CN103830739A - Medicine conveying system formed by ligand polypeptide PH1 and application thereof - Google Patents
Medicine conveying system formed by ligand polypeptide PH1 and application thereof Download PDFInfo
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Abstract
The invention relates to a medicine conveying system formed by ligand polypeptide PH1 and an application thereof. The medicine conveying system comprises a ligand polypeptide PH1, a medicine carrying system and at least one active substance, wherein the polypeptide PH1 is connected on the surface of the medicine carrying system. The invention also relates to the application of the ligand polypeptide PH1 in preparing a medicine capable of specifically binding tumor-related macrophage. Discovered and proved by long-time test, the polypeptide PH1 can well target tumor-related cells, induce the differentiation of the tumor-related cells and suppress the tumor growth; the composite of PH1 polypeptide lipidosome can well target the tumor-related macrophage and can be applied to target conveying of the tumor-related macrophage of small-molecular medicines and gene medicines. The medicine conveying system has the advantages that the transretinoic acid polypeptide lipidosome can induce the differentiation of the tumor-related macrophage and suppress the tumor growth effectively, and can be applied to inhibiting proliferation of the tumor cells and the neoplasm recurrence so as to have great medical practicability.
Description
Technical field
The present invention relates to a kind of target polypeptide Liposomal formulation and application thereof, specifically relate to delivery system of a kind of ligand polypeptide PH1 formation and uses thereof.
Background technology
In clinical practice for many years, people observe always usually a large amount of inflammatory cell infiltrations in tumour patient tumor tissue, but its biological significance is only just obtaining the attention of academia in recent years: these Tumor-assaciated inflammatory cells are considered to growth, the survival to tumor and shift have important facilitation.
The source of Tumor-assaciated inflammatory cell, is considered to the lymphoid precursor cell (Myeloid cells) from bone marrow.In normal structure, the precursor in these medullary system sources can be divided into granulocyte, macrophage and dendritic cell etc. very soon, but under the disease conditions such as tumor, the differentiation of these precursors is suppressed, but recruited in tumor tissues in a large number simultaneously, become medullary system and suppress cell.These tumor suppression cells comprise tumor-associated macrophages, express the mononuclear cell of tie2, Tumor-assaciated granulocyte etc.
In the microenvironment of entity tumor enrichment a large amount of inflammatory cells, and tumor-associated macrophages (TAMs) plays important regulating action between cancer and inflammation.TAMs has the effects such as short tumor cell proliferation, angiogenic growth, inhibition adaptive immunity.In the deterioration process of disease, play an important role.With other recruit tumor tissues medullary system source cell as (tie2-macrophages, MDSCs) together, targeting TAMs may become the new way of tumor biotherapy.
Tumor-associated macrophages is by the factor of a series of promotion tumor cell proliferations of expression and secretion and survival, suppressor T cell activity, and promote in the growth of promotion tumor and transfer, to play an important role tumor-blood-vessel growth etc., and be the main cause that causes oncotherapy failure.
Macrophage is effector lymphocyte and antigen presenting cell important in body fluid and cellular immunization, has the gathering of macrophage in tumor tissues.Tumor-associated macrophages participates in multiple stages of the generation development of tumor, the poor prognosis that infiltrates quantity and tumor is closely related, therefore, are study hotspot directions of current tumour immunity to the intervention of various biological behaviours in tumor evolution process, the ability of going back to the nest of tumor tissues is also for development and the clinical practice of some targeting oncolytic viruses taking macrophage as carrier provide possibility.
Macrophage mainly originates from the mononuclear cell of peripheral circulation, it is the Major Members of natural immune system, macrophage under the effect of monocyte chemoattractant protein VEGF, M-CSF etc. to tumor microenvironment displacement, be divided into tumor-associated macrophages, participate in the existence propagation infiltration metastasis of tumor, and relevant to prognosis mala, but be not the effect that all macrophages have short tumor, the macrophage in liver plays antitumor action by the tumor cell of engulfing in peripheral circulation; There is zoopery to show, knock out macrophage and can improve the rate of transform of tumor.
Angiogenesis is the critical event in growth and metastasis of tumours process, basement membrane of blood vessel and the extracellular matrix destruction that is degraded under the stimulating factor effects such as damage, then endothelial cell migration forms new vessels, for tumor cell provide the path of nutrient substance transport and diffusion by way of, in some human tumors, gathering often of tumor-associated macrophages is relevant to the expression of angiogenesis and VEGF, platelet derived growth factor.Also someone thinks, tumor-associated macrophages has the double effect of short angiogenesis inhibitor, but the effect of Angiogensis is preponderated.To breast carcinoma, pulmonary carcinoma, renal carcinoma, the research of basal cell carcinoma etc. is found, infiltrate with tumor tissues medium vessels and generate and be proportionate, hypoxia is the essential condition of revascularization and activation, under anoxia and tumor microenvironment signal stimulus, macrophage raises fiber mother cell growth factor, thymidine phosphorylase, the expression of the angiogenic factors such as plasma urokinase-type plasminogen activator, find by Study on Transgenic Animal, tumor macrophage participates in the transfer of tumor as the switch of angiogenesis, it is the main cell that produces angiogenic factors in tumor stroma, eliminate it and can slow down tumour progression, neutralize its inducer or surface receptor and can stop raising in tumor tissues, suppress angiogenesis, the research of human hela is found, the VEGF of tumor cell secretion participates in the lymphatic vessel regeneration of Tumor-assaciated and shifts with tumor cell lymphatic vessel.
Simple use antitumor drug is as Suo Feilani, after result shows Suo Feilani treatment, tumor easily recurs, and the therapeutic effect of associating macrophage removing medicine zoledronic acid, chlorine disodium hydrogen phosphate liposome can play the therapeutic effect that strengthens tumor, but this whole macrophages of optionally not knowing in body, toxicity is larger, destroy immunologic balance in body, in actual treatment, there is significant limitation.
