CN106421810A - Tumor-targeting cell drug carrier and application thereof - Google Patents
Tumor-targeting cell drug carrier and application thereof Download PDFInfo
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Abstract
The invention discloses a tumor-targeting cell drug carrier and an application thereof. The drug carrier is prepared from a cytomembrane-embedded drug bonding material and a carrier cell, the cytomembrane-embedded drug bonding material is prepared from hydrophilia polypeptide, an alkyl chain and a bonding drug, and embedded into the cytomembrane of the carrier cell, and drug delivery is achieved through the tropism of the carrier cell for tumor. The tumor-targeting cell drug carrier can simultaneously deliver any two bonding drugs of a photodynamics treatment photosensitizer, a chemotherapy drug and a fluorescent molecular to tumor; enrichment of drugs at the tumor region is achieved, the treatment effect of drugs is improved, and the side effect of drugs is reduced. A preparing method of the cytomembrane-embedded drug bonding material is simple, practicable, high in yield and easy to purify. The cell drug carrier is of great significance in promoting accurate treatment of tumor and achieving personalized treatment.
Description
Technical field
The invention belongs to biomedicine field is and in particular to a kind of cancer target cell drug carrier and its application.
Background technology
Tumor is the major disease threatening human health, realizes the targeted therapy of tumor, reduces medicine to normal group
Knit and be significant to capturing this great difficult problem of tumor disease with the injury of organ.Chemotherapeutics, radiotherapeutic drug, light power
Learn the treatment that medicine has been widely used in tumor, the growth of tumor can be suppressed to a certain extent.But, swollen
The medicine using during tumor treatment does not generally have selectivity, for realizing effective suppression of tumor, needs to increase curative
The consumption of thing.And the strong toxic and side effects in tumor therapeutic procedure not only greatly reduce the effect of oncotherapy, also drop simultaneously
The low quality of life of tumor patient.Therefore, realize the targeting transport of anti-tumor medicine, to reach the effective suppression to tumor
System, the always biomedical study hotspot with technical field of biological material.
Tumor-targeting drug transport generally can be divided into active targeting and passive target.Expressed by tumor cell specific
Albumen or receptor, are referred to as active targeting using the transport that antibody or other small molecule targeting groups realize tumor cell.Profit
With " enhancing penetrates and is detained " effect of tumor region, nano-medicament carrier can be realized becoming passive to the enrichment of tumor region
Targeting.But, the immune system of fine and close tumor environment, tumor region hyperosmosises and human body will greatly limit conventional target
To pharmaceutical carrier, the targeting of tumor is transported and penetrate, so that active targeting and passive target all suffer from great application hardly possible
Topic.
In recent years, by the use of circulating cells as pharmaceutical carrier, by the tropism to tumor environment for the carrier cell, circumvent and exempt from
The removing of epidemic disease system, realizes penetrating of tumor, thus realizing the targeting transport of drug on tumor.Traditional cell drug carrier is
Containing and discharging of medicine is realized by carrier cell to the endocytosis of medicine and diffusion, significantly limit this cell
The application value of pharmaceutical carrier.First, the requirement to cell and medicine for this cell drug carrier is higher, so being typically only capable to use
Mononuclear cell or macrophage, as cell carrier, can only be confined to the transport of photo-thermal therapy and some chemotherapeutics simultaneously；
Secondly, medicine leads to carrier cell not enable long body-internal-circulation the toxicity of carrier cell；Again, due to endocytosis medicine
Thing is in the intracellular environment of complexity, and the organized enzyme in such as lysosome and endosome in carrier cell will lead to medicine carrying
Degraded in somatic cell, is also difficult to escape from carrier cell subcellular organelle for the medicine simultaneously；Finally, the charging property of medicine
The carrying drug ratio of matter, hydrophilic and hydrophobic also strong influence cell carrier.Therefore, design a kind of general cell drug carrier, permissible
Realize the cancer target transport to various types of and multi-medicament simultaneously, to the Synergistic treatment realizing tumor, accurate treatment, reduce poison
Side effect and to realize personalized treatment significant.
