Use of F5-peptide and / or p-gp inhibitor in preparation of drugs crossing blood-testis barrier and oral targeted nanoparticles and drugs

Abstract

The application of F5-peptide and / or P-gp inhibitor in the preparation of drugs crossing blood-testis barrier and oral targeted nanoparticles and drugs, for the first time, F5-peptide is used to destroy the physical barrier of BTB, and Tariquidar is used to inhibit the biochemical barrier of BTB, so that the BTB is instantaneously opened and closed, the basal ES protein and the tight junction protein are no longer closely connected with the BTB near the basement membrane, and the P-gp transporter cannot pump the exogenous drug Adjudin out of the BTB, so that the Adjudin is successfully transported to the spermatogenic epithelium, and the FAF5T-LAN oral targeted nano-preparation carrying active Adjudin molecules is constructed, and is expected to become a high-compliance male contraceptive, and has important social significance and economic value.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, specifically to the application of F5-peptide and / or P-gp inhibitors in the preparation of drugs that cross the blood-testis barrier, and oral targeted nanoparticles and drugs. Background Technology

[0002] The seminiferous epithelium of the testis is the starting point of spermatogenesis and is mainly composed of supporting cells attached to the basement membrane and various germ cells. The various intercellular junctions between the supporting cells constitute the blood-testis barrier (BTB). The presence of the BTB prevents harmful substances from entering the seminiferous epithelium from the bloodstream, but it also prevents the translocation of most small-molecule and large-molecule drugs (such as peptides, proteins, and gene-based drugs). Studies have shown that the BTB not only hinders the transport of drugs (such as contraceptives and antiviral drugs) within the seminiferous tubules, preventing them from directly acting on the near-compartmental region of the seminiferous epithelium, but also prevents antiretroviral agents from completely clearing viruses from the seminiferous tubules. The testes thus become a potential reservoir for viruses, significantly increasing the risk of relapse of viral diseases such as Zika virus, HIV, and COVID-19, and severely limiting the treatment of testicular-related diseases (such as testicular cancer, testicular infection, and testicular injury).

[0003] In the mammalian testis, the adhesion junctions between Sertoli cells and sperm cells are called apical ectoplasmic specialization (apical ES), while the adhesion junctions between Sertoli cells are called basal ectoplasmic specialization (basal ES). The basement membrane of the seminiferous epithelium (BTB) is primarily composed of tight junctions between adjacent Sertoli cells located near the basement membrane of the seminiferous epithelium. In addition, basal ES, gap junctions, and desmosomes based on intermediate filaments also participate in the formation of the BTB. Figure 1 The seminiferous tubules (BTB) divide the seminiferous epithelium into basal and luminal chambers, creating relatively independent microenvironments for spermatogenesis. In rodent spermatogenesis stages VIII-IX, which correspond to human seminiferous epithelial stages VI-VII, spermatocytes, as a specialized cell group, cross the BTB via intercellular bridges. Simultaneously, mature sperm are released near the lumen of the seminiferous tubules. Although these two cellular events occur at the same stage of the spermatogenesis cycle, they are located at opposite ends of the seminiferous tubules.

[0004] The lipid bilayer (BTB) functions as both a physical and biochemical barrier, significantly restricting drug transport to the testes. Firstly, while the BTB provides a favorable microenvironment for spermatogenesis, it also restricts the diffusion and transport of water, electrolytes, ions, hormones, paracrine factors, and other substances via bypass and transcellular pathways. This also greatly limits drug transport across the barrier, for example, small hydrophilic drugs and large molecules that cannot easily diffuse across the lipid bilayer (such as antibodies and antibody-drug conjugates). Therefore, inhibiting the physical barrier function of the BTB, momentarily "opening" it, is crucial for drug transport across the BTB. Summary of the Invention

[0005] To address the technical deficiencies of existing technologies, this invention provides an application of F5-peptide and / or P-gp inhibitors in the preparation of drugs that cross the blood-testis barrier, as well as oral targeted nanoparticles and drugs. This promotes the drug's entry into the seminiferous epithelial lumen after BTB to exert its effects, providing a new approach for the development of barrier-crossing drugs and possessing significant social and economic value.