Jejune myeloid cell can suppressor T cell reaction and angtigen presentation immunoreation.Retinoid medicine comprises the derivant of the natural and synthetic of vitamin A, after vitamin A (retinol) enters human body, is transformed into retinal, then becomes retinoic acid through oxidation.Retinoic acid is to maintain the indispensable material of growth promoter, especially plays an important role promoting epithelial tissue differential growth and maintain aspect its normal function.Research shows can greatly reduce after all-trans-retinoic acid administration the quantity of the immunosuppressant class cell in spleen, but use cannot suppress the growth of tumor separately.
In sum: tumor-associated macrophages (TAMs) is as a subgroup of immunosuppressant cell (MDSCs).By secreting the factor of a series of short tumor growths, at generation, development and the transfer important role of tumor.And the Drug therapy of conventional needle to tumor cell, often prognosis poor effect, easily recurrence, this and tumor-associated macrophages have an important effect.The present invention's trial not directly for the angle of tumor cell, is divided into from induced tumor associated macrophages the new method of normally angle of antineoplastic macrophage being explored oncotherapy from immunomodulating.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, delivery system of a kind of ligand polypeptide PH1 formation and uses thereof is provided.
The present invention is achieved by the following technical solutions,
First aspect, the present invention relates to a kind of ligand polypeptide PH1 in the purposes of preparing in specific binding tumor-associated macrophages medicine, the structure of described ligand polypeptide PH1 is threonine-methionine-Gly-Phe-threonine-Ala-Pro-arginine-phenylalanine-proline-histidine-tyrosine (T-M-G-F-T-A-P-R-F-P-H-Y).
Second aspect, the present invention relates to a kind of delivery system, and described system comprises aforesaid ligand polypeptide PH1, drug-loading system, at least one active substance; Described polypeptide PH1 is connected in described drug-loading system surface.
Preferably, described drug-loading system is following arbitrary structure or its combinative structure: nanoparticle, polymer, liposome, vesicle, solid lipid microgranule, micelle, CNT, lipoprotein.
Preferably, described active substance is antitumor drug, antimetabolite, cytotoxicity/antibiotic, photosensitizer, inhibitors of kinases, anti-inflammatory drug, immunosuppressant, medicine or antiviral drugs for infection.
Preferably, described active substance is the diagnostic reagent for ultrasonic contrast, pneumoradiography, nuclear medicine contrast agent, genes of interest, antisense gene, suicide gene, apoptosis gene, cytokine gene, siRNA, mRNA, or the combination of said gene, or containing the carrier for expression of eukaryon DNA of said gene.
Preferably, described active substance is all-trans-retinoic acid.
The third aspect, the present invention relates to the aforementioned method of stating delivery system, comprises the steps:
Preferably, described matrix material is egg yolk lecithin, cholesterol and PEG-DSPE 2000; Wherein, the mol ratio of egg yolk lecithin, cholesterol and PEG-DSPE 2000, polypeptide lipid is (15-20): (10-12): (0.5-1.1): (0.5-1.1);
Described all-trans-retinoic acid and described matrix material mass ratio are (18-22): (1-1.2);
The Merlon membrane aperture of described filter membrane extruder is followed successively by 400nm, 200nm, 80nm.
Fourth aspect, the present invention relates to the purposes of aforementioned delivery system in the specific target tumor associated macrophages of preparation, the differentiation of induced tumor associated macrophages, inhibition tumor proliferation and recurrence medicine.
Preferably, described tumor-associated macrophages is the associated macrophages of breast carcinoma, colon cancer, ovarian cancer, pulmonary carcinoma, renal carcinoma, gastric cancer, hepatocarcinoma, cervical cancer, carcinoma of endometrium, bladder cancer, carcinoma of prostate cancer of pancreas, colorectal cancer, basal cell carcinoma, melanoma, folliculus lymphatic cancer, small lymphocyte tumor.
Compared with prior art, beneficial effect of the present invention is as follows: found and proved that PH1 polypeptide can well target tumor relevant cell by long term test, induce its differentiation and suppress tumor growth, the complex of PH1 polypeptide liposome can well target tumor associated macrophages, can be applied to the targeted of the tumor-associated macrophages of small-molecule drug and genomic medicine, all-trans-retinoic acid polypeptide liposome in the present invention is the differentiation of induced tumor associated macrophages and the growth of inhibition tumor effectively, can be applied to inhibition tumor cell propagation and tumor recurrence, therefore there is great medical practicality.
Brief description of the drawings
By reading the detailed description of non-limiting example being done with reference to the following drawings, it is more obvious that other features, objects and advantages of the present invention will become:
Fig. 1 is PH1 polypeptide liposome combined coefficient Liquid Detection result.
Fig. 2 is PH1 polypeptide liposome MALDI-TOF-MS(ground substance assistant laser desorption ionization flight time mass spectrum) testing result.
Fig. 3 is the particle diameter result that is loaded with the different prescription of ATRA liposome.
Fig. 4 is tumor-associated macrophages characterization result.
Fig. 5 is the combination experimental result of external PH1 polypeptide liposome and PEG liposome and tumor-associated macrophages.
Fig. 6 is the combination experimental result of the interior PH1 polypeptide liposome of body and tumor-associated macrophages.
Fig. 7 is the differentiated result that external different prescription is loaded with the liposome-induced tumor-associated macrophages of ATRA polypeptide.
Fig. 8 is the differentiated result that in body, different prescriptions are loaded with the liposome-induced tumor-associated macrophages of ATRA polypeptide.
Fig. 9 is the result of variations that is loaded with induced tumor associated macrophages phenotype in ATRA polypeptide liposome and PEG liposome body.
Figure 10 be different proportion polypeptide lipid-modified carry the different cationic-liposomes of siRNA and tumor-associated macrophages (TAMs), normal mononuclear cell (Monocyte) combined with fluorescent intensity schematic diagram.
Figure 11 be the lipid-modified Evacet of different proportion polypeptide to MDSCs cytotoxicity situation schematic diagram.
