Peptide molecules for specific recognition of HIV gp120 protein and their applications

Abstract

The application provides a polypeptide molecule for specifically recognizing HIV gp120 protein and an application thereof, and belongs to the technical field of biological medicine. The application provides a lead polypeptide derivative for specifically targeting and combining HIV gp120, wherein the lead polypeptide comprises an amino acid sequence shown in SEQ ID No. 1. The application also provides a lead polypeptide derivative prepared based on the lead polypeptide, and a polypeptide nanoparticle constructed by the lead polypeptide derivative, and both of them have gp120 protein combining capacity. The derivative and the nanoparticle have good virus neutralization inhibition effect. The series of polypeptide molecules can specifically combine with free HIV virus, HIV infected cells and free gp120, so as to block HIV infected cells, delay the AIDS disease process, realize AIDS treatment drug targeted delivery, detection and diagnosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to polypeptide molecules for the specific recognition of HIV gp120 protein and their applications. Background Technology

[0002] Acquired Immunodeficiency Syndrome (AIDS), also known as HIV / AIDS, is caused by the Human Immunodeficiency Virus (HIV) attacking the human immune system, leading to a reduction in immune cells, decreased immune function, and ultimately, infection and cancer, resulting in death. HIV is spherical, with its outer lipid envelope derived from the host cell's phospholipid bilayer. Embedded within this envelope are viral surface glycoprotein gp120 and viral transmembrane glycoprotein gp41, which non-covalently bind to form the viral envelope glycoprotein gp160. The HIV gp120 protein is a key subunit of the HIV surface spike glycoprotein (gp160), playing a crucial role in HIV infection, immune evasion, diagnosis, and vaccine development.

[0003] During HIV infection, gp120 is responsible for recognizing and binding to specific receptors such as the CD4 receptor on the surface of host cells. After binding to CD4, gp120 undergoes a conformational change, further exposing its binding site, and binds to co-receptors (CCR5 or CXCR4), which is a core step for viral entry into cells. After binding to the receptor, gp120 activates another subunit, gp41, which promotes the fusion of the viral envelope with the host cell membrane, allowing viral genetic material to enter the cell. Therefore, gp120 is a key "key" to HIV infection.

[0004] When HIV replicates and assembles into new viral particles within host cells, a large number of unassembled gp120 / gp41 protein complexes are shed or secreted into the extracellular environment, producing free gp120. Simultaneously, infected immune cells (such as CD4 cells)... + When T cells die or lyse, they release viral protein components, including gp120, into the bloodstream, forming free gp120. Free gp120 is not a harmless byproduct; it plays the role of a "toxic molecule" in the progression of HIV disease.

[0005] Free gp120 can act as a "decoy," binding to CD4 receptors on the surface of uninfected immune cells and transmitting false "infected" signals. This leads to persistent and abnormal activation of the immune system, depleting immune cell resources, triggering harmful inflammatory responses, and ultimately causing immune system failure. The binding of gp120 to CD4 receptors can also trigger an intracellular "suicide" signaling pathway, known as the "bystander effect," which is a CD4 signaling pathway... + One of the key mechanisms underlying the reduction in T cell numbers is the presence of a large amount of free gp120 in the blood. This free gp120 can bind to anti-gp120 antibodies produced by the body, forming antigen-antibody complexes. This reduces the number of antibodies that can actually neutralize intact viral particles, weakening the humoral immune response. Free gp120 can cross or affect the blood-brain barrier, acting on nerve cells and immune cells (such as microglia) in the brain, triggering inflammation and oxidative stress, leading to neuronal damage and death. This is a significant cause of HIV-related neurocognitive impairment. Therefore, free gp120 in the blood is far more than just a simple metabolic waste product; it is one of the core weapons HIV uses to achieve immune destruction and escape, and a crucial link between viral infection and immune system collapse.

