Growth hormone receptor targeting polypeptide and application thereof

Abstract

The invention relates to the field of polypeptides, in particular to a polypeptide targeting a growth hormone receptor and application of the polypeptide. The invention provides a polypeptide for targeting a growth hormone receptor, which is characterized in that the polypeptide is selected from a group consisting of SEQ ID NOs: 1-15. The polypeptide provided by the invention can effectively target adipocytes and adipose tissues (such as groin fat and / or gonad fat), and the enrichment degree in main organs (such as liver, lung, kidney and other organs) is low.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of polypeptides, in particular to a polypeptide targeting growth hormone receptor and uses thereof. BACKGROUND

[0002] Growth hormone (GH) is a hormone essential for growth and is secreted by growth hormone-secreting cells in the anterior pituitary, which participates in multiple biological processes such as growth, development, reproduction, metabolism and immunity. Growth hormone is secreted into the blood when it is produced in the pituitary, binds to the growth hormone receptor (GHR), and then exerts its function.

[0003] Current research on growth hormone and growth hormone receptor mainly focuses on the signal pathways (such as STATs, PI3K-Akt and MEK-Erk, etc.) activated after growth hormone binds to growth hormone receptor. SUMMARY

[0004] In a first aspect, the present application provides a polypeptide for targeting growth hormone receptor, characterized in that the polypeptide is selected from the group consisting of SEQ ID NOs: 1-15.

[0005] In some embodiments, the polypeptide is used for targeting growth hormone receptor of adipocytes.

[0006] In some embodiments, the polypeptide is further linked to a fluorescent group.

[0007] In some embodiments, the fluorescent group comprises FITC (fluorescein isothiocyanate).

[0008] In a second aspect, the present application provides use of the above-mentioned polypeptide in the preparation of an adipocyte detection reagent.

[0009] In some embodiments, the detection reagent is used for detecting the expression level of growth hormone receptor of the adipocytes.

[0010] In some embodiments, the detection reagent is used for qualitative and / or quantitative detection of the adipocytes.

[0011] In some embodiments, the adipocytes are derived from visceral fat and / or subcutaneous fat.

[0012] In some embodiments, the adipocytes are derived from a mammal.

[0013] In some embodiments, the adipocytes are in a single cell state.

[0014] Thirdly, the present invention provides the use of the above-mentioned polypeptide in the preparation of a drug delivery system targeting adipose tissue.

[0015] In some embodiments, the drug delivery system includes the peptide, the nanoparticle carrier, and the drug, wherein the drug is loaded inside the nanoparticle carrier, and the peptide is linked to the nanoparticle carrier via an amide bond.

[0016] In some embodiments, the drug includes a weight-loss drug and / or a lipophilic fluorescent dye.

[0017] In some embodiments, the nanoparticle carrier includes liposomes.

[0018] In some embodiments, the weight-loss drug includes one or more of rosiglitazone, capsaicin, insulin, and smegglutide.

[0019] In some embodiments, the liposomes comprise phospholipids, DSPE-PEG, and cholesterol.

[0020] In some embodiments, the phospholipids include soybean lecithin.

[0021] In some embodiments, the drug delivery system is prepared by the following steps:

[0022] S101 incorporates phospholipids, DSPE (distearate phosphatidylethanolamine)-PEG (polyethylene glycol), and cholesterol. Sterols and drugs are dissolved in a container containing an organic solvent to form a first mixture;

[0023] In some embodiments, the mass ratio of the phospholipid, the DSPE-PEG, the cholesterol, and the drug comprises 4:1:1:(0.1-1).

[0024] In some embodiments, the mass ratio of the phospholipid, the DSPE-PEG, the cholesterol, and the drug comprises 4:1:1:0.2.

[0025] In some embodiments, the organic solvent includes chloroform or trichloromethane.

[0026] In some embodiments, the concentration of the drug in the first mixture includes 0.1-10 mg / mL.

[0027] In some embodiments, the concentration of the drug in the first mixture includes 0.1-1 mg / mL.

[0028] In some embodiments, the concentration of the drug in the first mixture includes 0.2 mg / mL.