Figure 12 is the standard curve of all-trans-retinoic acid blank plasma.
Figure 13 is for being loaded with all-trans-retinoic acid PH1 polypeptide liposome and PEG liposome half-life.
Figure 14 is for being loaded with all-trans-retinoic acid polypeptide liposome drug effect experimental result.
Detailed description of the invention
Below in conjunction with specific embodiment, further set forth the present invention.These embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, for example Sambrook equimolecular clone: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
synthesizing of embodiment 1, PH1 polypeptide lipid
Hydrogenated soybean lecithin (HSPC) and 1,2-DSPE 2000-maleate (DSPE-PEG2000-Mal) are purchased from Nof Corp. (NOF Corporation);
PH1 polypeptide is purchased from the biochemical biological company limited of Shanghai gill; Other reagent is purchased from Chinese traditional Chinese medicines chemical reagents corporation;
All-trans-retinoic acid (ATRA) is purchased from company of Sigma of the U.S.;
Synthesizing of step 1, PH1 polypeptide lipid (DSPE-PEG2000-Mal-PH1)
(1) preparation of degasification HEPES solution: ultrapure H2O 50ml ebuillition of heated 10min, fill N2 and be saturated to coolingly, add HEPES salt 595.75mg, obtaining concentration is 50mM, regulates pH to 7.0 with 2mol/L NaOH, whole process N2 protection, 4 DEG C save backup;
(2) take DSPE-PEG2000-MAL powder 10mg, add degasification HEPES salt 10ml hydration adipose membrane to become micellar solution; Be 2:1 according to the mol ratio of polypeptide PH1:DSPE-PEG-MAL, under N2 protection, PH1 added in this micellar solution.Reaction is 10 DEG C in temperature, and rotating speed is constant temperature oscillation under 200rpm condition, after reaction 24h, obtains end product DSPE-PEG2000-Mal-PH1;
(3) the most all solution is collected in bag filter, at 4 DEG C, and 2000ml deionized water, dialysis, changed one time water every 2 hours, repeatedly after 8 times, continue dialysed overnight, to eliminate free polypeptide and salt;
(4) finally collect in 15ml centrifuge bottle, continue to add pure water in bag filter, rinse bag filter wall, final standardize solution is to 12ml, divides to install to lyophilizing in 10 2ml centrifuge bottles and, after 36 hours, save backup;
The combined coefficient high performance liquid chromatography of step 2, PH1 polypeptide lipid detects and synthetic product ground substance assistant laser desorption ionization flight time mass spectrum (MALDI-TOF-MS) detects.Liquid-phase condition (as shown in table 1), mobile phase: A: pure water; B: methanol/oxolane (94:5) wavelength is 210nm; Sample size: 20 μ l; Flow velocity 1ml/min; 35 DEG C of column temperatures.
Table 1
| Time/minute | Water | Methanol/ |
| 0 | 20% | 80% |
| 5 | 0% | 100% |
| 20 | 0% | 100% |
| 23 | 0% | 80% |
Result as depicted in figs. 1 and 2, combined coefficient > 85%.
preparation and the qualification of the complex of embodiment 2, all-trans-retinoic acid polypeptide liposome
Ovum Gallus domesticus Flavus lecithin (EPC) is purchased from NOF Corp, Lot No:108057-3;
Cholesterol and Pegylation phosphatidase 11,2-mono-PEG-DSPE 2000(DSPE-PEG2000) purchased from Avanti Polar Lipids company of the U.S.;
The preparation of step 1, all-trans-retinoic acid, polypeptide liposome complex
Lipid ratio in eight liposome prescriptions (as shown in table 2): Ovum Gallus domesticus Flavus lecithin (EPC): cholesterol (Chol): polypeptide lipid: Pegylation phosphatidase 11,2-mono-PEG-DSPE 2000(DSPE-PEG2000);
Table 2
| Prescription | Constituent mol |
| 2%PEG | ATRA:EPC:CHOL:DSPE-PEG2000=3:15：10：0.52 |
| 4%PEG | ATRA:EPC:CHOL:DSPE-PEG2000=3.3:15：10：1 |
| NON-ATRA | PH1:EPC:CHOL:DSPE-PEG2000=0.52：15：10：0.52 |
| 2%PH1+2%PEG | ATRA:PH1:EPC:CHOL:DSPE-PEG2000=3.4:0.52：15：10：0.52 |
| 2%PH1+4%PEG | ATRA:PH1:EPC:CHOL:DSPE-PEG2000=3.7:0.52：15：10：1.1 |
| 4%PH1+2%PEG | ATRA:PH1:EPC:CHOL:DSPE-PEG2000=3.8:1.1：15：10：0.52 |
| 2%PH1 | ATRA:PH1:EPC:CHOL=3.2:0.52：15：10 |
| 4%PH1 | ATRA:PH1:EPC:CHOL=3.6:10：15：10 |
Lipid and all-trans-retinoic acid mass ratio are 20:1;
(1) dissolve: matrix material and all-trans-retinoic acid
After putting in eggplant-shape bottle and dissolving with chloroform, rotary evaporation is removed chloroform, and the product obtaining is denoted as to lysate.This step produces adipose membrane, and adipose membrane and all-trans-retinoic acid form mixture;
(2) aquation: (be PBS, pH is 7.4, and it is by 137mM NaCl, 2.7mM KCl, 8mM Na to adding appropriate phosphate buffer in eggplant type bottle
2hPO
4, 2mM KH
2pO
4with water composition) 6～7 little beades continue under evacuation condition, not rotate hydration 30 minutes;
It is 400nm that gained liposome vesicle is pressed through to aperture successively, 200nm, and each 3 times of the poly-carbon ester film of 100nm, finally obtains the all-trans-retinoic acid liposome about mean diameter 100nm;
The sign of step 2, all-trans-retinoic acid, polypeptide liposome complex
1, all-trans-retinoic acid polypeptide liposome encapsulation detects;