Content of the invention
For solving the deficiencies in the prior art, the first object of the present invention is to provide a kind of cancer target cell drug carrier,
Described cell drug carrier is made up of embedding cell membrane medicine key compound and carrier cell, and embedding cell membrane medicine key compound embeds and carries
Somatic cells film, realizes drug delivery by carrier cell to the tropism of tumor.
Further, it is a further object to provide a kind of embedding cell membrane medicine key compound, essence is gathered by hydrophilic
Propylhomoserin, alkyl chain and bonding medicine composition, its chemical structural formula formula is as shown in formula I：
Wherein m=5-8；N=1-6；R is bonding medicine, is that photodynamic therapy photosensitizer, chemotherapeutics or fluorescence divide
Any one of son.
Further, it is a further object to provide a kind of embedding cell membrane medicine key compound, it is Palmic acid-rely
Propylhomoserin (camptothecine)-Arg-Arg-Arg-Arg, Palmic acid-lysine (protoporphyrin)-Arg-Arg-
Any one in Arg-Arg and Palmic acid-lysine (fluorescein)-Arg-Arg-Arg-Arg.
Further, it is a further object to provide a kind of cancer target cell drug carrier, described cell
Pharmaceutical carrier is made up of two kinds of embedding cell membrane medicine key compounds and carrier cell, and two kinds of embedding cell membrane medicine key compounds are simultaneously embedded in
Carrier cell cell membrane, the bonding medicine of two kinds of described embedding cell membrane medicine key compounds is photodynamic therapy photosensitizer, change
Treat any two kinds in medicine or fluorescence molecule.
Further, it is a further object to provide the carrier cell of cancer target cell drug carrier is circulation
Cell.
Further, it is a further object to provide the carrier cell of cancer target cell drug carrier, it is red
Cell, macrophage, mononuclear cell, leukocyte.
Application in the reagent preparing tumor imaging for the cancer target cell drug carrier that the present invention provides.
Application in preparing chemotherapeutics for the cancer target cell drug carrier that the present invention provides.
Application in preparing photodynamic therapy photosensitizer for the cancer target cell drug carrier that the present invention provides.
The cancer target cell drug carrier that the present invention provides can complete the medicine of tumor imaging and chemotherapy in preparation simultaneously
Application in thing.
The present invention provide cancer target cell drug carrier preparation can complete simultaneously photodynamic therapy for cancer and
Application in the medicine of chemotherapy.
The present invention provide cancer target cell drug carrier preparation can complete simultaneously photodynamic therapy for cancer and
Application in the medicine of tumor imaging.
The principle of the present invention is：By having the alkyl chain of embeddeding action to cell membrane and having adsorbing band to cell membrane
Positive electricity polypeptide chain combines to form cell membrane block polymer, by small-molecule drug by be chemically bonded to block polymer side base formed embedding thin
After birth medicine key compound.Embedding cell membrane medicine key compound is embedded in the cell membrane of circulating cells, realizes the delivery of medicine.Embedding
Cell membrane medicine key compound will not affect that the tropism to tumor for the carrier cell after being embedded into carrier cell cell membrane.Meanwhile,
Using the tropism to tumor for the circulating cells, realize the targeting transport of drug on tumor.
One embodiment of the present of invention confirms that in cancer target cell drug carrier, embedding cell membrane medicine key compound is permissible
The embedded and transport on carrier cell as chemotherapeutics, and there is certain stability within a certain period of time.
One embodiment of the present of invention confirms that in cancer target cell drug carrier, embedding cell membrane medicine key compound is permissible
The embedded and transport on carrier cell as photodynamic therapy photosensitizer, and there is certain stability within a certain period of time.
One embodiment of the present of invention confirms that in cancer target cell drug carrier, embedding cell membrane medicine key compound is permissible
The embedded and transport on carrier cell as fluorescence imaging molecule, and there is certain stability within a certain period of time.
One embodiment of the present of invention confirms that in cancer target cell drug carrier, embedding cell membrane medicine key compound is permissible
It is embedded on the cell membrane of macrophage, stem cell and erythrocyte.