[0029] S102 causes the first mixture to form a film in the container;

[0030] In some embodiments, the film is made by nitrogen blowing or by vacuum evaporation of organic solvents using a rotary evaporator.

[0031] S103 The film is resuspended to form a suspension and sonicated until the suspension is clear, forming a nanoparticle carrier loaded with the drug.

[0032] S104 The first solution and the second solution are added to the drug-loaded nanoparticle carrier and shaken to form the drug delivery system, wherein the first solution comprises an aqueous solution of EDAC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and a polypeptide, and the second solution comprises an aqueous solution of N-hydroxythiosuccinimide sodium salt.

[0033] In some embodiments, the concentration of the polypeptide in the first solution includes 0.1-10 mg / mL.

[0034] In some embodiments, the concentration of the polypeptide in the first solution includes 2.5 mg / mL.

[0035] In some embodiments, the mass ratio of the polypeptide to the drug includes 1:(0.1-1).

[0036] In some embodiments, the lipophilic fluorescent dye includes a DID fluorescent dye.

[0037] In some embodiments, the adipose tissue includes inguinal fat and / or gonadal fat.

[0038] In some embodiments, the particle size of the drug delivery system includes 100-200 nm.

[0039] In some embodiments, the drug delivery system is used to treat obesity-related diseases.

[0040] In some embodiments, the obesity-related diseases include one or more of diabetes, obesity, and metabolic syndrome.

[0041] Compared with the prior art, the beneficial effects of the present invention include at least the following aspects:

[0042] Current research on growth hormone and its receptor mainly focuses on the signaling pathways activated after growth hormone binds to its receptor (e.g., STATs, PI3K-Akt, and MEK-Erk). This invention demonstrates through extensive experiments that the growth hormone receptor is a membrane protein gene specifically highly expressed in adipocytes, and that the growth hormone receptor can serve as a marker for adipocytes.

[0043] Based on this, the present invention creatively screens small molecule peptides that target adipocytes. These peptides are all 12-peptides and possess high affinity for growth hormone receptors in mammalian (e.g., human, pig, mouse) adipocytes (e.g., affinity for growth hormone receptors in adipocytes is above 0.8). They can specifically target adipocytes, exhibiting significant advantages in adipocyte detection. Compared to traditional adipocyte detection methods (e.g., surface plasmon resonance (SPR) and biomembrane interferometry (BLI)), the small molecule peptides provided by the present invention exhibit extremely high specificity while being less expensive, making them suitable for qualitative and / or quantitative detection of adipocytes from various sources.

[0044] Furthermore, and unexpectedly, the small molecule peptides provided by this invention can not only effectively target adipose tissue (e.g., groin fat and / or gonadal fat) in organisms, but also exhibit low enrichment levels in major organs (e.g., liver, lungs, kidneys, etc.). Compared to existing reported peptides targeting adipose tissue (e.g., Posi peptides targeting prohibitin and thus adipose tissue), the small molecule peptides provided by this invention can precisely deliver drugs (e.g., weight-loss drugs) to the adipose tissue of organisms, thereby promoting drug efficacy to a certain extent while reducing potential side effects and adverse effects on major organs. This allows for more targeted inhibition of fat accumulation and the obesity process, leading to better weight-loss results. Attached Figure Description

[0045] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. The elements or parts in the drawings are not necessarily drawn to scale. Obviously, the drawings described below are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0046] Figure 1 Single-cell nuclear transcription maps of adipose tissue from 17 different sites in pigs;

[0047] Figure 2a The figure shows the results of single-cell nuclear transcriptome analysis of human adipose tissue to identify high expression of GHR in adipocytes;

[0048] Figure 2b The figure shows the results of single-cell nuclear transcriptome analysis of porcine adipose tissue to identify high expression of GHR in adipocytes;

[0049] Figure 2c The figure shows the results of single-cell transcriptome analysis of mouse adipose tissue to identify high expression of GHR in adipocytes;

[0050] Figure 3 This is a graph showing the affinity results of the small molecule peptides screened in Example 2;

[0051] Figure 4 This is a diagram showing the results of the small molecule peptides screened in Example 2 being able to target adipocytes;

[0052] Figure 5a The results of GHR-A8 targeting are shown in the figure.