The complex of the all-trans-retinoic acid of above-mentioned acquisition and liposome is crossed to sephadex G-50(Sephadex G-50) microtrabeculae is (purchased from Beijing Baeyer enlightening biotech company, affiliated company: Pharmacia, Code:17-0042-01), the complex of separated free all-trans-retinoic acid and all-trans-retinoic acid and liposome, mobile phase is PBS (PH7.4), collects eluting peak and is the alltrans after purification;
The complex suspension of retinoic acid and liposome, wherein the concentration of all-trans-retinoic acid is 2mg/ml.The complex suspension of the all-trans-retinoic acid after purification and liposome is destroyed by 9 times of volumes methanol, with the high-performance liquid chromatogram determination envelop rate of UV-detector.Condition determination: ODS post (Diamonsil, 5 μ m, 250 × 4.6mm); Detected temperatures is 25 DEG C; Detection wavelength is 340nm; Flow velocity is 1.0ml/min; Mobile phase is acetonitrile/methanol (volume ratio is 95: 5).The envelop rate (EE) of all-trans-retinoic acid calculates as follows: EE=(Wi/Wtotal) × 100%, wherein, Wi was the quality of all-trans-retinoic acid in the complex being destroyed by methanol after the post of sephadex G-50 (Sephadex G-50); Wtotal is the cross all-trans-retinoic acid quality in the complex that through methanol destroy post before identical with the liposome volume of crossing post; Result shows that the envelop rate of the complex of all-trans-retinoic acid and liposome is 85%;
The particle diameter of 2 all-trans-retinoic acid polypeptide liposomees
The average in triplicate ± standard deviation of particle diameter experiment of the complex of all-trans-retinoic acid and liposome.Result (as shown in Figure 3) shows that the mean diameter of all-trans-retinoic acid polypeptide liposome complex is 100 left and right, and particle size distribution (PDI) is <0.3 all.
Ovum Gallus domesticus Flavus lecithin (EPC) is purchased from NOF Corp, Lot No:108057-3;
Cholesterol and Pegylation phosphatidase 11,2-mono-PEG-DSPE 2000(DSPE-PEG2000) purchased from Avanti Polar Lipids company of the U.S.;
Fluorescent lipid FITC DHPE is purchased from invitrogen company of the U.S.
Polypeptide lipid (DSPE-PEG2000-maleimid-PH1) is synthetic voluntarily;
Anti-mouse FITC-CD11b antibody, Anti-mouse PE-Gr1 antibody, Anti-mouse Percp/cy5.5-F4/80 antibody, Anti-mouse FITC-CD11b antibody and homotype control antibodies thereof are all purchased from ebiosciecce company of the U.S.;
By the trypsinization of 1x106 4T-1 cell, and be resuspended in 200 μ L PBS buffer; At the Balb/c white mice mammary gland in 6 week age, subcutaneous injection of tumor cells suspension; In 2 week, tumor is grown to diameter 10mm, can use;
The preparation of step 2, PH1 polypeptide fluorescent lipid body
The each lipid ratio of PH1 polypeptide liposome is Ovum Gallus domesticus Flavus lecithin (EPC): cholesterol (Chol): FITC fluorescent lipid: polypeptide lipid: Pegylation phosphatidase 11,2-mono-PEG-DSPE 2000(DSPE-PEG2000) mol ratio is 1.5:1:0.31:0.066:0.066;
The each lipid ratio of PEG liposome is Ovum Gallus domesticus Flavus lecithin (EPC): cholesterol (Chol): FITC fluorescent lipid: Pegylation phosphatidase 11,2-mono-PEG-DSPE 2000(DSPE-PEG2000) mol ratio is 1.5:1:0.31:0.066;
(1) dissolve: after matrix material is put in eggplant-shape bottle and to be dissolved with chloroform, rotary evaporation is removed chloroform, and the product obtaining is denoted as to lysate, and this step produces adipose membrane;
(2) aquation: (be PBS, pH is 7.4, and it is by 137mM NaCl, 2.7mM KCl, 8mM Na to adding appropriate phosphate buffer in eggplant type bottle
2hPO
4, 2mM KH
2pO
4with water composition) 6～7 little beades continue under evacuation condition, not rotate hydration 30 minutes.It is 400nm that gained liposome vesicle is pressed through to aperture successively, 200nm, and each 3 times of the poly-carbon ester film of 100nm, finally obtains the polypeptide liposome of mean diameter 100nm left and right and does not contain the PEG liposome of polypeptide;
(1) dislocation of tumor-bearing mice cervical vertebra is put to death, carefully take out tumor, shred tumor tissues on ice.Be transferred to containing in the 15ml centrifuge tube of 1ml collagenase (collagense IV) Digestive system, vortex gently mixes (37 degrees Celsius, 200 revs/min), digests after 2 hours, crosses 40 μ m films to 50ml centrifuge tube.The centrifugal supernatant of abandoning, adds erythrocyte cracked liquid 5ml, 37 cracking 5 minutes, and PBS washes twice, i.e. the tumor individual cells suspension of system;
(2) tumor-associated macrophages phenotype analytical
1. cell counting, adjustment cell concentration is 107/ml; Divide three parallel test groups, every group of 5 sample cells (streaming pipe), numbering 1-1～1-5; 2-1～2-5; 3-1～3-5;
2. in each streaming pipe, add 100 μ l cells (10
6individual);
3. in the 5th sample cell of first, add CD11b by number, GR1, F4/80 three's homotype control antibodies; Second sample cell adds CD11b antibody; The 3rd sample cell adds GR1 antibody; The 4th sample cell adds F4/80; Second sample cell adds CD11b, GR1 and F4/80 antibody;
4.4 degree lucifuges are hatched 30min, PBS washed cell 2 times, and every pipe adds 400 μ l PBS;
5. flow cytometer detects (noting the adjusting of each fluorescence compensation);