[0006] The technical solution adopted in this invention is the application of F5-peptide in the preparation of drugs that cross the blood-testis barrier.

[0007] The amino acid sequence of the F5-peptide is shown in SEQ ID NO. 1.

[0008] The aforementioned transblood-testis barrier drug includes the DNA sequence of an F5-peptide, which is synthesized in vivo via delivery.

[0009] Application of P-gp inhibitors in the preparation of drugs that cross the blood-testis barrier.

[0010] The P-gp inhibitor mentioned is the selective, non-competitive P-gp inhibitor Tariquidar.

[0011] Application of F5-peptide and P-gp inhibitor Tariquidar in the preparation of drugs that cross the blood-testis barrier.

[0012] An oral targeted nanoparticle for contraception, the oral targeted nanoparticle comprising the aforementioned F5-peptide and P-gp inhibitor Tariquidar, pharmaceutical component FSHβ-Adjudin conjugate, and nanomaterials.

[0013] The nanomaterials mentioned are one or more of liposomes or alginate.

[0014] A method for preparing the oral targeted nanoparticles includes the following steps:

[0015] (1) Preparation of FSHβ-Adjudin coupling: FSHβ was dissolved in DMF, Adjudin was dissolved in chloroform, DCC was dissolved in DMSO, and DMAP was dissolved in DMSO. The completely dissolved FSHβ and Adjudin were stirred evenly at 37°C. DCC was then added and reacted. DMAP was then added and reacted in the dark. The mixture was dialyzed in a 1.0 kDa dialysis bag in the dark and then lyophilized for later use.

[0016] (2) Preparation of FAF5T-LAN nanoparticles: The plasmid DNA of F5-peptide was dissolved in DEPC water, and Tariquidar was diluted to 2 μM / L. 40 mg / ml of DPPC, cholesterol, and DSPE-PEG were dissolved in chloroform at a molar ratio of 2:1:0.15. The solution was placed in a round-bottom evaporation flask and evaporated under negative pressure at room temperature to completely remove the solvent components. The plasmid DNA of F5-peptide, Tariquidar and FSHβ-Adjudin conjugate were added to the liposome components and mixed by rotating and sonicating. Then, the solution was resuspended in sodium alginate solution and 1 mM CaCl2 was slowly added dropwise at 4 °C. The mixture was polymerized and crosslinked to obtain FAF5T-LAN oral targeted nanoparticle formulation.

[0017] An oral targeted drug for contraception, said oral targeted drug comprising the oral targeted nanoparticles of claim 7.

[0018] Mechanism: Transport is crucial. During phase VIII of the spermatogenic epithelial cycle, Sertoli cells express a large amount of matrix metalloproteinase 2, which mediates the hydrolysis of lamininγ3 (apical ES linker protein), releasing domain IV. The 50 amino acid residues cloned from this fragment are called F5-peptide, which allows for reversible "leakage" of BTB and promotes the degradation of apical ES, facilitating sperm maturation and release. Figure 2 The project applicant previously discovered that overexpression of F5-peptide in the testes of male SD rats resulted in the release of numerous immature sperm cells into the lumen of seminiferous tubules or confinement near the basement membrane. F5-peptide modulates the integrity of the cytoskeleton by affecting actin regulatory proteins (such as Eps8 and Arp3) and microtubule regulatory proteins (such as MARK4 and EB1), thereby altering the assembly process, morphology, and function of the cytoskeleton. Therefore, endogenous F5-peptide could be an ideal molecule to "open" the physical barrier of the BTB.

[0019] In addition, the testicular tract barrier (BTB) contains many drug transport proteins, such as permeability glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MDR1), which pump drugs out of the BTB in the testes. P-gp, also known as MDR1, consists of two hydrophobic domains, one of which is a drug-binding domain with 12 transmembrane α-helical structures responsible for transporting toxins and drugs out of the cell. P-gp is highly expressed in testicular Sertoli cells, germ cells, peritubular myoid cells of the kidneys, and endothelial cells of microvessels. P-gp works synergistically with other efflux drug pumps to actively pump exogenous drugs that have permeated into the BTB out of the testes, preventing drugs from entering the seminiferous epithelium. Therefore, blocking the drug efflux function of P-gp is crucial for the development of drugs that cross the BTB barrier. Tariquidar is a selective, non-competitive P-gp inhibitor that binds to P-gp with high affinity, blocking P-gp from pumping exogenous drugs out of the tissue. Therefore, Tariquidar can be another effective target for drug entry into the testis. After the drug completes its transport process, Tariquidar can be used to weaken the biochemical barrier function of BTB, causing BTB to "shut down" momentarily, reducing drug efflux and increasing drug utilization.

