CN118290517B - A short peptide derivative and its application, a short peptide derivative hydrogel and its application - Google Patents
A short peptide derivative and its application, a short peptide derivative hydrogel and its application Download PDFInfo
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- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 2
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 2
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- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 description 1
- 208000003950 B-cell lymphoma Diseases 0.000 description 1
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- 102000004127 Cytokines Human genes 0.000 description 1
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- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
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- QRUDEWIWKLJBPS-UHFFFAOYSA-N benzotriazole Chemical compound C1=CC=C2N[N][N]C2=C1 QRUDEWIWKLJBPS-UHFFFAOYSA-N 0.000 description 1
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- -1 hexafluorophosphate Chemical compound 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1002—Tetrapeptides with the first amino acid being neutral
- C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
- C07K5/1008—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Organic Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Epidemiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Inorganic Chemistry (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明提供了一种短肽衍生物及其应用、短肽衍生物水凝胶及其应用，属于生物医药技术领域。本发明提供了一种短肽衍生物，序列为Z‑GFFXaa，其中Z为封端基团，Xaa为氨基酸。本发明提供的短肽衍生物可活化Vγ9Vδ2T细胞，简便地与Vγ9Vδ2T细胞细胞混合后即可有效活化T细胞，使其活化为抗癌、抗病毒等表型，增强其癌细胞杀伤活性，提高其癌细胞杀伤活性，短肽衍生物所形成的水凝胶作为递送载体可以控释细胞药物。
The present invention provides a short peptide derivative and its application, a short peptide derivative hydrogel and its application, and belongs to the field of biomedicine technology. The present invention provides a short peptide derivative, the sequence of which is Z-GFFXaa, wherein Z is a capping group and Xaa is an amino acid. The short peptide derivative provided by the present invention can activate Vγ9Vδ2T cells, and can effectively activate T cells after simply mixing with Vγ9Vδ2T cells, so that they are activated into anti-cancer, anti-viral and other phenotypes, and their cancer cell killing activity is enhanced, and their cancer cell killing activity is improved. The hydrogel formed by the short peptide derivative can be used as a delivery carrier to control the release of cell drugs.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a short peptide derivative and application thereof, and a short peptide derivative hydrogel and application thereof.
Background
Because of the progress of chimeric antigen receptor T cell (CAR-T) therapy in B cell lymphoma treatment, immune cell therapy has become one of the most powerful treatment regimens for malignant patients. Among them, vγ9vδ2t cells are attracting increasing attention as a new discovered promising anticancer candidate. vγ9vδ2T cells are a subset of T lymphocytes, and recognition of foreign antigens is independent of major histocompatibility complexes (not MHC restricted), conferring broader anti-tumor cytotoxicity; their anti-cancer activity is insufficient to meet clinical demands, and in order to increase the anti-cancer activity of vγ9vδ2t cells, antibody-based agonists and genetic engineering strategies have been developed, but these methods are costly in time and money. In addition, while adoptive vγ9vδ2t cell therapy is expected to treat many cancers, its clinical application is limited by how to efficiently target vγ9vδ2t cells for delivery to tumor sites. Therefore, there is an urgent need to develop more effective methods to increase vγ9vδ2t cell anti-tumor activity and to deliver it efficiently to tumor tissue to meet unmet clinical needs.
Disclosure of Invention
In view of the above, the present invention aims to provide a short peptide derivative and application thereof, a short peptide derivative hydrogel and application thereof. The short peptide derivative provided by the invention can activate and locally transport the V gamma 9V delta 2T cells, improves the killing capacity to various tumor cells, and can be used as a delivery carrier to slowly release cell medicines.
In order to achieve the above object, the present invention provides the following technical solutions:
The invention provides a short peptide derivative, which has a sequence of Z-GFFXaa, wherein Z is a blocking group, xaa is amino acid.
Preferably, the sequence is Z-GFFF or Z-GFFY.
Preferably, Z is an aromatic ring-containing end-capping group.
Preferably, Z is Nap.
Preferably, the short peptide derivative is in the D configuration.
Preferably, the sequence is Nap-G DFDFD F or Nap-G DFDFD Y.
The invention also provides application of the short peptide derivative in preparation of the V gamma 9V delta 2T cell activator.