One embodiment of the present of invention confirms that in cancer target cell drug carrier, embedding cell membrane medicine key compound is permissible
It is simultaneously embedded on carrier cell as fluorescence imaging molecule and photodynamic therapy photosensitizer and transports, thus realizing tumor
Imaging and treatment.
One embodiment of the present of invention confirms that in cancer target cell drug carrier, embedding cell membrane medicine key compound is permissible
Embedded and transport, thus realizing the association of tumor while as chemotherapeutics and photodynamic therapy photosensitizer on carrier cell
With treatment.
One embodiment of the present of invention confirms embedding cell membrane medicine key compound conduct in cancer target cell drug carrier
Embedded and transport on carrier cell for the photodynamic therapy photosensitizer, does not have toxicity to carrier cell, with respect to endocytosis form
Transport have more biocompatibility.
One embodiment of the present of invention confirms embedding cell membrane medicine key compound conduct in cancer target cell drug carrier
Embedded and transport on carrier cell for the photodynamic therapy photosensitizer, the drug loading of photosensitizer is bonded with embedding cell membrane photosensitizer
The co-cultivation time of thing and carrier cell increases and increases.
One embodiment of the present of invention confirms embedding cell membrane medicine key compound conduct in cancer target cell drug carrier
Embedded and transport on carrier cell for the photodynamic therapy photosensitizer, the drug loading of photosensitizer is with carrier cell and co-cultivation
The concentration of embedding cell membrane photosensitizer key compound increases and increases.
One embodiment of the present of invention confirms embedding cell membrane medicine key compound conduct in cancer target cell drug carrier
Embedded and transport on carrier cell for the photodynamic therapy photosensitizer, right after the embedded medicine key compound in carrier cell film surface,
Carrier cell is unaffected to the tropism of tumor cell.
One embodiment of the present of invention confirms embedding cell membrane medicine key compound conduct in cancer target cell drug carrier
Embedded and transport on carrier cell for the photodynamic therapy photosensitizer, embedding theca cell carrier has targeting to tumor.
One embodiment of the present of invention confirms embedding cell membrane medicine key compound conduct in cancer target cell drug carrier
Embedded and transport on carrier cell for the photodynamic therapy photosensitizer, embedding theca cell vehicle treatment has suppression to tumour growth
Effect.
One embodiment of the present of invention confirms embedding cell membrane medicine key compound conduct in cancer target cell drug carrier
Embedded on carrier cell of photodynamic therapy photosensitizer and transporting, embedding theca cell carrier in tumor therapeutic procedure, mice
Body weight is constant, has less toxic and side effects.
One embodiment of the present of invention confirms embedding cell membrane medicine key compound conduct in cancer target cell drug carrier
Embedded and transport on carrier cell for the photodynamic therapy photosensitizer, embedding theca cell vehicle treatment has to tumor volume growth
Inhibition.
One embodiment of the present of invention confirms embedding cell membrane medicine key compound conduct in cancer target cell drug carrier
Embedded and transport on carrier cell for the photodynamic therapy photosensitizer, embedding theca cell vehicle treatment has to tumor weight growth
Inhibition.
It is an advantage of the invention that：1) cell toxicant for carrier cell for the medicine can be reduced using cell membrane medicine block polymer
Property.2) this cell membrane block polymer has versatility, can serve as chemotherapeutics, the delivery of photosensitizer and fluorescence molecule.3) embedding
Theca cell medicine key compound does not affect on the tumor tropism of carrier cell.4) embedding theca cell pharmaceutical carrier can realize tumor
Targeting transport.5) the cell membrane block polymer preparation in this cancer target cell drug carrier is simple, and easily, carrier is thin for purification
The acquisition of born of the same parents is relatively easy to.6) this embedding theca cell carrier can be realized tumor being diagnosed simultaneously and being treated or Synergistic treatment
Deng.
Brief description
Fig. 1：Embedding cell membrane camptothecine key compound is to the embedding film of macrophage and its stability diagram to the time.
Fig. 2：Embedding cell membrane protoporphyrin key compound is to the embedding film of macrophage and its stability diagram to the time.