[0020] The beneficial effects of this invention are as follows: This invention provides an application of F5-peptide and / or P-gp inhibitor in the preparation of drugs that cross the blood-testis barrier, as well as oral targeted nanoparticles and drugs. It is the first invention to propose using F5-peptide to disrupt the physical barrier of BTB, and Tariquidar to inhibit P-gp to disrupt the biochemical barrier of BTB, causing BTB to "open" and "close" instantaneously. The basal ES protein and tight junction protein are no longer tightly connected to the BTB near the basement membrane, and the P-gp transporter protein cannot pump the exogenous drug Adjudin out of BTB, thereby allowing Adjudin to be successfully transported to the seminiferous epithelium. It proposes to construct an oral targeted nanoformulation of FAF5T-LAN carrying active Adjudin molecules, which is expected to become a highly compliant male contraceptive, and has important social and economic value. Attached Figure Description

[0021] Figure 1 This image shows the location and morphology of BTB in the seminiferous epithelium.

[0022] Figure 2 2. F5-peptide inhibits apical ES and basal ES / BTB.

[0023] Figure 3 A scientific hypothesis for the regulation of BTB barrier function and spermatogenesis by FAF5T-LAN nanoparticles.

[0024] Figure 44. The coupling principle of Adjudin and FSHβ.

[0025] Figure 5 This is a technical roadmap for the present invention.

[0026] Figure 6 (A) Particle size distribution of FAF5T-LAN, (B) Transmission electron microscopy of FAF5T-LAN, (C) Cytotoxicity test of FAF5T-LAN.

[0027] Figure 7 (A) RT-PCR detection of F5-peptide overexpression level, (B) semi-quantitative level of F5-peptide overexpression, (C) WB detection of P-gp protein level, (D) semi-quantitative level of P-gp, (E) immunofluorescence detection of red fluorescence near cell nucleus (DAPI) (FAF5T-LAN containing Cy5.5), confirming that FAF5T-LAN was successfully transfected into Sertoli cells (green fluorescent GFP).

[0028] Figure 8 (A) TER (transepithelial electrical resistance) assay to detect FAF5T-LAN’s inhibition of BTB barrier function in Sertoli cells in vitro; (B) WB assay to detect the level of BTB tight junction protein inhibited by FAF5T-LAN; (C) Semi-quantitative level of BTB tight junction protein.

[0029] Figure 9 The FIF assay was used to detect the inhibition of BTB tight junction protein distribution by FAF5T-LAN.

[0030] Figure 10 To assess the inhibition of F-Actin, α-Tubulin, and Vimentin expression and distribution by FAF5T-LAN using immunofluorescence or Phalloidin staining. Detailed Implementation

[0031] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] Adjudin's interference with spermatogenesis and its limitations in application.

[0033] Adjudin, also known as AF2364 (MW: 335.18), is a derivative of chlordamine. It works by affecting the expression and distribution of actin regulatory proteins (Eps8, Arp3, Palladin, Formin1, etc.) and microtubule regulatory proteins (CAMSAP2, EB1, MARK4, etc.) in the seminiferous epithelium. This prevents the normal polymerization and depolymerization of actin and microtubules, interfering with the anchoring junction between Sertoli cells and spermatids. It also affects the morphology and function of their adhesion protein complexes, leading to the shedding of immature germ cells, including elongated spermatids, round spermatids, and spermatozoa, from the seminiferous epithelium (while spermatogonia remain unaffected), thus achieving male contraception. Adjudin may also interfere with spermatogenesis by regulating the expression levels of p-FAK-Y407 and N-WASP, thus affecting cytoskeleton stability.