[0006] Given its crucial role in HIV infection and the progression of AIDS, gp120 has long been considered an important target for AIDS / HIV vaccine design, treatment, diagnosis, and detection. Currently, research on HIV gp120 protein-targeting molecules is one of the most active and promising directions in the field of AIDS. In recent years, this field has rapidly transitioned from basic research to clinical applications, achieving several breakthroughs in broad-spectrum neutralizing antibodies, small molecule / peptide inhibitors, and vaccine design. Broad-spectrum neutralizing antibodies represent the fastest-progressing and most significant area of ​​research. Researchers have isolated bNAbs capable of neutralizing multiple HIV strains from the blood of rare elite controllers and, through genetic engineering optimization, have advanced them to clinical trials. Currently, broad-spectrum neutralizing antibody combinations such as VRC01 / LS, IIIBNC117+10-1074, and CAP256V2LS+PGDM1400 have entered Phase II clinical trials. They directly target different conserved epitopes of gp120, demonstrating strong viral inhibition capabilities. Compared to antibodies, small molecule drugs have advantages in terms of cost, oral administration, and storage, but developing highly effective inhibitors targeting the large and flat protein-protein interaction interface of gp120 is extremely challenging. Summary of the Invention

[0007] This invention provides a polypeptide molecule for specifically recognizing the HIV gp120 protein and its application. The polypeptide molecule can specifically target and bind to HIV gp120, thereby neutralizing HIV, HIV-infected cells, and free gp120 in the blood, and realizing the functions of prevention, detection, diagnosis, and treatment of AIDS.

[0008] The present invention provides a lead polypeptide derivative that specifically targets and binds to HIV gp120, which is obtained by linking the lead polypeptide with other substances; the amino acid sequence of the lead polypeptide includes any of the following: (1) SEQ ID No.1; (2) A mutation of one or more amino acids is performed based on SEQ ID No. 1; The other substances include at least one of the following: (a) a polypeptide; (b) Protein; (c) Nucleic acid; (d) Small molecule compounds; (e) Active molecules such as derivatives of polypeptides, proteins, nucleic acids, or small molecule compounds; (f) Precursor components of polypeptides, proteins, nucleic acids or small molecule compounds.

[0009] In one specific embodiment of the present invention, the connection includes covalent or non-covalent connections.

[0010] In one specific embodiment of the present invention, the lead polypeptide derivative includes any of the following structures: C16-LVFF-KR--linker--AGAEAPYARYRY, C16-LVFF-KRK--linker--AGAEAPYARYRY, and Cy7-C16-LVFF-KR--linker--AGAEAPYARYRY.

[0011] This invention also provides the application of the above-mentioned lead polypeptide derivative in the preparation of delivery vectors.

[0012] The present invention also provides a delivery carrier, comprising the above-mentioned lead polypeptide derivative and raw materials for preparing the delivery carrier; The raw materials for preparing the delivery carrier include at least one of the following: carrier peptides, phospholipids, lipids, PLGA, PLA, polyethylene glycol, and novel polymers.

[0013] The present invention also provides a microparticle comprising the above-mentioned lead polypeptide derivative and other materials; The other materials include at least one of the following: polypeptides, phospholipids, lipids, and polymers.

[0014] In one specific embodiment of the present invention, the polymer includes at least one of the following: PLGA, PLA, polyethylene glycol, polylactic acid, polylactic acid-glycolic acid copolymer, novel nanocarrier preparation polymer, and novel micron carrier preparation polymer.

[0015] The present invention also provides the use of the above-mentioned lead polypeptide derivative, the above-mentioned delivery carrier or the above-mentioned microparticles in the preparation of medicaments or kits for the detection, diagnosis, prevention and / or treatment of AIDS.

[0016] The present invention also provides a medicament for the prevention and / or treatment of AIDS, wherein the active ingredient comprises the above-mentioned lead polypeptide derivative, the above-mentioned delivery carrier or the above-mentioned microparticles.

[0017] The present invention also provides a kit for detecting and / or diagnosing HIV, comprising the above-mentioned lead polypeptide derivative, the above-mentioned delivery carrier or the above-mentioned microparticles; The lead polypeptide derivative, delivery carrier, or microparticle is labeled with a fluorescent molecule.