In Fig. 4, can find out that tumor-associated macrophages accounts for 50% left and right of tumor stroma, and its phenotype is CD11b
+gR1
+f4/80
+cell;
The external combination test of step 4, PH1 polypeptide fluorescent lipid body and tumor-associated macrophages
Design PH1, PEG, tri-large group of PBS, each large group is divided into again 3 groups, two sample cells of every group, the tumor-associated macrophages that each sample cell adds 106 step 2 to prepare, then each sample cell adds FITC fluorescent lipid body and PEG liposome room temperature prepared by step 1 to hatch 1h, PBS washed twice detects the external joint efficiency of liposome and tumor-associated macrophages with drain cell instrument, in Fig. 5, can find out, relative PBS matched group PEG figure PH1 polypeptide liposome all has with tumor-associated macrophages is combined, but PEG group relatively, PH1 polypeptide group and Tumor-assaciated cell obviously have stronger joint efficiency,
Prepare PH1 fluorescent lipid body 10mg/ml by step 2.Two groups of design PH1, PBS, every group of 3 tumor-bearing mices, two groups of difference tail vein injection PBS, PH1 fluorescent lipid body 200ul; After 4h, put to death mice, prepare tumor single cell suspension by the method for step 3.The analysis of running off: every mice arranges a sample cell, each sample cell adds 106 tumor single cell suspensions of corresponding tumor-bearing mice, first sample cell (PBS organizes mice) adds F4/80 homotype control antibodies, and second sample cell (PH1 organizes mice) adds F4/80 antibody.4 degree are hatched 30min, and PBS washed twice detects the Binding in vivo efficiency of liposome and tumor-associated macrophages with drain cell instrument.In Fig. 6, can find out tail vein injection after 4 hours PH1 fluorescent polypeptide liposome a large amount of be gathered in tumor tissues and be F4/80+ tumor-associated macrophages in conjunction with upper cell, illustrate that PH1 polypeptide liposome can well be targeted to tumor-associated macrophages in vivo.
the differentiation of induced tumor associated macrophages in embodiment 4, the external body of retinoid acid polypeptide liposome
The sorting of step 1, tumor-associated macrophages
1. cell counting 300g, 10min, removes supernatant;
2. in every 107 cells, add 90 μ l buffer;
3. in every 107 cells, add 10 μ l CD11b microbeads;
4. mix, 4 DEG C, 15min;
5. in every 107 cells, add 1～2ml buffer washing, 300g, 10min, removes supernatant;
6. resuspendedly obtain cell suspension to 500 μ l buffer;
7. MS post is put into magnetic field, separately get 500 μ l buffer profit MS posts;
After 8.buffer stream sky, cell suspension is crossed to post, after stream is empty, 3*500 μ l buffer rinses (buffer adds sorting post after first rinsing specimen test tube again), collects and obtains unlabeled cells suspension;
9. take off MS post, be put into (50ml centrifuge tube) in positive cell collecting pipe, add 1ml buffer to push fast, can obtain the CD11b+ tumor-associated macrophages of purity >95%.
The differentiation of step 2, the external evoked tumor-associated macrophages of retinoid acid polypeptide liposome
(1) prepare 8 kinds of different liposomees of writing out a prescription
Press the method for embodiment 1, prepare eight different prescription liposomees and be respectively 2%PH1+2%PEG, 2%PH1+4%PEG, 4%PH1+2%PEG, 2%PH1,4%PH1,2%PEG, NON-ATRA, 4%PEG (2mg/ml);
(2) be divided into 9 large groups, every large component is 3 groups, 5 sample cells of every component, numbering 1-1～1-5; 2-1～2-5; 3-1～3-5; 4-1～4-5; 5-1～5-5; 6-1～6-5; 7-1～7-5; 8-1～8-5; The every pipe of 9-1～9-5 adds the method sorting of the step 1 of 106 embodiment 4 to obtain CD11b+ tumor-associated macrophages.Every group adds respectively PBS, 2%PH1+2%PEG, and 2%PH1+4%PEG,, 4%PH1+2%PEG, 2%PH1,4%PH1,2%PEG, NON-ATRA, 4%PEG20 μ l.Room temperature is hatched 12 hours, adds CD11b by number, GR1, F4/80 three's homotype control antibodies in the 5th sample cell of first; Second sample cell adds CD11b antibody; The 3rd sample cell adds GR1 antibody; The 4th sample cell adds F4/80; Second sample cell adds CD11b, and GR1 and F4/80 antibody 4 are spent lucifuge and hatched 30min.PBS washed cell 2 times, every pipe adds 400 μ l PBS, and flow cytometer detects (noting the adjusting of each fluorescence compensation).From Fig. 7 (* p < 0.05, * p < 0.01, * * p < 0.005) can find out PBS, 2%PEG, NON-ATRA, 4%PEG group does not quantitatively have difference in minimizing tumor-associated macrophages, and NON-ATRA group does not have difference to illustrate that PH1 is not a treatment polypeptide with PBS matched group, the differentiation of induced tumor associated macrophages, reduces Tumor-assaciated cell quantity with regard to all-trans-retinoic acid in action.All the other groups that contain PH1 polypeptide are reducing the significant difference of having compared with PBS group in the quantity of tumor-associated macrophages, illustrate that PH1 polypeptide can well target tumor associated macrophages, and only have all-trans-retinoic acid to be transported to and in tumor-associated macrophages, just can bring into play the significantly effect of differentiation of induction.