The invention also provides a short peptide derivative hydrogel which is prepared by mixing the short peptide derivative according to the technical scheme with water and gelling.
The invention also provides application of the short peptide derivative hydrogel in preparation of the V gamma 9V delta 2T cell activator.
The invention also provides application of the short peptide derivative hydrogel in preparation of a cell drug delivery carrier.
The invention provides a short peptide derivative, which has a sequence of Z-GFFXaa, wherein Z is a blocking group, xaa is amino acid.
Compared with the prior art, the invention has the following beneficial effects:
The short peptide derivative provided by the invention can activate the V gamma 9V delta 2T cells, can effectively activate the T cells after being simply mixed with the V gamma 9V delta 2T cells, enables the T cells to be activated into anti-cancer, anti-virus and other phenotypes, enhances the killing activity of cancer cells, improves the killing activity of the cancer cells, and can control the release of cell medicines by taking hydrogel formed by the short peptide derivative as a delivery carrier.
The invention increases the variety of the V gamma 9V delta 2T cell activator and the delivery tool, and provides valuable information for developing the hydrogel with the V gamma 9V delta 2T cell activating and releasing functions, which can be applied to human bodies.
Drawings
FIG. 1 shows the percentage of intracellular TNF- α positive cells from different short peptide derivatives treated with V.gamma.9V.delta.2T cells for 6 h;
FIG. 2 shows the killing activity of different short peptide derivatives on cancer cells by treating V gamma 9V delta 2T cells for 6h, and different treatment groups V gamma 9V delta 2T cells;
FIG. 3 is a graph showing the percent release of V.gamma.9V.delta.2T cells from hydrogels based on the short peptide derivative D-Nap-GFFF.
Detailed Description
The invention provides a short peptide derivative, which has a sequence of Z-GFFXaa, wherein Z is a blocking group, xaa is amino acid, and the structural formula is shown as follows:
The term "short peptide derivative" as used in the present invention is a term commonly used in the art and refers to a short chain peptide consisting of 3 to 9 amino acid residues.
The amino acid sequence of the present invention is not particularly limited in its configuration unless otherwise specified.
In the present invention, the sequence is preferably Z-GFFF or Z-GFFY.
In the present invention, the Z is preferably an aromatic ring-containing end-capping group, more preferably Nap.
In the invention, the sequence is preferably Nap-G DFDFDF、Nap-GLFLFL Y or Nap-G DFDFD Y, and the structural formula of Nap-G DFDFD F is shown in formula I:
the structural formula of Nap-G DFDFD Y is shown as formula II:
the structural formula of Nap-G LFLFL Y is shown in formula III:
in the present invention, the short peptide derivative is preferably in the D configuration.
The preparation method of the short peptide derivative is not particularly limited, and the short peptide derivative can be synthesized by adopting a known FMOC-solid phase synthesis method.
The invention also provides application of the short peptide derivative in preparation of the V gamma 9V delta 2T cell activator.
The invention also provides a short peptide derivative hydrogel which is prepared by mixing the short peptide derivative according to the technical scheme with water and gelling.
The invention also provides application of the short peptide derivative hydrogel in preparation of the V gamma 9V delta 2T cell activator.
The invention also provides application of the short peptide derivative hydrogel in preparation of a cell drug delivery carrier.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
In the following examples, the formation of hydrogels was checked by the method of inverting vials as is commonly used in the art.
The sources of the formulations referred to in the examples below are as follows:
medium, RMPI 1640, available from Semerle Feier technology (ThermoFisher Scientific), sterile;
fetal bovine serum, purchased from zemoer feishier technology (ThermoFisher Scientific), sterile;
2-cl-Trt resin was purchased from Tianjin Nankai and technology Co., ltd, activity 1.2mmol/mL;
N, N-diisopropylethylamine (hereinafter referred to as DIEPA) available from Sigma Aldrich, 99% purity;
benzotriazole-N, N' -tetramethylurea hexafluorophosphate (hereinafter HBTU) purchased from gill biochemistry (shanghai) limited with a purity of 98%;
Trifluoroacetic acid (hereinafter referred to as TFA), commercially available from Sigma-Aldrich, having a purity of 99%;
Triisopropylsilane (hereinafter referred to as TIS), purchased from Sigma Aldrich, 99% purity;
l-configuration and D-configuration amino acids were purchased from Jier Biochemical (Shanghai) limited with a purity of 98%;
Aluminum adjuvant was purchased from sameiser technology (ThermoFisher Scientific), 99% pure;
naphthalene acetic acid, pamidronate from Sigma Aldrich, 99% pure;
human recombinant interleukin-2 was purchased from sameimer's technology (ThermoFisher Scientific);
The remaining reagents were all commercially available analytically pure reagents.