[0018] Beneficial effects: This invention provides a lead polypeptide that specifically targets and binds to HIV gp120, comprising the amino acid sequence shown in SEQ ID No. 1, wherein the lead polypeptide binds to gp120. 40404 and gp120 40405 The dissociation constants KD values ​​were 0.08±0.018 μM and 0.05±0.007 μM, respectively; and lead peptide derivatives AY20 and LY20 were constructed. AY20 and gp120... 40404 The dissociation constant KD is 0.42 ± 0.02 μM.

[0019] This invention mixes AY20 and LY20 at different molar ratios to construct peptide nanoparticles. The dissociation constant KD of gp12040404 is 0.70~0.90 μM, proving that the derivative and peptide nanoparticles still possess gp120 protein binding ability. In one embodiment, peptide nanoparticles constructed with LY-20:AY-20 = 6:4 were used for verification. The peptide nanoparticles showed IC50 resistance to various HIV pseudoviruses. 50 The concentration is 30~140μg / mL, exhibiting good neutralizing and inhibitory effects against pseudoviruses; the IC50 against various HIV viruses is [not specified]. 50 The concentration is 40~110 μg / mL.

[0020] The series of polypeptide molecules described in this invention can specifically recognize and bind to the gp120 protein. With the help of this specific recognition ability, they can specifically bind to free HIV virus, HIV-infected cells, and free gp120, thereby blocking HIV infection of cells, delaying the progression of AIDS, achieving targeted delivery of AIDS treatment drugs, and realizing the detection and diagnosis of AIDS / HIV. Attached Figure Description

[0021] Figure 1 The molecular formula of AY20; Figure 2 The molecular formula of LY20; Figure 3 This is a mass spectrometry result of the lead peptide derivative AY20; Figure 4 This is a mass spectrometry result of the lead peptide derivative LY20; Figure 5 The binding curves of the lead peptide and gp120 protein are shown. Figure 6 Binding curves of AY20, LY20 and their co-assembled nanoparticles with gp120 protein; Figure 7 This is a transmission electron microscope image of polypeptide nanoparticles. Figure 8 The results of HIV pseudovirus neutralization and inhibition effects of different proportions of polypeptide nanoparticles are shown in the figure. Figure 9 The curves showing the neutralization and inhibition of HIV pseudoviruses by polypeptide nanoparticles (LY-20:AY-20=6:4); Figure 10 The curves showing the neutralization and inhibition of HIV eukaryotic antibodies by polypeptide nanoparticles (LY-20:AY-20=6:4); Figure 11 The graph shows the cytotoxicity results of the polypeptide nanoparticles (LY-20:AY-20=6:4). Figure 12 The image shows the binding results of polypeptide nanoparticles (LY-20:AY-20=6:4) with HIVNL4-3 infected cells. Detailed Implementation

[0022] The present invention provides a lead polypeptide derivative that specifically targets and binds to HIV gp120, which is obtained by linking the lead polypeptide with other substances; the amino acid sequence of the lead polypeptide includes any of the following: (1) SEQ ID No.1; (2) A mutation of one or more amino acids is performed based on SEQ ID No. 1; The other substances include at least one of the following: (a) a polypeptide; (b) Protein; (c) Nucleic acid; (d) Small molecule compounds; (e) Active molecules such as derivatives of polypeptides, proteins, nucleic acids, or small molecule compounds; (f) Precursor components of polypeptides, proteins, nucleic acids or small molecule compounds.

[0023] The amino acid sequence of the lead peptide of the present invention is shown in SEQ ID No. 1 in one embodiment: AGAEPYARYRY. Multiple embodiments have demonstrated that the lead peptide can specifically recognize HIV gp120 and bind to HIV gp120, thereby neutralizing HIV, HIV-infected cells, and free gp120 in the blood, thus realizing the functions of prevention, detection, diagnosis, and treatment of AIDS.