The differentiation of induced tumor associated macrophages and the variation of phenotype in step 3, retinoid acid polypeptide liposome body
From the experimental result of above-mentioned steps two, optimize the differentiation test that 5 prescriptions carry out induced tumor associated macrophages in body.These 5 prescriptions are respectively 2%PH1+2%PEG, 2%PH1+4%PEG,, 2%PH1,4%PH1,2%PEG also prepares these 5 kinds of liposomees of concentration 15mg/ml.Balb/c mice is divided into 6 groups at random, 3 every group.Every mouse inoculation 1x1064T1 breast cancer tumour cell.Treat that tumor grows to 100mm
3start, by the administration of every group of prescription, within every two days, to be administered once, every mice 200 μ l.After 4 administrations, the dislocation of tumor-bearing mice cervical vertebra is put to death, carefully take out tumor, shred tumor tissues on ice.Be transferred to containing in the 15ml centrifuge tube of 1ml collagenase (collagense IV) Digestive system, vortex gently mixes (37 degrees Celsius, 200 revs/min), digests after 2 hours, crosses 40 μ m films to 50ml centrifuge tube.The centrifugal supernatant of abandoning, adds erythrocyte cracked liquid 5ml, 37 cracking 5 minutes, and PBS washes twice, i.e. the tumor individual cells suspension of each mice of system.Carry out antibody labeling by the colouring method of the step 4 of example four.Fig. 8 (* p < 0.05, * p < 0.01, * * p < 0.005) show the significant difference of having compared with PBS group containing the group that has a PH1 polypeptide with PBS matched group and 2%PEG group in vivo in the quantity that reduces tumor-associated macrophages, illustrate that PH1 polypeptide can well target tumor associated macrophages, and only have all-trans-retinoic acid to be transported to and in tumor-associated macrophages, just can bring into play the significantly effect of differentiation of induction.Fig. 9 can find out has the group of PH1 tumor-associated macrophages (CD11b+GR1+F4/80+) CD11b phenotype quantity to reduce, GR1 phenotype quantity reduces, F4/80 phenotype quantity is constant, illustrates that all-trans-retinoic acid polypeptide liposome can well be divided into normal macrophage by induced tumor associated macrophages.Thereby reduce on the one hand growth, the transfer of the short tumor of tumor-associated macrophages, have an effect killing and wounding to tumor on the other hand the normal macrophage of increase.The new way of having explored oncotherapy from immunoregulatory angle instead of traditional direct tumor cell aspect of this novelty.
The present embodiment relates to two kinds of siRNA cationic-liposomes, and siRNA cationic-liposome is that tumor-associated macrophages target polypeptide is modified siRNA cationic-liposome, and its preparation method comprises the steps:
1) take out L1 cation lipid, cholesterol (CHOL), dipalmitoyl phosphatidyl choline (DPPC), PH1-polypeptide storing solution, balance half an hour under room temperature;
2) prepare by corresponding mol ratio liposome that PEG content 0,2%, 4% content of peptides are 7% respectively and not containing the contrast liposome of polypeptide.As: according to L1:DPPC:CHOL:PH1-polypeptide :=50:40:10:7(mol ratio, PH1-polypeptide 7%) be mixed in eggplant type bottle, fully dissolve and mix;
3) get HEPES buffer (20mM pH4.0) in 5ml centrifuge tube, 30 DEG C of preheatings, 5～10min.Vortex instrument is adjusted to touch II shelves, under vortex stirs, will adds lipid alcohol mixeding liquid slowly to add in 20Mm pH4.0 HEPES buffer, vortex stirs 1～2min, and preparation is containing the liposome turbid liquor of 30% ethanol;
4) use and extrude instrument, liposome is crossed respectively to 0.2 μ m, 0.1 μ m, 0.08 μ m polycarbonate membrane 11 times, prepare the little and blank liposome that is evenly distributed of particle diameter, 4 DEG C keep in Dark Place for subsequent use;
5) laser light scattering Particle Size Analyzer (PCS) is measured nano particle diameter distribution;
6) instrument using is the ZetaSizer3000H laser particle analyzer of Ma Erwen company of Britain, using He-Ne ion (λ=633nm) is incident illumination, kinetics light scattering test is carried out at 25 DEG C, angle of reflection is 1.33, angle is 90 °, and the meansigma methods that continuous detecting is three times is as the data that obtain;
7) press siRNA/ lipid total amount=1/10, get appropriate cy5-luciferase-siRNA solution.The blank liposome preparing is placed on vortex instrument, slowly adds siRNA solution, hatch 2h for 37 DEG C;
8) complex preparing is placed in HEPES buffer (20mM pH4.0) in, 4h dialyses at 4 DEG C.Then take out, continue in pH7.0PBS solution, 12h dialyses at 4 DEG C.Dialysis finishes to be collected in centrifuge tube, is lipid gene composite, and 4 DEG C save backup;
9) laser light scattering Particle Size Analyzer (PCS) is measured nano particle diameter distribution;
10) instrument using is the ZetaSizer3000H laser particle analyzer of Ma Erwen company of Britain, and using He-Ne ion laser (λ=633nm) is incident illumination, and kinetics light scattering test is carried out at 25 DEG C, and angle of reflection is 1.33, and angle is 90 °.The meansigma methods that continuous detecting is three times is as the data that obtain.
Compliance test result:
(1) sign of tumor-associated macrophages target polypeptide modification siRNA liposome is as shown in table 3 below:
Table 3
(2) the siRNA cationic-liposome of different proportion polypeptide lipid and Cell binding ability are investigated
1) separate tumor-associated macrophages (TAMs), normal mouse mononuclear cell (Monocyte) cell, counting, 1640 adjust cell concentration to 2 × 106/ml.24 orifice plates, every hole 200 μ l cell suspension;
2) under lucifuge condition, add different liposome 50 μ l (the every hole 0.5 μ g of siRNA, final concentration 2 μ g/ml) in various cells to be measured, lucifuge leaves standstill and hatches 4.5 hours at 37 DEG C;
3) hatch after end, discard cell culture fluid, add PBS+2%FBS buffer 400 μ l/ holes, 300g, 10min, centrifuge washing thoroughly cleans for 3 times;
4) utilize stream type cell analyzer, count 10,000 cells under the exciting light passage of FL-4, analysis of cells fluorescence intensity distributes and meansigma methods;
As shown in Figure 10: contrast liposome higher than the MC3 without peptide modified being combined the Cy5-siRNA fluorescence intensity of peptide modified MC3 liposome in experiment with tumor-associated macrophages.And both no significant differences in control cells Monocyte.Illustrate that PH1 liposome can wrap a year siRNA, and produce specific binding or endocytosis with tumor-associated macrophages cell.