[0034] Adjudin, a small-molecule lipophilic compound, cannot freely diffuse across the blood-brain barrier (BTB) to the seminiferous epithelium; instead, it makes the BTB more compact and difficult to cross. Adjudin may enter the seminiferous epithelium via drug inflow and outflow pumps located at the base of Sertoli cells and within the BTB. This results in a small difference between the effective dose and toxic concentration of Adjudin, with a bioavailability of less than 1%. The applicant previously co-administered Adjudin with F5 peptides in rat Sertoli cells to reduce the effective contraceptive dose of Adjudin, but still failed to achieve oral administration of Adjudin. This study will use Adjudin as a drug research model to evaluate its distribution in the testes and utilize F5-peptide and the P-gp inhibitor Tariquidar to disrupt the physical and biochemical barriers of the BTB, promoting the trans-BTB transport of Adjudin within the seminiferous epithelium and increasing its bioavailability. In-depth animal model studies will elucidate the mechanism of drug trans-BTB transport and provide new insights for other drug trans-tissue-blood barrier studies.

[0035] FSHβ peptide targets Sertoli cells

[0036] Follicle-stimulating hormone (FSH) is a glycosylated protein hormone synthesized by the anterior pituitary gland. It binds to the FSH receptor (FSHr) on Sertoli cells, stimulating these cells to provide structural and nutritional support to germ cells at different developmental stages. FSH consists of two subunits, α and β, which form a heterodimer through non-covalent bonds. The FSHβ subunit is the main active component of FSH, composed of 118 amino acid residues, with the binding sites for FSHr located between amino acids 33-53 and 81-95. FSHr is primarily found on Sertoli cells in the testes of male mammals, but its expression is very low in germ cells and other tissue cells. Therefore, the FSHβ subunit is a preferred carrier for targeted drug delivery to the testes. In fact, the FSH mutant occludin conjugate can specifically disrupt BTB, and the melphalan and FSHβ conjugate can induce testicular damage in mice to achieve male contraceptive effects. Based on the binding site of FSHr, the applicant's research group synthesized a short peptide of FSHβ (FSHβ for short). By binding to FSHβ, the contraceptive efficacy of Adjudin was increased by approximately 10,000 times without adverse reactions. However, once the FSHβ-Adjudin conjugate is taken orally, it is hydrolyzed and inactivated by various proteases in the stomach. Therefore, this conjugate is limited to intraperitoneal injection. How to administer the FSHβ-Adjudin conjugate orally and induce a reversible effect remains unsolved.

[0037] Nanomedicine sustained release system

[0038] Nanomedicines are mostly distributed in the size range of 50-500 nm. By adjusting the release time and rate of nanomedicines, controlled drug release can be achieved, prolonging the duration of drug action, reducing dosage, and decreasing drug toxicity and side effects. The applicant has previously prepared multifunctional silk fibroin nanoparticles containing 5-FU carrying cyclic cRGDfk peptides and Chlorin e6, which can effectively target and achieve multimodal therapy for gastric cancer. In the first 4 hours, free 5-FU was completely released (>97%), with 60% of the 5-FU released from 5-FU @ SF (particle size 226.0 nm), while only about 33% of the 5-FU was released from 5-FU @ SF-cRGDfk-Ce6 NP (particle size 364.9 nm), suggesting that the release rate can be effectively controlled after the drug is reassembled by the nanocomposite. Therefore, preparing Adjudin as a nano-sustained-release drug and targeting its delivery to the testicular seminiferous epithelium has certain potential application value and social significance.