Preparation example 1
Preparation of short peptide derivative hydrogel Nap-G DFDFD F (formula I, D-Nap-GFFF) by FMOC-solid phase Synthesis at pH7.0 and room temperature of 20deg.C
The method comprises the following specific steps:
1) Weighing 0.5mmol of 2-cl-Trt resin, adding 10mL of anhydrous dichloromethane (hereinafter referred to as DCM) into a solid phase synthesizer, and shaking on a shaker for 5min to fully swell the 2-cl-Trt resin;
2) The DCM is removed from the solid phase synthesizer filled with 2-cl-Trt resin by pressing with ear washing ball;
3) Dissolving 0.75mmol of Fmoc-protected amino acid in 10mL of anhydrous DCM, adding 0.75mmol of DIEPA, transferring to the solid phase synthesizer, adding 0.75mmol of DIEPA, and reacting for 1h at room temperature;
4) Closing: removing the reaction solution in the solid phase synthesizer by using an ear washing ball, then washing with 10mL of anhydrous DCM for 1min each time, washing for 5 times, adding 20mL of prepared solution with the volume ratio of anhydrous DCM to DIEPA to methanol=17:1:2, and reacting for 10min at room temperature;
5) Removing the reaction liquid in the solid phase synthesizer by using an ear washing ball, washing with anhydrous DCM for 1min at 10mL each time, washing with N, N-dimethylformamide (hereinafter referred to as DMF) for 5 times, washing with 10mL each time and washing with 1min each time, washing with 5 times, adding 10mL of DMF containing 20% by volume of piperidine, reacting for 25min, reacting with 10mL of DMF containing 20% by volume of piperidine for 5min, washing with DMF, washing with 10mL each time and washing with 1min each time, washing with 5 times, and performing the next reaction;
6) Adding 1mmol of the second Fmoc-protected amino acid, 1.5mmol of HBTU, 2mmol of DIEPA and 10mLDMF, adding the prepared solution into the solid phase synthesizer, and reacting for 2h;
7) Repeating the method of steps 5) and 6) to sequentially add the required amino acid or end capping group; then washing with DMF for 5 times and dichloromethane for 5 times, and carrying out the next reaction;
8) 10mL of a solution consisting of 95% TFA,2.5% TIS and 2.5% H 2 O by volume percent is added into the solid phase synthesizer to react for half an hour (or the volume ratio of TFA to DCM is 1:99 to prepare a TFA solution with the volume percent concentration of 1%, 3mL of the TFA solution is taken and added into the solid phase synthesizer each time, the total adding time is ten times, the reaction time is 1 min), the product is cut off from 2-cl-Trt resin, the solvent is removed by vacuum concentration, and crude products are obtained, and then HPLC separation and purification are carried out.
The structural characterization data are as follows:
1H NMR(400MHz,DMSO)δ8.29(d,J＝7.3Hz,1H),8.23(t,J＝5.7Hz,1H),8.17(d,J＝7.9Hz,1H),8.02(d,J＝8.6Hz,1H),7.89-7.79(m,3H),7.75(s,1H),7.49-7.39(m,3H),7.26-7.11(m,12H),7.08(d,J＝7.9Hz,2H),4.60-4.40(m,4H),3.71(dd,J＝16.9,4.9Hz,2H),3.57(dd,J＝16.4,5.6Hz,3H),2.99-2.82(m,5H),2.81-2.75(m,1H),2.69-2.61(m,1H).[M+1]＝684.3
(2) 1mg Nap-G DFDFD F is placed in a 1.5mL glass bottle, 400 mu LPBS solution (pH=7.0) is added, the pH value is regulated to 7.0 by sodium carbonate solution, PBS solution is used for fixing the volume to 500 mu L, the solution is heated to boiling to completely dissolve the compound, and the short peptide hydrogel is obtained after cooling to room temperature.