embodiment 6, tumor-associated macrophages target polypeptide are modified Evacet
The present embodiment relates to a kind of Evacet, and this liposome is that MDSCs target polypeptide is modified Evacet, and its preparation method comprises the steps:
1) take out EPC, CHOL, the storing solution of PH1, balance is to room temperature;
2) according to EPC: cholesterol=2:1(mol ratio) ratio is mixed in eggplant type bottle, adds appropriate chloroform, and fully dissolve and mix;
3) under room temperature, use Rotary Evaporators rotary evaporation, chloroform is volatilized, form lipid membrane;
4) add (NH
4)
2sO
4-HEPES buffer (ammonium sulfate (NH
4)
2sO
4125mM, HEPES 20mM, pH4.0) to 10mg/ml TL concentration, vibration ultrasonic about 3～5min disperse adipose membrane;
5) water-bath is ultrasonic transparent to liposome solutions, utilizes and extrudes instrument and 100nm aperture polycarbonate membrane, reduces particle diameter;
6) blank liposome with the bag filter of 3500 molecular weight according to 1:150(liposome: dialysis solution) the volume ratio 2h that dialyses in HEPES (20mM, PH7.5) buffer, 4 DEG C of preservations.Thoroughly remove ammonium sulfate and free polypeptide composition in the outer water of liposome, make blank liposome;
7) doxorubicin hydrochloride is dissolved in to HBS(sodium chloride nacl 150mM, HEPES 20mM, pH7.5) in to 5mg/ml;
8) blank liposome is transferred in centrifuge tube, according to the ratio concussion mix homogeneously of lipid: amycin=10:1 (w/w), in isothermal vibration device, 60 DEG C of concussions are hatched 1 hour, obtain Evacet;
9) utilize laser particle analyzer to measure particle diameter and the distribution of liposome, be placed in 4 DEG C for subsequent use;
Compliance test result:
(1) sign of tumor-associated macrophages target polypeptide modification Evacet is as shown in table 4 below:
Table 4
| Prescription | Particle diameter Diameter (nm) | Polydispersity coefficient (PDI) |
| 2%PEG | 132.37±2.57 | 0.17±0.01 |
| 2%PEG+ amycin | 140.00±3.83 | 0.07±0.05 |
| 2%PH1 | 126.53±2.67 | 0.06±0.01 |
| 2%PH1+ amycin | 130.07±2.31 | 0.17±0.02 |
(2) the CCK-8 Study of cytotoxicity of tumor-associated macrophages target polypeptide drug-loaded liposome
Adopt CCK-8 method to detect the cytotoxic effect that cell survival rate represents amycin.Detect according to following steps, each experiment at least repeats 3 times;
1) MDSC cell suspension is inoculated in to 96 well culture plates with the density in 2 × 105/hole, cultivating base unit weight is 100 μ l/ holes, in 37 DEG C, 5%CO2 incubator, cultivates;
2) add the test substance of 10 μ l variable concentrations to culture plate.Each experiment arranges free amycin medicine group, contrast liposomal doxorubicin medicine group (2%PEG), polypeptide liposomal doxorubicin medicine group (2%PH1), matched group (kind has cell, adds the not culture medium containing medicine) and blank group (acellular adds culture medium);
By calculating, it is the relative medicine solution (serum-free medium dilution) of 50 μ g/ml, 25 μ g/ml, 10 μ g/ml, 5 μ g/ml, 1 μ g/ml, 0.1 μ g/ml, 0.01 μ g/ml that each drug component does not add doxorubicin concentration, totally 7 Concentraton gradient, each sample concentration repeats 3 holes, each hole adds rapidly medicine to mix, and continues to cultivate 2～4h;
3) the centrifugal supernatant that goes, after PBS washed cell 1 time, adds containing 10% hyclone fresh culture, continues to cultivate 24h;
4) add 10 μ l CCK-8 solution (note not generating bubble in hole, they can affect the reading of O.D value) to every hole, in incubator, hatch 1～4 hour (concrete incubation time will be observed, and looks at color every a period of time taking-up cell);
5) microplate reader detects each hole absorbance value (OD) value, and detection wavelength is 450nm, records result.Drug dose logarithm mapping is drawn to drug level-cell survival rate curve with cell survival rate.
As seen from Figure 11, the cytotoxic effect of the liposomal doxorubicin that PH1 modifies is all better than non-targeted contrast Evacet, and closer to free amycin.Free amycin is the small-molecule drug that as easy as rolling off a log permeate through cell membranes enters cell, but lack in vivo targeting and be distributed in each organ of whole body, normal cell, tissue are had to very large toxic and side effects, and this can be by causing that with liposome amycin passive target effect reduces toxic and side effects.But in Study of cytotoxicity in vitro, as our cytotoxicity result, show that the cytotoxicity of liposomal doxorubicin is than there being free amycin to reduce, this may be because free amycin enters in cell than the easier permeates cell membranes of Evacet.Therefore, the modification of polypeptide PH1 can strengthen liposomal doxorubicin cytotoxicity in vitro, show can effectively lead drug-loaded liposome special target tumor-associated macrophages promote the endocytosis of cell to it of part PH1, not only improved the selectivity administration effect to cell of liposome but also strengthened the cytotoxicity of liposomal doxorubicin.