[0024] The lead peptide described in this invention can also be further optimized through mutation based on SEQ ID No. 1, and named the lead peptide mutation-optimized sequence. The mutation optimization described in this invention includes mutating one or more amino acid sequences of the sequence described in SEQ ID No. 1 to obtain superior performance, such as stronger gp120 recognition / binding function and physicochemical properties, including hydrophilicity and stability.

[0025] The present invention can further covalently or non-covalently link the lead polypeptide sequence or the lead polypeptide mutant optimized sequence with other substances such as peptides, proteins, nucleic acids, small molecule compounds and corresponding derivative active molecules or prodrugs to form lead polypeptide derivatives. Other substances may also be polymers and excipients used to prepare delivery carriers, including carrier polypeptides, phospholipids, lipids, PLGA, PLA, polyethylene glycol, and novel polymers.

[0026] In one embodiment of the present invention, a lead peptide derivative AY20 was constructed. The structure of AY20 is shown below: C16-LVFF-KR--linker--AGAEAPYARYRY, wherein the linker can be 6-aminohexanoic acid, and the chemical structural formula of AY20 is as follows: Figure 1 As shown. In one embodiment of the present invention, a lead peptide derivative LY20 was constructed. The structure of LY20 is shown below: C16-LVFF-KRK--linker--AGAEAPYARYRY, wherein the linker can be 6-aminohexanoic acid, and the chemical structural formula of LY20 is shown below. Figure 2 As shown.

[0027] The AY20 of the present invention can be further modified with fluorescein Cy7 to become a fluorescently labeled lead peptide derivative Cy7-AY20, the sequence of which is: Cy7-C16-LVFF-KR--linker--AGAEAPYARYRY.

[0028] This invention also provides the application of the above-mentioned lead polypeptide derivative in the preparation of delivery vectors.

[0029] The present invention also provides a delivery carrier, comprising the above-mentioned lead polypeptide derivative and raw materials for preparing the delivery carrier; The raw materials for preparing the delivery carrier include at least one of the following: carrier peptides, phospholipids, lipids, PLGA, PLA, polyethylene glycol, and novel polymers.

[0030] The delivery carrier described in this invention can be nanoparticles or microparticles, used to deliver small molecules, polypeptides, proteins, nucleic acids, and even vaccines.

[0031] The present invention also provides a microparticle comprising the above-mentioned lead polypeptide derivative and other materials; The other materials include at least one of the following: polypeptides, phospholipids, lipids, and polymers.

[0032] The polymers described in this invention include at least one of the following: PLGA, PLA, polyethylene glycol, polylactic acid, polylactic acid-glycolic acid copolymer, novel nanocarrier preparation polymers, and novel micron carrier preparation polymers.

[0033] The optimized peptides and their derivatives described in this invention can be used alone in molecular form, or assembled into nano or micron particles, either alone or in combination with other materials. They can even be assembled into particles and used in conjunction with reagents / substances for therapeutic, detection, or diagnostic purposes. Other materials described in this invention can be one or more of peptides, phospholipids, lipids, and polymers, wherein the polymers can be PLGA, PLA, polyethylene glycol, polylactic acid, polylactic-co-glycolic acid copolymer, novel nanocarrier-based polymers, or novel micron-based carrier-based polymers.

[0034] The present invention also provides the use of the above-mentioned lead polypeptide derivative, the above-mentioned delivery carrier or the above-mentioned microparticles in the preparation of medicaments or kits for the detection, diagnosis, prevention and / or treatment of AIDS.

[0035] The lead peptides, optimized peptides, and their derivatives described in this invention can be used for the prevention and treatment of HIV or AIDS. They can also be used in combination with photosensitive materials, thermosensitive materials, and magnetic materials, and in conjunction with phototherapy, thermotherapy, or magnetotherapy for the prevention and treatment of HIV or AIDS. The lead peptides, optimized peptides, and their derivatives described in this invention can also be further modified with fluorescently labeled molecules or other labeling molecules in a covalent or non-covalent manner for the diagnosis and detection of gp120, HIV, HIV-infected cells, and HIV reservoir cells.