[0053] Figure 5b The results of GHR-A9 targeting are shown in the figure.

[0054] Figure 5c The results of GHR-A18 targeting;

[0055] Figure 6 A diagram showing the result of a delivery system for loading weight-loss drugs;

[0056] Figure 7 This is a diagram showing the specific delivery results of the delivery system of the present invention. Detailed Implementation

[0057] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, 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 some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0058] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0059] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0060] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0061] As used in this specification, the term "approximately" typically means + / - 5% of the stated value. More typically, the value is + / -4%, more typically, the value is + / -3%, more typically, the value is + / -2%, even more typically, the value is + / -1%, even more typically, the value is + / -4%. + / - 0.5%.

[0062] In this specification, certain embodiments may be disclosed in a range-bound format. It should be understood that this "range-bound" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered as specifically disclosing all possible subranges and the individual numerical values ​​within that range. For example, a description of the range 1-6 should be considered as specifically disclosing subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and the individual numbers within this range, such as 1, 2, 3, etc. 4, 5, and 6. The above rules apply regardless of the breadth of the scope.

[0063] Example 1

[0064] This embodiment constructed single-cell nuclear transcriptional atlases of adipose tissue from 17 different sites in pigs (1-2 replicates per site, totaling 192,006 nuclei) and identified five major cell types common to pig adipose tissue (which can be further divided into 11 cell types and 20 clusters), including immune cells, adipocytes, vascular cells, mesenchymal stromal cells, and mesothelial cells, such as... Figure 1 As shown.

[0065] exist Figure 1 The four outer circles, from the outside in, represent: celltype, which indicates the five major cell types mentioned above; tissue, which indicates the origin of the five major cell types (for example, adipocytes have 17 different origins with varying proportions); group, which indicates the classification of 17 adipose tissues (i.e., subcutaneous (4), visceral (10), and others (3)); and cell_type20, which represents a further subdivision of the five major cell types, including adipocytes, myoblastic adipocyte progenitor cells, preadipocytes, endothelial cells, smooth muscle cells, macrophages, B cells, T cells, dendritic cells, and mesothelial cells. In addition, adipocytes can be further divided into subclasses.

[0066] Next, based on the above results, this embodiment compared the gene expression of 43,707 porcine adipocytes with that of four other cell types, identifying 160 adipocyte-specifically highly expressed genes, of which 37 could be annotated as membrane protein genes. Furthermore, this embodiment further analyzed single-cell nuclear transcriptome data from human and mouse adipose tissue, finding that the 37 porcine-specifically highly expressed membrane protein genes were largely conserved in both humans and mice. The results are as follows... Figure 2a , Figure 2b and Figure 2c As shown in the violin diagram (which represents the expression levels of GHR in adipocytes, mesenchymal cells, immune cells, vascular cells, and mesothelial cells, with the horizontal axis representing different cell types and the vertical axis representing normalized and logarithmic gene expression levels), GHR (growth hormone receptor) is a membrane protein gene that is specifically highly expressed in adipocytes (i.e., it has the best consistency and the highest expression level), and therefore can be used as a marker for adipocytes.

[0067] Example 2

[0068] 2.1 Biological Screening

[0069] (1) Spread GHR antigen (10 μg / ml, CBS buffer (pH 9.6), 200 μl / well) onto ELISA plates and incubate overnight at 4°C.

[0070] (2) Discard the plating buffer and pat the plate with a sterile paper towel. Wash the plate three times with PBS.

[0071] (3) Add blocking buffer (5% milk / PBS), incubate at 37°C for 2 hours, 300 μL / well, and discard the blocking buffer. Wash the plate 3 times with PBS.

[0072] (4) Dilute Ph.D. with 5% milk / PBS. TM -12 phage display peptide library, so that the amount of phage input is approximately 1 to 2 × 10⁻⁶. 12 PFU. Incubate at 37°C for 2 hours.