Fig. 3：Embedding cell membrane key compound is respectively embedded into macrophage, stem cell and erythrocyte and embedding cell membrane fluorescein key
Compound embeds the stability diagram to the time for the macrophage.
A embedding cell membrane camptothecine key compound, embedding cell membrane fluorescein key compound, embedding cell membrane protoporphyrin key compound is embedding respectively
Enter macrophage figure；
B embedding cell membrane camptothecine key compound, embedding cell membrane fluorescein key compound, embedding cell membrane protoporphyrin key compound is embedding respectively
Enter stem cell figure；
C embedding cell membrane camptothecine key compound, embedding cell membrane fluorescein key compound, embedding cell membrane protoporphyrin key compound is embedding respectively
Enter erythrogram；
D embedding cell membrane fluorescein key compound embeds the stability diagram to the time for the macrophage.
Fig. 4. the embedding cell membrane fluorescein key compound and embedding cell membrane protoporphyrin key compound embedding film figure to macrophage.
Fig. 5：The embedding cell membrane camptothecine key compound and embedding cell membrane protoporphyrin key compound embedding film figure to macrophage.
Fig. 6：The embedding cell membrane protoporphyrin key compound and protoporphyrin cytotoxicity figure to macrophage.
Fig. 7：Embedding cell membrane protoporphyrin key compound to macrophage cell membrane embedding film ability with incubation time variation diagram.
Fig. 8：Embedding cell membrane protoporphyrin key compound is bonded with embedding cell membrane protoporphyrin to macrophage cell membrane embedding film ability
The variation diagram of thing Concentraton gradient.
Fig. 9：Embedding cell membrane protoporphyrin key compound macrophage carrier tropism aptitude tests figure.
Figure 10：Embedding cell membrane protoporphyrin key compound macrophage carrier can be tried hard to the targeting of tumor.
Figure 11：Gross tumor volume under the conditions of various is schemed over time.
Figure 12：Mouse Weight under the conditions of various changes over figure.
Figure 13：The pictorial diagram of tumor is peeled off after mice is cultivated 12 days under the conditions of various.
Figure 14：Tumor weight (grams) figure after mice is cultivated 12 days under the conditions of various.
Specific embodiment
By combination accompanying drawing described further below it will be further appreciated that the features and advantages of the invention.The enforcement being provided
Example is only the explanation to the inventive method, and limits remaining content of present invention announcement never in any form.
【Embodiment 1】The synthesis of embedding cell membrane key compound
Embedding cell membrane key compound (Palmic acid-lysine (camptothecine)-Arg-Arg-Arg-Arg), (palm fibre
Palmitic acid acid-lysine (protoporphyrin)-Arg-Arg-Arg-Arg) and (Palmic acid-lysine (fluorescein)-essence ammonia
Acid-Arg-Arg-arginine) synthesis：
(1) add 0.5g chlorine resin toward in the reactor of the N,N-dimethylformamide steamed equipped with 10mL weight
(1.01mmol/g), extract DMF after chlorine resin after swelling 2 hours in DMF.
(2) FMOC is protected amino and Pbf to protect the arginine (4 times of equivalents in resin activity site) of guanidine radicals, N, N- bis-
Wopropyl ethyl amine (8 times of equivalents of aminoacid) is dissolved in 10mL DMF, is then added to room temperature reaction in reactor
Cysteine was bonded on resin in 2 hours, extracts solvent, DMF washs 2～4 times.
(3) 20% (V/V) piperidines/N,N-dimethylformamide (i.e. piperidines and N, N- dimethyl methyl are added toward in reactor
Amide volume ratio is 2:8) solution 10mL, room temperature reaction, after 15 minutes, extracts solvent；Repeat to add piperidines/N, N- dimethyl methyl
Amide solution is reacted to cut FMOC protection group, after reaction terminates, extracts solvent, washs tree with DMF
Fat 2～4 times.
(4) by Arg (4 times of equivalents in resin activity site), BTA-N, N, N', N'- tetramethylurea hexafluoro
Phosphate (4.8 times of equivalents in resin activity site), 1- hydroxy benzo triazole (4.8 times of equivalents in resin activity site), N,
N- diisopropylethylamine (8 times of equivalents in resin activity site) is dissolved in DMF, adds room temperature in reactor
Lysine bonding is got on by reaction for 2 hours, extracts solvent, with DMF washing resin 2～4 times.
