1α / PSS31@Fuco showed the best silencing effect on HIF-1α protein in HPH rat lung tissue, significantly superior to siHIF-1α / PSS31. Simultaneously, the immunofluorescence staining results were consistent with the Western blot results. These results indicate that siHIF-1α / PSS31@Fuco can effectively improve the transfection efficiency of siHIF-1α and reduce HIF-1α protein expression.

[0150] As can be seen from the above embodiments, the present invention provides a method for preparing a polymitochondrial targeting peptide PSS31 and a lung-targeting nucleic acid drug delivery system, as well as its applications. The polymitochondrial targeting peptide PSS31 provided by the present invention is polymerized by the amino group of SS31 and the carboxyl group of TK. The provided siHIF-1α / PSS31@Fuco is composed of PSS31, siHIF-1α, and Fuco. The PSS31 can efficiently load nucleic acid drugs through electrostatic adsorption and can also efficiently release nucleic acid drugs under intracellular ROS stimulation. The siHIF-1α / PSS31@Fuco can efficiently load siHIF-1α and improve the stability of siHIF-1α, reduce pulmonary vascular remodeling, reduce pulmonary artery pressure, and provide multi-pathway efficient treatment for hypoxic pulmonary hypertension.

[0151] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing the polymitochondrial targeting peptide PSS31, characterized in that, Includes the following steps: (1) After dissolving SS31, sodium bicarbonate was added, and after stirring, 9-fluorenylmethyl-N-succinimide carbonate was added. The reaction was carried out, water was added, extraction was performed, and separation was carried out to obtain compound 1 as shown in Formula I. (2) Mix and dissolve compound 1 and ketithiolide, add benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate and diisopropylethylamine, react for 8-12 h to obtain compound 2 as shown in formula II; (3) Mix and dissolve compound 2 and compound 3 as shown in formula III, add benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate and diisopropylethylamine, and react for 8-12 h to obtain compound 4 as shown in formula IV. (4) Compound 4 was added to a solution of N,N-dimethylformamide containing piperidine and reacted for 1 to 3 hours to obtain compound 5 as shown in formula V; (5) Mix and dissolve compound 5 and compound 2, add benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate and diisopropylethylamine, and react for 8-12 h to obtain compound 6 as shown in formula VI; (6) Compound 6 was dissolved in a solution of N,N-dimethylformamide containing piperidine and reacted for 1 to 3 h to obtain the polymitochondrial targeting peptide PSS31 as shown in Formula VII; 2. The preparation method according to claim 1, characterized in that, In step (1), the molar mass ratio of SS31, sodium bicarbonate and 9-fluorenmethyl-N-succinimide carbonate is 630-640:80-90:330-340; the reaction is carried out under nitrogen protection.

3. The preparation method according to claim 1, characterized in that, In step (2), the molar ratio of compound 1 to ketethiocyanate is 830–840:250–260; the molar ratio of compound 1 to benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate and diisopropylethylamine is 830–840:370–380:100–110; the reaction is carried out under nitrogen protection.

4. The preparation method according to claim 1, characterized in that, In step (3), the molar ratio of compound 2 to compound 3 is 1090–1100:860–870; the molar ratio of compound 2 to benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate and diisopropylethylamine is 1090–1100:370–380:120–130; the reaction is carried out under nitrogen protection.

5. The preparation method according to claim 1, characterized in that, In step (5), the molar ratio of compound 5 to compound 2 is 1490–1500:1090–1100; the molar ratio of compound 5 to benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate and diisopropylethylamine is 1490–1500:370–380:120–130; the reaction is carried out under nitrogen protection.

6. The preparation method according to claim 1, characterized in that, The N,N-di-methylformamide solution contains 15-25% piperidine by volume; the solvent used for dissolution is dichloromethane.

7. The use of the polymitochondrial targeting peptide PSS31 according to any one of claims 1 to 6 in the delivery of nucleic acid drugs.

8. A method for preparing a lung-targeted nucleic acid drug delivery system siHIF-1α / PSS31@Fuco, characterized in that, Includes the following steps: Hypoxia-inducible factor-1α-specific small interfering RNA was mixed with the polymitochondrial targeting peptide PSS31 according to any one of claims 1 to 6, and fucoidan was added. The mixture was then incubated to obtain the lung-targeting nucleic acid drug delivery system siHIF-1α / PSS31@Fuco.

9. The use of the lung-targeted nucleic acid drug delivery system siHIF-1α / PSS31@Fuco as described in claim 8 in the preparation of a drug for treating hypoxic pulmonary hypertension.

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