[0073] (5) Wash the plate 6 times with 0.1% PBST, and then wash it 3 times with PBS to remove unbound phages.

[0074] (6) Elute the bound phages with 200 μL of Glycine-HCl for 8 minutes, then rinse with Tris-HCl. Neutralize to pH 7.0.

[0075] (7) In order to increase the number of high-affinity phages, strict conditions will be applied in subsequent rounds, such as reducing the amount of coating antigen, increasing the washing time and the concentration of Tween-20, or using competitive elution.

[0076] The selection criteria for each round are as follows:

[0077] 2.2 Detection of titer of eluted bacteriophages

[0078] (1) Culture Escherichia coli ER2738 to the logarithmic phase.

[0079] (2) Dilute the eluent, and then mix 10 μL of the diluted solution with 200 μL of Escherichia coli ER2738. Incubate the mixture at 37°C for 5 minutes.

[0080] (3) Transfer the mixture to an IPTG-Xgal plate. Invert the plate and incubate overnight at 37°C.

[0081] 2.3 Amplification of eluted bacteriophages

[0082] (1) Culture Escherichia coli ER2738 to the logarithmic phase. Add 200 μL of the effusive phage to 40 mL of Escherichia coli ER2738 culture medium and shake at 250 rpm for 4-5 hours.

[0083] (2) Centrifuge at 10000 rpm for 2 minutes. After centrifugation, transfer the supernatant to a sterile test tube. Mix. Place the mixture on ice for 1–2 hours.

[0084] (3) Centrifuge the solution and suspend the precipitate in 1 mL of PBS. After centrifugation, transfer the supernatant to a sterile test tube. This is the amplified phage, which can be used for the next step of biological culture.

[0085] 2.4 Polyclonal phage ELISA

[0086] 1) Add 100 μL of target protein (1 μg / ml, HIS-tagged GHR protein) to each well. Incubate overnight at 4°C to allow for complete binding.

[0087] 2) Wash each well three times with 300 μL PBS.

[0088] 3) Block with 300 μL of blocking buffer (5% milk / PBS) at room temperature for 1.5 hours.

[0089] 4) Shake the blocking buffer and wash the plate three times with washing buffer (PBS), tapping the plate face down on a clean paper towel each time.

[0090] 5) Add 100 μL of diluted amplified phage to the well. Incubate at 37°C for 1-2 hours.

[0091] 6) Wash the plate three times with 300 μL of 0.1% PBST or PBS.

[0092] 7) Dilute M13 (M13 Bacteriophage Antibody)-HRP (1:1000) with blocking buffer. Add 100 μL of the diluted conjugate to each well and incubate at room temperature for 40 minutes to 1 hour.

[0093] 8) Wash 6 times with washing buffer. Add 100 μL TMB to each well and incubate at 37°C for 15 minutes. Then add 50 μL of 0.5 mol / L 98% H2SO4 to each well.

[0094] 9) Use an ELISA reader set to a wavelength of 450nm-630nm to read the plate.

[0095] 2.5 Monoclonal phage ELISA

[0096] 1) Pick 1×96 clones from the 2-YT plate and inoculate 800 μL into each well of a 96-well deep-well plate. In 2xYT medium (containing Escherichia coli ER2738 cultured to the logarithmic phase), shake at 250 rpm and 37°C for 4.5-5 hours.

[0097] 2) Centrifuge at 4000 rpm for 8-10 minutes in a plate centrifuge, transfer the supernatant to a new 96-well plate and store at 4°C.

[0098] 3) Spread the antigen (1 μg / mL, CBS buffer (pH 9.6), 100 μL / well) on a 96-well plate. Incubate overnight at 4°C.

[0099] 4) Wash each well three times with 300 μL PBS.

[0100] 5) Block with 300 μL of blocking buffer (5% milk / PBS) at 37°C for 1.5 hours.

[0101] 6) Shake the blocking buffer and wash the plate three times with washing buffer (PBST), patting the plate face down onto a clean paper towel each time.