[0039] Liposomes consist of a lipid bilayer with a particle size of approximately 100 nm. Compared to viral vectors, the gene-binding process of liposomes is simpler; after fusion with the cell membrane, the target gene is introduced into the cell, and the lipids are degraded immediately, exhibiting no immunogenicity and effectively protecting DNA or RNA from degradation by plasma ribozymes. Kevin and Luca et al. successfully used DNA-based triskelion structures on the surface of liposomes for targeted drug delivery; Jennifer and Andrew et al. co-encapsulated spherical nucleic acids and drugs within liposomes, facilitating delivery to tissues outside the liver. These studies demonstrate that genes carried by liposomes can be transported to specific sites, and the transfection process is convenient, easy, and reproducible. Alginic acid, a natural anionic polymer derived from brown algae, possesses characteristics such as non-immunogenicity and good biocompatibility. The three-dimensional network nanogels or nanoparticles formed by its cross-linking with Ca2+ can be widely used for loading drugs, plasmids, and linear nucleic acids. Furthermore, alginate nanogels exhibit high pH sensitivity, remaining highly stable in acidic environments (such as gastric juice) but gradually degrading in weakly alkaline environments (such as intestinal juice). This pH-responsive characteristic is highly advantageous for the preparation of oral formulations of small molecule drugs. Patil and Devarajan et al. prepared insulin-containing alginate nanoparticles using nicotinamide as a permeabilizer, achieving a bioavailability of up to 80%. Therefore, liposome- and alginate-based nanomaterials can be effectively used for targeted drug delivery.

[0040] Preparation and characterization of FAF5T-LAN

[0041] (1) Preparation of FSHβ-Adjudin conjugates Figure 4 ): Dissolve 1 mg FSHβ in 10 ml DMF, 2 mg Adjudin in 20 ml chloroform, 1 mg DCC in 0.5 ml DMSO, and 1.5 mg DMAP in 1.0 ml DMSO. Stir the completely dissolved FSHβ and Adjudin at 37°C until homogeneous; then add DCC and react for 30 minutes; then add DMAP and react overnight (protected from light); dialyze the mixture in a 1.0 kDa dialysis bag protected from light for 48 hours, and lyophilize for later use.

[0042] (2) Preparation of FAF5T-LAN nanoparticles: 1 mg of F5-peptide plasmid DNA (i.e., F5) was dissolved in 2.5 ml of DEPC water and diluted to 2 μM / L with 10 mM / L Tariquidar. 40 mg / ml of DPPC, cholesterol, and DSPE-PEG were dissolved in 2.5 ml of chloroform at a molar ratio of 2:1:0.15 and placed in a 15 ml round-bottom evaporation flask. The solvent components were completely removed by rotary evaporation under negative pressure for 1 hour at room temperature. F5, Tariquidar, and 0.5 mg of FSHβ-Adjudin conjugate were added to the lipid components and mixed by rotary sonication. The mixture was then resuspended in 100 ml of 1% sodium alginate solution, and 1 mM CaCl2 was slowly added dropwise at 4 °C. Polymerization was carried out for about 1 hour, followed by centrifugation, washing, purification, and lyophilization. FSHβ-LAN, Adjudin-LAN, F5-LAN, Tariquidar-LAN, and blank LAN were prepared as controls for subsequent experiments.

[0043] (3) Material characterization of FAF5T-LAN nanoparticles: Detection of material characterization parameters such as zeta potential, particle size, encapsulation efficiency, molecular group distribution and chemical structure, and in vitro sustained release kinetics.

[0044] 1. Zeta potential and particle size: The size and morphology of FAF5T-LAN nanoparticles were evaluated using transmission electron microscopy, and the hydrodynamic diameter and zeta potential of FAF5T-LAN nanoparticles were detected using a Zetasizer Nano ZSP.

[0045] 2. Encapsulation efficiency: The standard and FAF5T-LAN were added to the HPLC analyzer in fractions, and linear gradient analysis was performed using acetonitrile / water in a linear reversed phase to determine the encapsulation efficiency and cumulative drug release curve of Adjudin.

[0046] 3. Molecular group distribution and chemical structure: The molecular group distribution and chemical structure of FAF5T-LAN were detected by X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy and Raman confocal microscopy.

[0047] 4. In vitro sustained-release kinetics: 0.5 mg FAF5T-LAN was added to 100 ml of pH 1.8 buffer (simulated gastric fluid) and pH 8.0 buffer (simulated intestinal fluid), respectively, and stirred at 37 °C. Samples were taken at time intervals of 5, 10, 30 minutes and 1, 2, 4, 8, 12, 24, 48 hours. 0.5 ml of the supernatant was collected by centrifugation, and the release of Adjudin from FAF5T-LAN was determined by HPLC-MS / MS.