[0036] The present invention also provides a medicament for the prevention and / or treatment of AIDS, wherein the active ingredient comprises the above-mentioned lead polypeptide derivative, the above-mentioned delivery carrier or the above-mentioned microparticles.

[0037] The present invention also provides a kit for detecting and / or diagnosing HIV, comprising the above-mentioned lead polypeptide derivative, the above-mentioned delivery carrier or the above-mentioned microparticles; The lead polypeptide derivative, delivery carrier, or microparticle is labeled with a fluorescent molecule.

[0038] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of the polypeptide molecules provided by the present invention for specifically recognizing HIV gp120 protein and their applications, should not be construed as limiting the scope of protection of the present invention.

[0039] The nine HIV pseudoviruses (AE34, B14, B16, B121, C11, C15, BC28, BC29, and BC43) and four live viruses (GX2005002, IIIB, NL4-3, and AD8) used in the embodiments of this invention have all been disclosed in previous articles (Jinbang Zhang, Jingwan Han, Hui Li, et al. Lymphocyte Membrane- and 12p1-Dual-Functionalized Nanoparticles for Free HIV-1 Trapping and Precise siRNA Delivery into HIV-1-Infected Cells. Adv. Sci. 2023, 10, 2300282.).

[0040] Example 1: Synthesis and Identification of Lead Peptide Derivatives AY20 and LY20 Synthesized by a biotechnology company Figure 1 The AY20 (C16-LVFF-KR--linker--AGAEAPYARYRY) shown and Figure 2 The LY20 shown is (C16-LVFF-KRK--linker--AGAEAPYARYRY), and AY20 is modified with fluorescein Cy7 to obtain fluorescently labeled lead peptide-derived Cy7-AY20.

[0041] The synthesized AY20 and LY20 were analyzed by mass spectrometry, and the results are as follows: Figure 3 and Figure 4 As shown.

[0042] Example 2: Investigation of the binding affinity of the lead peptide AGAEAPYARYRY to gp120 protein ① Dilute gp120 protein powder (purchased from Sino Biological, 40404-V08H / 40405-V08H) of subtypes 40404 and 40405 with 400 μL of Watson's water to 0.25 mg / mL, dilute with ELISA coating buffer to 2 μg / mL, add 100 μL per well to a high-adsorption ELISA plate, and incubate overnight at 4°C until the gp120 protein is adsorbed on the bottom of the entire ELISA plate.

[0043] ② Wash twice with PBST, add 200 μL of 4% goat serum to each well, and block at 37°C for 2 h.

[0044] ③ After 2 hours, wash twice with PBST, dilute the fluorescent peptide target in half, add 100 μL to each well, and incubate for 2 hours.

[0045] ④ After 2 hours, wash twice with PBST solution and read the plate using an ELISA reader (Rhodamine excitation wavelength: 530nm; emission wavelength: 580nm).

[0046] ⑤ The measured data were fitted nonlinearly to the adsorption equation using Origin to obtain its KD (equilibrium dissociation constant) value.

[0047] The results are as follows Figure 5 As shown, the lead peptide AGAEAPYARYRY and gp120 40404 and gp120 40405 The dissociation constants KD values ​​are 0.08±0.018μM and 0.05±0.007μM, respectively.

[0048] Example 3: Investigation of the binding affinity between lead peptide derivatives AY20 and LY20 and their co-assembled nanoparticles and gp120 protein. ① Dilute the gp120 protein powder of subtype 40404 with 400 μL of Watson's water to 0.25 mg / mL, dilute with ELISA coating buffer to 2 μg / mL, add 100 μL per well to a strong adsorption ELISA plate, and incubate overnight at 4°C until the gp120 protein is adsorbed on the bottom of the entire ELISA plate.