Preparation example 2
Preparation of short peptide derivative hydrogel Nap-G DFDFD Y (formula II, D-Nap-GFFY) by FMOC-solid phase Synthesis at pH7.0 and room temperature of 20deg.C
1Mg Nap-G DFDFD Y is placed in a 1.5mL glass bottle, 400 mu LPBS solution (pH=7.0) is added, the pH value is regulated to 7.0 by sodium carbonate solution, PBS solution is used for fixing the volume to 500 mu L, the solution is heated to boiling to completely dissolve the compound, and the short peptide hydrogel is obtained after cooling to room temperature.
The structural characterization data are as follows:
1H NMR(400MHz,DMSO-d6)δ9.19(s,1H),8.29-8.07(m,3H),7.98(d,J＝8.3Hz,1H),7.89-7.77(m,3H),7.75(s,1H),7.59-7.35(m,3H),7.32-7.07(m,10H),7.02(d,J＝8.2Hz,2H),6.66(d,J＝8.2Hz,2H),4.59-4.42(m,1H),4.42-4.33(m,1H),3.71(dd,J＝16.9,5.7Hz,1H),3.65-3.30(m,3H),3.05-2.89(m,2H),2.80(ddd,J＝23.7,14.1,9.0Hz,2H),2.71-2.60(m,1H),2.54(s,1H).[M+1]＝701.8.
preparation example 3
Preparation of short peptide derivative hydrogel Nap-G LFLFL Y (formula III, L-Nap-GFFY) by FMOC-solid phase Synthesis at pH7.0 and room temperature of 20deg.C
1Mg Nap-G LFLFL Y is placed in a 1.5mL glass bottle, 400 mu LPBS solution (pH=7.0) is added, the pH value is regulated to 7.0 by sodium carbonate solution, PBS solution is used for fixing the volume to 500 mu L, the solution is heated to boiling to completely dissolve the compound, and the short peptide hydrogel is obtained after cooling to room temperature.
The structural characterization data are as follows:
1H NMR(400MHz,DMSO)δ9.21(s,1H),8.38(d,J＝8.2Hz,1H),8.26(t,J＝5.4Hz,1H),8.10(d,J＝8.4Hz,1H),8.02(d,J＝8.2Hz,1H),7.83(dd,J＝9.2,3.7Hz,3H),7.76(s,1H),7.53-7.39(m,4H),7.16(m,9.3Hz,10H),7.04(d,J＝8.2Hz,5H),6.66(d,J＝8.3Hz,3H),4.63-4.47(m,2H),4.44-4.32(m,1H),3.73(m,5.5Hz,1H),3.62(m,3H),3.56(d,J＝5.5Hz,1H),2.68(m,11.1Hz,5H).[M+1]＝701.8.
Preparation example 4
Preparation of short peptide derivative Nap-G DFD F (D-Nap-GFF) solution at pH7.0 and at 20deg.C, the concentration of polypeptide derivative used cannot be prepared as a hydrogel.
Synthesizing Nap-G DFD F by using an FMOC-solid phase synthesis method, wherein the structural formula is shown in formula IV:
1mg of Nap-G DFD F was placed in a 1.5mL glass bottle, 400. Mu. LPBS solution (pH=7.0) was added, the pH was adjusted to 7.0 with sodium carbonate solution, the volume was fixed to 500. Mu.L with PBS solution, and the mixture was heated to boiling to dissolve the compound completely, and the hydrogel could not be prepared.