[0048] Effects of FAF5T-LAN on the structure and function of BTB in Sertoli cells in vitro

[0049] (1) Isolation and primary culture of Sertoli cells: Diluted Matrigel (dilution ratio of 1:5 or 1:7) was evenly spread on cell culture plates and incubated for 1 hour, then placed in an incubator for later use. Sterile mixtures of trypsin, STI, collagenase 20, collagenase 40, and hyaluronidase containing DNase were prepared. Testes from 10 20-day-old male SD mice were digested sequentially with the above enzyme mixtures, centrifuged, and purified to obtain Sertoli cells, which were then seeded onto Matrigel-coated culture plates. Sertoli cells were cultured in DMEM / F12 medium supplemented with growth factors (including EGF, Transferin, Insulin, and Gentamicin), with fresh medium changed daily.

[0050] (2) FAF5T-LAN interference with BTB structure and function in vitro: On the third day of Sertoli cell culture in vitro, 50, 100, 500, and 1000 nmol / L FAF5T-LAN and corresponding controls were added to the culture system and incubated for 24 hours. The culture medium was replaced with fresh medium, and the cells were incubated for another 24 hours before the cells were collected. The expression levels of BTB-related proteins (N-cadherin, β-catenin, ZO-1, and CAR), cytoskeletal proteins and their regulatory proteins (F-actin, Eps8, Arp3, Palladin, Formin1, α-tubulin, CAMSAP2, EB1, and MARK4) and their interactions were detected by qPCR, WB, CO-IP, and IF. The expression differences of BTB regulatory molecules (p-FAK-Y407, p-FAK-Y397, and N-WASP) were evaluated. For Sertoli cells used in TER assays, after incubation in FAF5T-LAN for 24 hours, fresh DMEM / F12 was used, and TER was measured daily to detect the tight junction level of Sertoli cell BTB in vitro.

[0051] (3) Mechanism of FAF5T-LAN interference with in vitro BTB: The FAK and N-WASP genes were knocked out using CRISPR / Cas9 technology to obtain FAK- / - N-WASP- / - FAK- / - N-WASP- / - FAK- / - N-WASP- / - FAK- / - N-WASP- / - FAK- / - N-WASP- / - FAK- / - N-WASP- / - FAK- / - N-WASP- / - FAK- / - N-WASP- / - FAK- / - N-WASP- / - FAK- / - N-WASP- / - FAK- / - N-WASP- / - FAK- / - N-WASP- / - FAK- / - N-WASP- / - F and ​ Sertoli cells were treated with 50, 100, 500, and 1000 nmol / L FAF5T-LAN. ​ and ​Sertoli cells were incubated for 24 hours, then the culture medium was changed, and the cells were incubated for another 24 hours before collection. The expression and distribution of BTB-related proteins (as above), cytoskeletal proteins, and their regulatory proteins (as above) were detected using qPCR, WB, IF, and CO-IP techniques. Actin dynamics and microtubule dynamics were detected using F-actin bundling assay and Microtubule spin-down assay, respectively. TER was measured daily to detect continuous changes in tight junctions of BTB in vitro, exploring the role of FAK / N-WASP in the regulation of BTB by FAF5T-LAN.

[0052] The role of FAF5T-LAN in interfering with spermatogenesis in rats and its mechanism of trans-BTB transport.

[0053] (1) Animal model: 300g adult male SD rats were divided into 8 groups (8 rats in each group), namely FAF5T-LAN (containing Adjudin 1, 5, and 50mg / kg respectively), FSHβ-LAN, Adjudin-LAN, F5-LAN, Tariquidar-LAN and blank LAN. They were orally fed once every other day for 3 consecutive days.

[0054] (2) FAF5T-LAN interference with rat testicular BTB function and spermatogenesis: On day 12, 100 μl of biotin solution was injected into the testes. After 30 minutes, the testes were harvested and sectioned. Alexa Fluor 555-streptavidin was added and incubated at room temperature for 1 hour. The degree of BTB "leakage" was accurately assessed by calculating the percentage of the diffusion distance of biotin from the base of the seminiferous tubules to the lumen relative to the corresponding seminiferous tubule radius. On day 21, testicular, liver, and kidney tissues were harvested, dehydrated, embedded, sectioned, and stained with hematoxylin and eosin (HE) to assess spermatogenesis defects and toxic effects on tissues and organs. Semen was collected for routine testing to assess sperm motility defects.