[0049] ② Wash twice with PBST, add 200 μL of 4% goat serum to each well, and block at 37°C for 2 h.

[0050] ③ After 2 hours, wash twice with PBST, dilute the fluorescent peptide in half, add 100 μL to each well, and incubate for 2 hours.

[0051] ④ After 2 hours, wash twice with PBST solution and read the plate using an ELISA reader (FITC excitation wavelength: 490nm; emission wavelength: 525nm).

[0052] ⑤ The measured data were fitted nonlinearly to the adsorption equation using Origin to obtain its KD value.

[0053] The results are as follows Figure 6 As shown, AY20 and gp120 40404 The dissociation constant KD is 0.42 ± 0.02 μM. When the molar ratios of AY20 and LY20 are 8:2, 6:4, 4:6, and 2:8, the mixture reacts with gp120. 40404 The dissociation constants KD were 0.80±0.04μM, 0.88±0.08μM, 0.73±0.08μM, and 0.89±0.17μM, respectively, indicating that the derivatives of the lead peptide AGAEPYARYRY still possess gp120 protein binding ability.

[0054] Example 4 Preparation of lead peptide derivative nanoparticles Weigh out LY-20, AY-20, or a mixture of LY-20 and AY-20 in different proportions, dissolve them in a small amount of DMSO (not exceeding 1% of the total volume), dilute with distilled water to 500 μg / mL, and vortex repeatedly 5 times to mix; then sonicate at 40 Hz for 20 min, and let stand for 1 h to obtain polypeptide nanoparticles.

[0055] Example 5: Preparation of oligonucleotide (ASO)-loaded lead peptide derivative nanoparticles Weigh out LY-20, AY-20, or a mixture of LY-20 and AY-20 in different proportions, dissolve them in a small amount of DMSO (not exceeding 1% of the total volume), dilute with distilled water to 500 μg / mL, and vortex mix; separately take a solution containing anti-HIV ASO (SEQ ID No. 2: 5'-GGAAGCGGAGACAGCGACGA-3'), mix it with the above peptide solution in equal volume, and let it stand for 1 h to obtain ASO-loaded nanoparticles.

[0056] Example 6: Appearance Analysis of Lead Peptide Derivative Nanoparticles The morphology of different nanoparticles was analyzed using transmission electron microscopy (TEM). A 10 μL sample was dropped onto a 300-mesh copper grid and allowed to settle and evaporate completely. Then, 10 μL of a 2% uranium acetate solution was used to stain the sample for approximately 1 minute. Excess stain was then removed, and the sample was dried under fluorescent light. The morphology of the nanoparticles was observed under TEM. Specific results are shown below. Figure 7 As shown, LY-20, AY-20, and different proportions of LY-20 and AY-20 can all form spherical or fibrous nanoparticles.

[0057] Example 7: Neutralizing and inhibiting effect of peptide nanoparticles on pseudoviruses TZM-bl cells (National Key Laboratory of Pathogenic Microbial Biosafety, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences) were plated at 10,000 cells per well and incubated overnight.

[0058] The peptide nanoparticles (LY-20:AY-20 = 6:4) were serially diluted 1.5-fold, resulting in 10 serial dilutions. The diluted peptide nanoparticles were then incubated with 200 TCID of nine pseudoviruses (AE34, B14, B16, B121, C11, C15, BC28, BC29, and BC43) in a medium containing 15 μg / mL DEAE (diethylaminoethyl dextran) for 2 h. PBS incubation with 200 TCID of pseudovirus was used as a virus control, and incubation medium without pseudovirus was used as a cell control.

[0059] Discard the original culture medium from overnight-cultured TZM-bl cells, add a peptide nanoparticle-virus mixture, and bring the volume to 100 μL. Incubate at 37°C for 6 hours. Then discard the culture medium, wash three times with PBS, add 100 μL of culture medium, and incubate for another 48 hours. Next, add 20 μL of Bright-glo lysis buffer to each well, vortex to mix, and incubate for 10 minutes. Measure the cell fluorescence value using a PerkinElmer multi-plate reader.