The structural characterization data are as follows:
1H NMR(400MHz,DMSO)δ8.36(d,J＝7.5Hz,1H),8.24(t,J＝5.1Hz,1H),8.04(d,J＝8.5Hz,1H),7.89-7.79(m,3H),7.75(s,1H),7.46(dt,J＝21.4,8.3Hz,3H),7.29-7.13(m,10H),4.55(m,1H),4.43(dd,J＝13.9,7.7Hz,1H),3.73(dd,J＝16.5,5.7Hz,1H),3.64-3.53(m,3H),3.09-2.87(m,3H),2.68(dd,J＝13.2,9.7Hz,1H).[M+1]＝538.2.
preparation example 5
Preparation and use of short peptide derivative Nap- DFD F (D-Nap-FF) solution at pH7.0 and room temperature of 20deg.C
Synthesizing Nap- DFD F by using an FMOC-solid phase synthesis method, wherein the structural formula is shown as formula V:
1mg Nap- DFD F is placed in a 1.5mL glass bottle, 400 mu LPBS solution (pH=7.0) is added, the pH value is regulated to 7.0 by sodium carbonate solution, PBS solution is used for fixing the volume to 500 mu L, the solution is heated to boiling to completely dissolve the compound, and the short peptide hydrogel is obtained after cooling to room temperature.
The structural characterization data are as follows:
1H NMR(400MHz,DMSO)δ8.35(d,J＝7.7Hz,1H),8.29(d,J＝8.6Hz,1H),7.85(d,J＝7.3Hz,1H),7.76(dd,J＝13.8,8.1Hz,2H),7.58(s,1H),7.51-7.42(m,2H),7.28-7.11(m,11H),4.62-4.54(m,1H),4.45(dd,J＝14.0,8.0Hz,1H),3.57(d,J＝13.9Hz,1H),3.48(d,J＝13.8Hz,1H),3.11-2.87(m,4H),2.73(m,1H).[M+1]＝481.2.
preparation example 6
Preparation of short peptide derivative hydrogel Nap-G DFDFDFD K (D-Nap-GFFFK) at pH7.0 and at room temperature of 20 ℃
Synthesizing Nap-G DFDFDFD K by using an FMOC-solid phase synthesis method, wherein the structural formula is shown in a formula VI:
The structural characterization data are as follows:
1H NMR(400MHz,DMSO)δ8.28(d,J＝6.3Hz,2H),8.18(d,J＝8.3Hz,1H),8.08(m,2H),7.89-7.72(m,7H),7.51-7.39(m,3H),7.24-7.05(m,13H),6.65(d,J＝8.4Hz,2H),4.49(m,3H),3.72(dd,J＝16.8,5.8Hz,1H),3.65-3.53(m,3H),3.01-2.88(m,3H),2.70(m,6H),1.66-1.49(m,3H),1.41-1.28(m,3H).[M+1]＝829.4.
1mg Nap-G DFDFDFD K is placed in a 1.5mL glass bottle, 200 mu L of pure water solution (pH=7.0) is added, the pH value is regulated to 7.0 by using sodium carbonate solution, the volume is fixed to 250 mu L by using the pure water solution, the solution is heated to boiling to completely dissolve the compound, 250 mu L of 2 XPBS solution is immediately added, and the solution is cooled to room temperature to obtain the short peptide hydrogel.
Human V gamma 9V delta 2T (V gamma 9V delta) 2T) cell culture expansion example
1) After collecting peripheral blood or tunica albuginea of healthy volunteers, they can be stored in a refrigerator at 4℃for a short period of time (within 24 hours) and allowed to return to room temperature before use.
A) Diluting blood: the physiological saline is used for preparing the medicine according to the volume ratio of 1:1, the blood was diluted and gently mixed.
B) And (3) density gradient separation: in a 50mL centrifuge tube according to Ficoll: the mass ratio of diluted blood was 1:1, adding a Ficoll lymph separating liquid in proportion, and lightly adding diluted blood to the upper layer of the Ficoll along an angle of 45 degrees; centrifuging at room temperature for 25min at 600 g; sucking the middle white membrane layer into a new centrifuge tube lightly, and diluting the white membrane with normal saline according to the volume ratio of 1:1; centrifuge at 1500rpm for 10min at room temperature.
C) Lysing erythrocytes: the supernatant was discarded, and 3mL of the erythrocyte lysate was used to lyse erythrocytes for 5min, and after completion of the lysis, 10 volumes of physiological saline was added to terminate the lysis, and the mixture was centrifuged at 1500rpm for 5min at room temperature.
2) In vitro purification culture of vγ9vδ2t cells
A) And (3) paving: PBMCs isolated were resuspended in 1640 medium containing 10wt% serum and plated into 24 well plates; and 50IU/mL human recombinant interleukin 2 (IL-2) and 9. Mu.g/mL pamidronate solution were added to stimulate for 3 days.