[0055] (3) Mechanism of FAF5T-LAN promoting the trans-BTB transport of adjudin: On day 21, an appropriate amount of testicular tissue was taken for QPCR, WB, CO-IP, IF and other experimental techniques to detect the expression and distribution levels of BTB linker proteins, cytoskeletal proteins and their regulatory proteins in the testes, and the expression changes of BTB regulatory molecules. Further exploration of the regulatory mechanism of FAF5T-LAN promoting the trans-BTB transport of adjudin in vivo.

[0056] Technology roadmap: (see) ​ ).

[0057] Particle size distribution and cytotoxicity assessment of FAF5T-LAN

[0058] Currently, we have preliminarily completed the chemical conjugation of FSHβ and Adjudin, the preparation of F5- and Tariquidar-containing liposomes and alginate-shell cross-linked particles, and preliminary characterization and cytotoxicity tests show that the particles are approximately 150 nm in size, uniformly distributed, and have no significant toxicity to Sertoli cells. ​ ).

[0059] FAF5T-LAN targets Sertoli cells

[0060] After FAF5T-LAN was applied to Sertoli cells in vitro, the gene level of F5-peptide was significantly increased. ​ A, B), P-gp protein levels were significantly downregulated ( ​ C, D), FAF5T-LAN (Cy 5.5, red fluorescence) was successfully localized near the nucleus of Sertoli cells ( ​ E).

[0061] FAF5T-LAN inhibits BTB barrier function in vitro.

[0062] After FAF5T-LAN was applied to Sertoli cells in vitro, TER (transepithelial resistance) was significantly downregulated, while Free Adjudin significantly upregulated TER levels. ​ A), the levels of BTB tight junction proteins (N-cadherin and β-catenin) decreased to varying degrees. ​ B, C).

[0063] FAF5T-LAN inhibited the expression and distribution of BTB tight junction protein in vitro.

[0064] After acting on Sertoli cells in vitro, FAF5T-LAN significantly inhibited the expression and distribution of BTB tight junction proteins N-cadherin (green fluorescence) and β-catenin (red fluorescence) compared to Free Adjudin. ​ ).

[0065] FAF5T-LAN interfered with the expression and distribution of cytoskeletal proteins in Sertoli cells.

[0066] Compared with Free Adjudin, FAF5T-LAN significantly downregulated the expression and distribution of cytoskeletal proteins F-Actin (green fluorescence), α-Tubulin (red fluorescence), and Vimentin (red fluorescence) in Sertoli cells after in vitro treatment. ​ ).

[0067] in conclusion

[0068] (1) It was first proposed to use F5-peptide to destroy the physical barrier of BTB, and Tariquidar to inhibit P-gp to destroy the biochemical barrier of BTB, so that BTB can be "opened" and "closed" instantly. The basal ES protein and tight junction protein are no longer tightly connected to the BTB near the basement membrane. The P-gp transporter protein cannot pump the exogenous drug Adjudin out of BTB, thus allowing Adjudin to be successfully transported to the spermatogenic epithelium.

[0069] (2) The first proposal to construct FAF5T-LAN oral targeted nano-formulation carrying active Adjudin molecules is proposed, which is expected to become a highly compliant male contraceptive and has important social and economic value.

[0070] (3) It is expected to reveal the molecular mechanism by which FAF5T-LAN interferes with BTB function and promotes the trans-BTB transport of Adjudin. This project aims to further reveal the role of p-FAK-Y407, p-FAK-Y397 and N-WASP in the process of FAF5T-LAN promoting the trans-BTB transport of Adjudin from the perspective of cytoskeletal proteins through CRISPR / Cas9 gene editing technology.

[0071] Please note to all technical personnel: Although the present invention has been described according to the specific embodiments above, the inventive concept of the present invention is not limited to this invention. Any modifications that utilize the inventive concept will be included within the scope of protection of this patent.