[0102] 7) Add 100 μL of phage (the supernatant from step 2) to the wells (experimental group and control group). Incubate at 37°C for 2 hours.

[0103] 8) Wash the plate three times with 300 μL of 0.1% PBST, and then wash the plate three times with PBS.

[0104] 9) Dilute M13-HRP (1:1000) with blocking buffer. Add 100 μL of the diluted conjugate to each well and incubate at room temperature for 40 minutes to 1 hour.

[0105] 10) Wash the plate three times with 300 μL of 0.1% PBST, then wash it three times with PBS. Add 100 μL of TMB to each well, incubate at 37°C for 15 minutes, and then add 50 μL of 0.5 mol / L 98% H2SO4 to each well.

[0106] 11) Use an ELISA reader to read the plate at a wavelength of 450nm-630nm.

[0107] Using phage display technology, this embodiment screened out 15 small molecule peptides (all 12 peptides) with potential high affinity GHR, such as... Figure 3 As shown, its amino acid sequence is as follows. Note: Data at GHR indicates affinity for GHR (relative to the negative control protein), and data at His indicates affinity for the negative control protein (e.g., His). The small polypeptides obtained by cloning GHR-A12 and GHR-A16 have identical sequences.

[0108] Example 3

[0109] Porcine adipose tissue was single-celled using physical disruption and enzymatic digestion. Adipocytes and SVFs were then separated by centrifugation (SVFs precipitate after centrifugation) and flotation (adipocytes are lighter than water and float after centrifugation). Subsequently, adipocytes and SVFs were incubated with 2 μM GHR-A8_FITC (a small molecule peptide modified with FITC) for 5 min each, and excess GHR-A8-FITC was eluted to remove excess GHR-A8-FITC before fluorescence imaging.

[0110] Figure 4 The first column shows images of bright-field adipocytes and SVFs; the second column shows cell mask staining. The first column shows cell membrane labeling; the second column shows DAPI-labeled cell nuclei; the third column shows GHR-A8_FITC signals. The fifth column shows the merged results of the above. In this example, GHR-A8 was screened and modified with FITC, and then incubated with adipocytes and SVFs (adipose-vascular matrix components, a collective term for cells other than adipocytes in adipose tissue). The results showed that GHR-A8 was enriched around adipocytes, compared to... GHR-A8 is not enriched around SVFs. These results indicate that GHR-A8 has the ability to target adipocytes and demonstrate that adipocytes highly express GHR compared to SVFs.

[0111] Example 4

[0112] The following fluids were administered intravenously via the tail vein to 10-week-old male C57 mice according to their respective groups (200 μL per mouse):

[0113] Control group: Mice were injected with saline; FITC group: Mice were injected with FITC alone. GHR-A8-F group: mice were injected with the small molecule peptide GHR-A8 modified with FITC; S1H group: mice were injected with S1H modified with FITC (an artificial peptide mimicking amino acids 36 to 51 of human growth hormone); Positive group: mice were injected with a peptide modified with FITC that targets prohibitin (PHB) on the surface of adipocytes (CKGGRAKDC, SEQ ID NO:16).

[0114] In the GHR-A8-F group, the S1H group, and the Positive group, FITC was coupled 1:1 at the N-terminus of the peptide. The FITC concentration in each group was 500 μM.

[0115] Three hours after injection, the mice were euthanized, and major organs, adipose tissue, and muscle tissue were collected. The distribution and enrichment of fluorescence signals were detected using a small animal in vivo imaging system to determine the precise targeting of the small molecule peptides.

[0116] Compared with the control group, FITC mainly accumulated in the lungs, kidneys, abdominal fat (groin fat) and gonads (gonadal fat), GHR-A8-F mainly accumulated in the groin fat and gonadal fat, and the two positive controls, S1H and Positive, mainly accumulated in the liver, lungs, kidneys, abdominal fat and gonads. Figure 5a The results also showed that GHR-A8-F can effectively target adipocytes in abdominal fat and gonads, while essentially not targeting organs such as the liver, lungs, and kidneys.