B) Liquid is changed in the third day: cells were collected by centrifugation at 1500rpm for 5min at room temperature, the supernatant was discarded, and the prepared complete medium was added and incubated for 3 days with 50IU/mLIL-2 and 9. Mu.g/mL pamidronate solution.
C) Liquid is changed in the sixth day: cells were collected by centrifugation at 1500pm for 5min at room temperature, and the supernatant was discarded and added to the prepared complete medium containing 50IU/mLIL-2 for 3 days.
D) Liquid change on the ninth day: cells were collected by centrifugation at 1500rpm for 5min at room temperature, the supernatant was discarded, and the prepared complete medium was added and cultured for 3 days at 50IU/mLIL-2, and the cells were collected for subsequent experiments.
Vγ9vδ2T cells activation examples
(1) Vγ9vδ2t cell stimulation
The magnetic beads were used to isolate and purify the Vγ9Vδ2T cell count, approximately 1× 6 cells were placed in a 1.5mL EP tube, 1mL 1×PBS buffer was added, and the pellet centrifuge was centrifuged at 3500rpm for 5min.
Meanwhile, a certain amount of RPMI-1640 culture medium containing 10wt% of Fetal Bovine Serum (FBS) is taken, a short peptide derivative (the final concentration is 50 mug/mL) and IL-2 (the final concentration is 50 IU/mL) are added, uniformly mixed, lightly blown and uniformly mixed, and the mixture is packaged into 24-hole cell culture plates and placed in a cell culture box at 37 ℃ for culture for 6 hours.
(2) Measurement of the index of V gamma 9V delta 2T cell activation
1) After 6h the plates were removed and the vγ9vδ2t cell suspension in the plates was placed in a new 1.5mL EP tube, and 1mL of 1×pbs buffer was added and centrifuged at 3500rpm for 5min.
2) Surface molecular staining is performed first, and then intracellular factor staining is performed.
3) After surface-stained cells were subjected to centrifugation at 3500rpm for 5min with a small centrifuge to remove supernatant, intracellular staining was performed using a fixed-well-punch kit, 200. Mu.L of Cytofix/Cytoperm punch solution was added to each well, and light was protected from light at 4℃for 30min.
4) Diluting 10× Washbuffer with double distilled water to 1× Washbuffer, washing twice with 1× Washbuffer (500 μl/tube) after the last step of punching, centrifuging at 3500rpm for 5min, carefully discarding the supernatant with a pipette to wash away formaldehyde in the fixed punching liquid so as to avoid affecting the dyeing, and if the next step of dyeing is not to be carried out, suspending the cell pellet with a proper amount of 1× Washbuffer after washing twice, and standing at 4deg.C in dark for about one week at maximum;
5) The number of samples to be subjected to intracellular staining was determined, the intracellular factor antibody was diluted with an appropriate amount of 1X Washbuffer (100-fold dilution), gently mixed with a gun so as not to cause air bubbles, and 50. Mu.L of the staining solution was dispensed per tube, and the cells were stained at 4℃for 30 minutes in a dark place.
6) The EP tubes were filled with 1X Washbuffer, and 500. Mu.L of each EP tube was washed once and centrifuged at 6500rpm for 5min.
7) The supernatant was carefully aspirated with a gun and washed once with 1 XPBS at 3500rpm and centrifuged for 5min.
8) The supernatant was discarded and the cell pellet was resuspended in 200. Mu.L/tube, 1 XPBS. And transferring the cell suspension to a flow tube, loading, detecting the proportion of target cells and the secreted cytokines by using a flow cytometer, and placing the cell suspension for about one week after the dyeing is finished and the cell suspension is resuspended by using 1 XPBS buffer.
9) The test was performed using different healthy volunteers, fig. 1 shows the results of treatment of vγ9vδ2t cells for 6h with different short peptide derivatives, the percentages of intracellular TNF- α positive cells (a and B in fig. 1)), from fig. 1: wherein the percentage of TNF-a positive V gamma 9V delta 2T cells in the D-configuration Nap-GFFY short peptide treatment group is 3.0 times that in the L-configuration Nap-GFFY short peptide group, the D-configuration short peptide derivative is considered to have better V gamma 9V delta 2T cell activation capability, and the D-configuration Nap-GFFF short peptide derivative has optimal V gamma 9V delta 2T cell activation capability, which are 9.0 times, 8.3 times, 1.1 times and 3.5 times that of the PBS control group, the D-configuration short peptide derivatives Nap-FF, nap-GFF, nap-GFFY and Nap-GFFYK respectively.