[0072] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention. sequence list <110> The Second Affiliated Hospital of Wenzhou Medical University (Wenzhou Medical University Yuying Children's Hospital) <120> Applications of F5-peptide and / or P-gp inhibitors in the preparation of drugs that cross the blood-testis barrier and oral targeted nanoparticles and drugs <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 63 <212> PRT <213> Artificial Sequence <400> 1 Leu Ser Leu Ala Leu Lys Pro Ser Asn Leu Pro Ser Ser Gln Asp Thr 1 5 10 15 Arg Gln Pro Gly Arg Val Gln Leu Gln Phe Leu Leu Gln Glu Thr Ser 20 25 30 Glu Glu Ala Glu Pro Pro Leu Pro Ala Phe His Phe Gln Arg Leu Leu 35 40 45 Ser Asn Leu Thr Thr Leu Ser Ile Trp Thr Ser Gly Gln Gly Leu 50 55 60

Claims

1. An oral targeted nanoparticle for contraception, characterized in that, The oral targeting nanoparticle comprises F5-peptide and P-gp inhibitor Tariquidar, a drug component FSHbeta-Adjudin coupling and a nanometer base material, wherein the nanometer base material is liposome and sodium alginate, and the FSHbeta-Adjudin coupling is prepared by the following steps: dissolving FSHbeta in DMF, dissolving Adjudin in chloroform, dissolving DCC in DMSO, dissolving DMAP in DMSO, stirring the completely dissolved FSHbeta and Adjudin uniformly at 37 DEG C, then adding DCC after reaction, then adding DMAP and avoiding light reaction, then dialyzing the mixture in a 1.0KDa dialysis bag under light avoidance, and freeze-drying for standby use. The oral targeting nanoparticle is prepared by the following steps: dissolving plasmid DNA of F5-peptide in DEPC water, diluting Tariquidar into 2uM / L, dissolving 40mg / ml DPPC, cholesterol and DSPE-PEG into chloroform according to a molar ratio of 2:1:0.15, placing them in a round-bottom evaporation flask, rotating evaporation under negative pressure at room temperature, completely removing solvent components, adding plasmid DNA of F5-peptide, Tariquidar and FSHbeta-Adjudin coupling into liposome components and rotating ultrasonic mixing, then resuspending in sodium alginate solution, slowly dropping 1mM CaCl2 at 4 DEG C, and polymerizing and cross-linking to obtain FAF5T-LAN oral targeting nano-preparation, wherein the amino acid sequence of the F5-peptide is shown as SEQ ID NO.

1.

2. A process for the preparation of the oral targeted nanoparticle as claimed in claim 1, wherein, The method comprises the following steps: (1) preparing FSHbeta-Adjudin coupling: dissolving FSHbeta in DMF, dissolving Adjudin in chloroform, dissolving DCC in DMSO, dissolving DMAP in DMSO, stirring the completely dissolved FSHbeta and Adjudin uniformly at 37 DEG C, then adding DCC after reaction, then adding DMAP and avoiding light reaction, then dialyzing the mixture in a 1.0KDa dialysis bag under light avoidance, and freeze-drying for standby use; (2) preparing FAF5T-LAN nanoparticle: dissolving plasmid DNA of F5-peptide in DEPC water, diluting Tariquidar into 2uM / L, dissolving 40mg / ml DPPC, cholesterol and DSPE-PEG into chloroform according to a molar ratio of 2:1:0.15, placing them in a round-bottom evaporation flask, rotating evaporation under negative pressure at room temperature, completely removing solvent components, adding plasmid DNA of F5-peptide, Tariquidar and FSHbeta-Adjudin coupling into liposome components and rotating ultrasonic mixing, then resuspending in sodium alginate solution, slowly dropping 1mM CaCl2 at 4 DEG C, and polymerizing and cross-linking to obtain FAF5T-LAN oral targeting nano-preparation, wherein the amino acid sequence of the F5-peptide is shown as SEQ ID NO.

1.

3. An oral targeted drug for contraception, characterized in that, The oral targeting drug comprises the oral targeting nanoparticle of claim 1.

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