[0060] Inhibition rate calculation formula: Inhibition rate = ((As-Ab) / (Ac-Ab)) 100%, where As is the luminescence value of the experimental group, Ab is the luminescence value of the cell control, and Ac is the luminescence value of the virus control.

[0061] The results of pseudovirus neutralization and inhibition are as follows Figure 8 As shown, the inhibition curve is as follows Figure 9 As shown, the IC50 values ​​of polypeptide nanoparticles (LY-20:AY-20 = 6:4) against HIV pseudoviruses AE34, B14, B16, B121, BC28, BC29, BC43, C11, and C15 are... 50 The values ​​were 67.03, 62.42, 99.89, 90.28, 135.6, 58.11, 59.02, 38.66, and 60.27 μg / mL, respectively.

[0062] Example 8: Neutralizing effect of peptide nanoparticles on true toxicity TZM-bl cells were seeded at a density of 25,000 cells per well and incubated overnight.

[0063] The peptide nanoparticles (LY-20:AY-20 = 6:4) were serially diluted 1.5-fold, resulting in 10 serial dilutions. The diluted peptide nanoparticles were then incubated with 500 TCID of four different viruses (GX2005002, IIIB, NL4-3, and AD8) in medium containing 15 μg / mL DEAE (diethylaminoethyl dextran) for 2 h. PBS and 500 TCID of virus incubation medium were used as virus controls, and incubation medium without pseudoviruses was used as cell controls. The original culture medium was discarded, and the self-assembled nanodelivery system / virus incubation medium was added, bringing the volume to 100 μL. The cells were incubated at 37°C for 6 h. The culture medium was then discarded, the cells were washed three times with PBS, and 100 μL of medium was added, and the cells were cultured for another 48 h. Bright-glo lysis buffer was then added to each well at 20 μL, vortexed, and incubated for 10 min. Cell fluorescence was measured using a Perkin Elmer multi-plate reader.

[0064] Inhibition rate calculation formula: Inhibition rate = ((As-Ab) / (Ac-Ab)) 100%, where As is the luminescence value of the experimental group, Ab is the luminescence value of the cell control, and Ac is the luminescence value of the virus control.

[0065] HIV true virus neutralization inhibition results are as follows: Figure 10 As shown, the IC50 values ​​of polypeptide nanoparticles (LY-20:AY-20 = 6:4) against HIV evoviruses GX2005002, IIIB, NL4-3, and AD8 are... 50 The values ​​were 74.63, 38.2, 109.2, and 86.57 μg / mL, respectively.

[0066] Example 9 Cytotoxicity test of peptide nanoparticles MT-2 cells, THP-1 cells, TZM-bl cells, and 293T cells in the logarithmic growth phase were seeded at 6000 cells / well in 96-well plates and incubated overnight at 37°C. The peptide nanoparticles were diluted with culture medium (LY-20:AY-20 = 6:4) to final concentrations of 100, 50, 25, 12.5, 6.25, 3.13, 1.56, and 0.78 μM, and added to the cell-containing culture medium. Incubation was continued for 24 h. Subsequently, 10 μL of CCK-8 solution was added. After incubation at 37°C for 1 h, the absorbance at 450 nm was measured using a microplate reader. A blank control group (containing culture medium and CCK-8 reagent but no cells or drug solution) and a control group (containing culture medium and CCK-8 reagent but no drug solution) were also established. Cell viability was used to evaluate the cytotoxicity of the peptide nanoparticles. The survival rate was calculated using the following formula: Cell viability (%) = (Experimental group - Blank group) / (Control group - Blank group) 100%.

[0067] The results are as follows Figure 11 As shown, after administration of peptide nanoparticles at concentrations of 100 μM and below, the activity of the four cell types did not change significantly, indicating that the peptide nanoparticles had no significant cytotoxicity within this concentration range.