Example of killing cancer cells by vγ9vδ2T cells
1) Tumor cells were collected, centrifuged and resuspended in 2mL serum-free medium, 100. Mu.L was left as blank and 100. Mu.L was left as a single-stained PI tube.
2) Cells were washed 3 times with 3mL sterile PBS and serum was removed.
3) 1ML of resuspended tumor cells were removed from the 1.5mL EP tube, added with 0.4. Mu.L of CFSE (5 mM concentration) to a final concentration of 2. Mu.m, and incubated at 37℃for 10min. The remaining CFSE-free cells were returned to the dish.
4) After the staining was completed, 3 volumes of pre-chilled medium containing serum was added and placed on ice for 5min to terminate the staining. Then PBS was washed twice and medium was added for resuspension counting. Tumor cells that remained partially stained with CFSE were left as CFSE single stained tubes.
5) Vγ9vδ2t cells were collected and resuspended (fractions were added to the blanc in step 1).
6) Tumor cells were taken as 1×10 5, 3 control groups without vγ9vδ2t cells plus CFSE stained tumor cells alone were set, and then 10:1 effective target ratio, co-culture was performed in round bottom flow tubes.
7) The final volume of the medium at the time of co-cultivation was 150. Mu.L.
8) Co-culturing for 6h, taking out and washing once, and dyeing PI up-flowing type. CFSE, PI double positive cells are target cells that are killed.
9) The calculation formula is as follows: specific cytotoxic activity (%) = (experimental group target cell death rate-natural target cell death rate)/(100-natural target cell death rate) ×100%.
10 FIG. 2 shows the killing activity of different short peptide derivatives on Vγ9Vδ2T cells for 6h, and different treatment groups of Vγ9Vδ2T cells on cancer cells, as seen from the results in FIG. 2: wherein the V gamma 9V delta 2T cells of the D-configuration Nap-GFFF short peptide treatment group show better cancer cell killing activity.
Vγ9vδ2T cell sustained release example
1) On day 11, centrifugation at 1000rpm for 5min, V.gamma.9V.delta.2T cells were collected;
2) After counting, 5×10 6 cells were resuspended in 1000 μl of hydrogel, respectively;
3) 200 μl of cell suspension was added to a 48-well plate, 3 multiplex wells per group;
4) After the incubator at 37 ℃ is kept stand for 2 hours, 200 mu L of complete culture medium is slowly added along the wall, and a pore plate is placed in the incubator at 37 DEG C
5) At time points 2,4,6, 12, 24, 48h, 100 μl of supernatant was taken per well and cells were counted manually and mechanically. After 100. Mu.L of the medium was removed at each time point, 100. Mu.L of complete medium was then supplemented.
6) FIG. 3 is a graph showing the percent release of V.gamma.9V.delta.2T cells from hydrogels based on the short peptide derivative D-Nap-GFFF, the results of FIG. 3 show: about 20% of the cell medicine is released from the hydrogel within 48 hours, which shows that the hydrogel formed by the Nap-GFFF short peptide derivative based on the D configuration has slow release effect on the cell therapeutic medicine
The foregoing is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. It should be noted that modifications and adaptations to the present invention may occur to one skilled in the art without departing from the principles of the present invention and are intended to be comprehended within the scope of the present invention.
Claims (5)
1. A short peptide derivative, characterized in that the sequence is Nap-G DFDFD F.
2. Use of the short peptide derivative of claim 1 for the preparation of vγ9vδ2T cell activator.
3. A short peptide derivative hydrogel, which is prepared by mixing the short peptide derivative according to claim 1 with water and gelling.
4. Use of the oligopeptide derivative hydrogel of claim 3 in the preparation of vγ9vδ2T cell activator.
5. Use of the oligopeptide derivative hydrogel of claim 3 in the preparation of a cellular drug delivery vehicle.
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