[0117] Using a similar method, this embodiment also validated other small molecule peptides (e.g., GHR-A9 and GHR-A18) obtained in Example 2. The results are as follows: Figure 5b and Figure 5c As shown, GHR-A9 and GHR-A18 can also effectively target adipocytes in abdominal fat and gonads, but they do not target organs such as the liver, lungs, and kidneys.

[0118] Example 5

[0119] The experimental reagents and materials used in this embodiment are: soybean lecithin, cholesterol, rosiglitazone, DSPE-PEG2000, chloroform, EDAC, sodium N-hydroxythiosuccinimide, and GHR-A8. Round-bottom flask, beaker, ultrafiltration tube.

[0120] The experimental equipment used in this embodiment includes: a weighing balance, a rotary evaporator, an ultrasonic cell disruptor, a centrifuge, and an ultraviolet spectrophotometer.

[0121] 5.1 Preparation of rosiglitazone@liposomes:

[0122] Weigh 40mg of soybean lecithin, 10mg of cholesterol and 2mg of rosiglitazone into a 250ml round-bottom flask, add 10ml of chloroform to dissolve completely, and then evaporate under reduced pressure in a constant temperature water bath at 55℃ for 40min until a uniform and transparent film forms on the round-bottom flask.

[0123] Add 5ml of pure water to hydrate the lipid film for 10 minutes. The film will be cloudy. Sonicate at 150W power for 10 minutes until the liquid is clear to form a lipid nanocarrier.

[0124] The lipid nanocarrier was transferred into an ultrafiltration tube with a molecular weight of 3KD and centrifuged at 8000xg for 1.5h at 4°C to remove free drug (rosiglitazone) and water.

[0125] The absorbance of the lipid nanocarrier at 317 nm was measured using a UV spectrophotometer, and the concentration was calculated based on the standard curve.

[0126] 5.2 Preparation of rosiglitazone@liposomes@small molecule peptides:

[0127] Step 1: Weigh out 40mg of soy lecithin, 10mg of cholesterol, 2mg of rosiglitazone, and DSPE-PEG. 2000 10mg was dissolved in 10ml of chloroform in a 250ml round-bottom flask. The solution was then evaporated under reduced pressure in a constant temperature water bath at 55℃ for 40min until a uniform and transparent film was formed on the surface of the round-bottom flask.

[0128] Step 2: Add 5ml of pure water to hydrate the lipid film for 10 minutes. The film will be cloudy. Sonicate at 150W for 10 minutes until the liquid is clear, forming a lipid nanocarrier.

[0129] Step 3: Weigh 4 mg of EDAC and 5 mg of small molecule peptide (GHR-A8 in this example) and dissolve them in 2 ml of pure water, then vortex at 28.0 rpm for 30 min.

[0130] Step 4: Weigh 4 mg of N-hydroxythiosuccinimide sodium salt and dissolve it in 1 ml of pure water;

[0131] Step 5: Add the dissolved reagents from steps 3 and 4 to the lipid nanocarrier and rotate and shake at 28.0 rpm for 12 hours;

[0132] Step 6: Transfer the lipid nanocarrier into an ultrafiltration tube with a molecular weight of 3KD, and centrifuge at 8000xg for 1.5h at 4°C to remove free drug and water;

[0133] Step 7: Measure the absorbance of the lipid nanocarrier at 317 nm using a UV spectrophotometer. Calculate the concentration based on the standard curve.

[0134] The particle size of rosiglitazone@liposomes@GHR-A8 prepared in this embodiment was measured to be 124.03 nm.

[0135] This study included 30 spontaneously obese mice (10 weeks old), divided into three groups of 10 each. Three control groups were established for in vivo experiments: the NC group (blank control, injected with saline), the Rosi group (injected with 200 μM rosiglitazone@liposomes), and the GHR-A8 group (injected with 200 μM rosiglitazone@liposomes@GHR-A8 (liposomes containing GHR-A8 and internally loaded with rosiglitazone). Each mouse received 200 μL of injection every other day for 40 days. The average body weight of the mice in each group was recorded every two days.