[0068] Example 10: Photolabeled peptides for the detection and sorting of HIV-infected cells. The steps for using fluorescently labeled peptides to detect and sort HIV-infected cells are as follows: (1) Cell inoculation and cell infection The MT-2 cell suspension was cultured at 8.0 × 10⁻⁶ cells / mL according to the culture conditions. 5 Cells were seeded in 6-well plates and incubated overnight using a standard cell incubator. The cells were inoculated with HIV at an MOI of 0.02. NL4-3 After infecting MT-2 cells and culturing for another 6 hours, the cells were washed twice with PBS to remove free virus.

[0069] (2) Incubation Different concentrations of FITC-labeled self-assembled peptide nanoparticles were diluted with culture medium and added to cells in the above 6-well plates, and cultured for 2 hours. Then, the cells were washed twice with PBS to remove free self-assembled peptide nanoparticles, and then resuspended in 1 mL of PBS.

[0070] (3) Detection and data processing The fluorescence intensity of FITC was measured by flow cytometry, and the data were processed using FlowTo_v10.8.1 and Graphpad9.

[0071] Depend on Figure 12 The results showed that after administration of peptide nanoparticles at concentrations of 12 μM and above, the interaction with HIV was confirmed. NL4-3 There was a clear binding between the infected cells.

[0072] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A lead peptide derivative that specifically targets and binds to HIV gp120, characterized in that, The precursor peptide is obtained by linking it with other substances; the amino acid sequence of the precursor peptide includes any of the following: (1) SEQ ID No. 1; (2) A mutation of one or more amino acids is performed based on SEQ ID No. 1; The other substances include at least one of the following: (a) a polypeptide; (b) Protein; (c) Nucleic acid; (d) Small molecule compounds; (e) Active molecules such as derivatives of polypeptides, proteins, nucleic acids, or small molecule compounds; (f) Precursor components of polypeptides, proteins, nucleic acids or small molecule compounds.

2. The lead polypeptide derivative according to claim 1, characterized in that, The connections include covalent or non-covalent connections.

3. The lead polypeptide derivative according to claim 1 or 2, characterized in that, The lead peptide derivative includes any of the following structures: C16-LVFF-KR--linker--AGAEAPYARYRY, C16-LVFF-KRK--linker--AGAEAPYARYRY, and Cy7-C16-LVFF-KR--linker--AGAEAPYARYRY.

4. The use of the lead polypeptide derivative according to any one of claims 1 to 3 in the preparation of a delivery carrier.

5. A delivery carrier, characterized in that, Includes the lead polypeptide derivative as described in any one of claims 1 to 3 and the raw materials for preparing the delivery carrier; The raw materials for preparing the delivery carrier include at least one of the following: carrier peptides, phospholipids, lipids, PLGA, PLA, polyethylene glycol, and novel polymers.

6. A particle, characterized in that, Includes the lead polypeptide derivative as described in any one of claims 1 to 3 and other materials; The other materials include at least one of the following: polypeptides, phospholipids, lipids, and polymers.

7. The microparticle according to claim 6, characterized in that, The polymer includes at least one of the following: PLGA, PLA, polyethylene glycol, polylactic acid, polylactic acid-glycolic acid copolymer, novel nanocarrier preparation polymer, and novel micron carrier preparation polymer.

8. The use of the lead polypeptide derivative of any one of claims 1 to 3, the delivery carrier of claim 5, or the microparticle of claim 6 or 7 in the preparation of a medicament or kit for the detection, diagnosis, prevention, and / or treatment of AIDS.

9. A drug for the prevention and / or treatment of AIDS, characterized in that, The active ingredient includes the lead polypeptide derivative of any one of claims 1 to 3, the delivery carrier of claim 5, or the microparticle of claim 6 or 7.

10. A kit for detecting and / or diagnosing HIV, characterized in that, Includes the lead polypeptide derivative of any one of claims 1 to 3, the delivery carrier of claim 5, or the microparticle of claim 6 or 7; The lead polypeptide derivative, delivery carrier, or microparticle is labeled with a fluorescent molecule.

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