embodiment 7, retinoid acid polypeptide liposome Half-life in vivo and pharmacodynamics test
Acitretin is purchased from Shanghai Orient Company;
1) prepare 2%PH1+2%PEG by the step 1 of example two, 4%PEG two lipoid plastids;
2) drafting of standard curve
Measure respectively SD rat plasma 0.1mL, precision is mixed with the retinoic acid solution of a series of different quality concentration, press example two envelop rate chromatographic condition bar method operations, taking acitretin as internal standard substance, peak area ratio As/Ai with retinoic acid and internal standard substance does linear regression to retinoic acid mass concentration in sample, and Figure 12 has shown that and standard curve equation good in 0.1～8 μ g/mL internal linear relation shows;
3) the SD rat of 200g is divided into 2 groups at random and is respectively 2%PH1+2%PEG group, 4%PEG group, every group 3, by every tail vein injection 500 μ l two lipoid plastids of group, two kinds of all-trans-retinoic acid lipid nanoparticle solution of vena femoralis injection and all-trans-retinoic acid contrast liquid respectively, dosage is 10mg/kg.Before administration and after administration 0,15,1,2,4,6,8 and 12h extracting vein blood 0.16mL, the centrifugal 5min of 3000r/min, isolates blood plasma to be measured in-20 DEG C of Refrigerator stores;
4) sample treatment: plasma sample 0.1ml, add mark acitretin solution 10 μ L vortex vibration 5min in 2.5 μ g/mL, each sample adds 100 μ l acetonitriles, vortex vibration 5min, the centrifugal 10min of 10000r/min, get supernatant high effective liquid chromatography for measuring all-trans-retinoic acid drug level, sample size is 20 μ L, the assay method of chromatographic condition example two envelop rates;
5) draw all-trans-retinoic acid liposome half-life curve as shown in figure 13, utilizing kinetics to calculate half-life 2%PH1+2%PEG group is 4.30h, and 4%PEG group is 3.99h;
In sum, find and prove that PH1 polypeptide can well target tumor relevant cell by long term test, induce its differentiation and suppress tumor growth, the complex of PH1 polypeptide liposome can well target tumor associated macrophages, can be applied to the targeted of the tumor-associated macrophages of small-molecule drug and genomic medicine, all-trans-retinoic acid polypeptide liposome in the present invention is the differentiation of induced tumor associated macrophages and the growth of inhibition tumor effectively, can be applied to inhibition tumor cell propagation and tumor recurrence, therefore there is great medical practicality.
Above specific embodiments of the invention are described.It will be appreciated that, the present invention is not limited to above-mentioned specific implementations, and those skilled in the art can make various distortion or amendment within the scope of the claims, and this does not affect flesh and blood of the present invention.
Claims (10)
1. ligand polypeptide PH1 is in a purposes of preparing in specific binding tumor-associated macrophages medicine, and the structure of described ligand polypeptide PH1 is threonine-methionine-Gly-Phe-threonine-Ala-Pro-arginine-phenylalanine-proline-histidine-tyrosine.
2. a delivery system, is characterized in that, described delivery system comprises ligand polypeptide PH1 claimed in claim 1, drug-loading system and at least one active substance; Described polypeptide PH1 is connected in described drug-loading system surface.
3. delivery system as claimed in claim 2, is characterized in that, described drug-loading system is following arbitrary structure or its combinative structure: nanoparticle, polymer, liposome, vesicle, solid lipid microgranule, micelle, CNT, lipoprotein.
4. delivery system as claimed in claim 2, is characterized in that, described active substance is antitumor drug, antimetabolite, cytotoxicity/antibiotic, photosensitizer, inhibitors of kinases, anti-inflammatory drug, immunosuppressant, medicine or antiviral drugs for infection.
5. delivery system as claimed in claim 2, it is characterized in that, described active substance is the diagnostic reagent for ultrasonic contrast, pneumoradiography, nuclear medicine contrast agent, genes of interest, antisense gene, suicide gene, apoptosis gene, cytokine gene, siRNA, mRNA, or the combination of said gene, or containing the carrier for expression of eukaryon DNA of said gene.
6. delivery system as claimed in claim 2, is characterized in that, described active substance is all-trans-retinoic acid.
7. a method of preparing delivery system described in claim 6, is characterized in that, comprises the steps:
Step 1, dissolves PH1 polypeptide lipid, matrix material and all-trans-retinoic acid, obtains the mixture of adipose membrane and all-trans-retinoic acid;
Step 2, obtains the uniform adipose membrane product of one deck by the mixture rotary evaporation of described polypeptide lipid, matrix material and all-trans-retinoic acid;
Step 3, obtains hydrated product by described adipose membrane product hydration;
Step 4, extrudes described hydrated product by polycarbonate leaching film extruder, obtain the complex of all-trans-retinoic acid, polypeptide and liposome.
8. preparation method as claimed in claim 7, is characterized in that, described matrix material is egg yolk lecithin, cholesterol and PEG-DSPE 2000; Wherein, the mol ratio of egg yolk lecithin, cholesterol and PEG-DSPE 2000, polypeptide lipid is (15-20): (10-12): (0.5-1.1): (0.5-1.1);
Described all-trans-retinoic acid and described matrix material mass ratio are (18-22): (1-1.2);
The Merlon membrane aperture of described filter membrane extruder is for being followed successively by 400nm, 200nm, 80nm.
9. a delivery system as claimed in claim 2 breaks up, suppresses the purposes in tumor proliferation and recurrence medicine at the specific target tumor associated macrophages of preparation, induced tumor associated macrophages.
10. purposes as claimed in claim 9, it is characterized in that, described tumor-associated macrophages is the associated macrophages of breast carcinoma, colon cancer, ovarian cancer, pulmonary carcinoma, renal carcinoma, gastric cancer, hepatocarcinoma, cervical cancer, carcinoma of endometrium, bladder cancer, carcinoma of prostate cancer of pancreas, colorectal cancer, basal cell carcinoma, melanoma, folliculus lymphatic cancer, small lymphocyte tumor.
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| CN115501185B (en) * | 2022-09-26 | 2023-09-26 | 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） | Compound for treating osteosarcoma and preparation method thereof |
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Application publication date: 20140604 Assignee: HIGHFIELD BIOPHARMACEUTICAL Corp. Assignor: SHANGHAI JIAO TONG University Contract record no.: X2020980005209 Denomination of invention: Drug delivery system formed by ligand peptide Ph1 and its application Granted publication date: 20180731 License type: Exclusive License Record date: 20200820 |