[0136] Figure 6 The x-axis represents the number of injections, and the y-axis represents the mouse's weight (in g). The initial average weight of the mice in the three groups was similar, approximately 29 g. As the feeding and injection experiment progressed, the weight of the mice in each group increased. Figure 6 As shown, the average weight of mice in the Rosi group was similar to that in the NC group, indicating that liposomes internally loaded with rosiglitazone did not inhibit obesity. After the 10th injection, mice in the GHR-A8 group had a lower average weight compared to the NC and Rosi groups. Therefore, linking the small molecule peptides provided by this invention to liposomes internally loaded with rosiglitazone enables more efficient delivery of rosiglitazone to adipose tissue, allowing rosiglitazone to exert a better weight-loss effect and thus better inhibit the obesity process.

[0137] Example 6

[0138] 3T3L1 is a mouse preadipocyte cell line that can be induced to differentiate into mature adipocytes. Quantitative comparison of GHR expression levels before and after differentiation using qPCR revealed that differentiated 3T3L1 adipocytes (i.e., after adipogenesis) expressed higher levels of GHR compared to undifferentiated 3T3L1 cells (i.e., before adipogenesis). Figure 7As shown in figure a. This result further proves that GHR is a marker of adipocytes.

[0139] Subsequently, using a similar method to Example 5, a DID fluorescent agent@liposome@GHR-A8 (liposomes internally loaded with DID fluorescent dye and linked to GHR-A8) was constructed. This was added to 3T3L1 differentiated cell cultures. After 3 hours, the culture medium was washed away, and bright-field images were captured under a fluorescence microscope to indicate cell morphology; DID fluorescence channel signal; BODIPY (a lipophilic dye) indicating lipid droplets in adipocytes; and Merge to indicate the overlap of the three images. Figure 7 b shows that the DID signal specificity coincides with the location of the lipid droplets. At the same time, there are also incompletely differentiated / undifferentiated 3T3L1 cells (BODIPY not stained) in the same field of view, indicating that the delivery system of the present invention can specifically deliver the DID fluorescent dye to adipocytes and no non-specific delivery into non-adipocytes has been observed.

[0140] The experimental results above demonstrate that the drug delivery system constructed based on the small molecule peptides of this invention can specifically and precisely deliver drugs (e.g., weight-loss drugs, fluorescent dyes) to adipocytes. Furthermore, because the small molecule peptides of the drug delivery system provided by this invention have high targeting and affinity, and suitable particle size, they can target adipose tissue via blood vessels. Compared to existing reported peptides targeting adipose tissue (e.g., Posi peptides that target adipose tissue by targeting prohibitin), the drug delivery system provided by this invention can, to some extent, enhance the efficacy of drugs (e.g., weight-loss drugs) while mitigating potential side effects and adverse effects on major organs, thereby more specifically inhibiting fat accumulation and the obesity process, and achieving better treatment for obesity-related diseases (e.g., diabetes, obesity, and metabolic syndrome).

[0141] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A polypeptide for targeting the growth hormone receptor, characterized in that, The polypeptide is selected from the group consisting of SEQ ID NOs:1-15.

2. The polypeptide according to claim 1, characterized in that, The polypeptide is used to target the growth hormone receptor in adipocytes.

3. The polypeptide according to claim 1, characterized in that, The polypeptide is also linked to a fluorescent group.

4. The polypeptide according to claim 3, characterized in that, The fluorescent group includes FITC.

5. Use of the polypeptide according to any one of claims 1 to 4 in the preparation of an adipocyte detection reagent.

6. The use as described in claim 5, characterized in that, The detection reagent is used to detect the expression level of growth hormone receptor in the adipocytes.

7. The use as described in claim 6, characterized in that, The detection reagent is used for qualitative and / or quantitative detection of the adipocytes.

8. The use as described in claim 5, characterized in that, The fat cells are derived from visceral fat and / or subcutaneous fat.

9. The use as described in claim 5, characterized in that, The fat cells are derived from mammals.

10. The use as described in claim 5, characterized in that, The fat cells are in a single-cell state.

Images