(5) by remaining arginine, arginine is bonded according to step (3) (4) one by one successively.
(6) remove FMOC protection group according to step (3), FMOC is protected amino and Dde to protect the aminoacid of pendant amino group
(4 times of equivalents in resin activity site), BTA-N, N, N', N'- tetramethylurea hexafluorophosphate (resin activity site
4.8 times of equivalents), 1- hydroxy benzo triazole (4.8 times of equivalents in resin activity site), N, N- diisopropylethylamine (resin
8 times of equivalents of avtive spot) it is dissolved in DMF, add in reactor room temperature reaction 2 hours by lysine key
Close, extract solvent, with DMF washing resin 2～4 times.
(7) remove FMOC protection group according to step (3), by Palmic acid (4 times of equivalents in resin activity site), benzo three nitrogen
Azoles-N, N, N', N'- tetramethylurea hexafluorophosphate (4.8 times of equivalents in resin activity site), 1- hydroxy benzo triazole (tree
4.8 times of equivalents of fat avtive spot), N, N- diisopropylethylamine (8 times of equivalents in resin activity site) is dissolved in N, N- dimethyl
In Methanamide, add room temperature reaction in reactor lysine bonding to get in 2 hours, extract solvent, with N, N- dimethyl formyl
Amine washing resin 2～4 times.
(8) remove Dde protection group, add 2% (V/V) hydrazine hydrate/DMF (i.e. piperidines toward in reactor
It is 2 with N,N-dimethylformamide volume ratio:98) solution 10mL, room temperature reaction, after 7 minutes, extracts solvent；Repeat to add hydration
Hydrazine/DMF, to cut Dde protection group, after reaction terminates, extracts solvent, is washed with DMF
Resin 2～4 times.
(9) by protoporphyrin or carboxyl camptothecin or 5 (6)-CF 5(6)-Carboxyfluorescein, (4 times of resin activity site are worked as
Amount), BTA-N, N, N', N'- tetramethylurea hexafluorophosphate (4.8 times of equivalents in resin activity site), 1- hydroxy benzeness
And triazole (4.8 times of equivalents in resin activity site), N, N- diisopropylethylamine (8 times of equivalents in resin activity site) is dissolved in
In DMF, it is separately added into 4 hours photosensitizer of room temperature reaction or chemotherapeutics or fluorescence in reactor and divides
Sub-key is closed, and extracts solvent, with DMF washing resin 2～4 times.
(10) DMF washing resin 2～4 times, methanol washs 2～4 times, and dichloromethane washs 2～4 times.
(11) add toward in reactor the solution being grouped into by the group of following volumn concentration act at room temperature 2h with
Cut many peptide bonds compound and the side base on ammonia resin：95% trifluoroacetic acid, 5% water.
(12) collect and cut liquid, revolving, vacuum drying obtains product, keeps in Dark Place in -20 DEG C.
In example 2 below -12 all taking the embedding cell membrane key compound of synthesis in embodiment 1 as a example.
【Embodiment 2】Embedding cell membrane camptothecine key compound is to the embedding film of macrophage and its stability to the time
By macrophage with 1 × 105The density inoculation of individual cells/well, cultivates under the conditions of 37 DEG C in 1mL culture medium.24
After hour, embedding cell membrane camptothecine key compound is dissolved in the medium, add 1mL to contain embedding cell membrane happiness in macrophage
The culture medium of tree alkali key compound (30 micromoles per liter).After culture 2 hours, by the culture containing embedding cell membrane camptothecine key compound
Base suctions out, and has PBS buffer solution that three times cell washing is added new culture medium afterwards, uses confocal laser scanning microscope cell
The fluorescence intensity of interior camptothecine.
Result is as shown in figure 1, embedding cell membrane camptothecine key compound can effectively embed in macrophage cell membrane, same
When, incubation time increases, and the fluorescence intensity of surface of cell membrane is only weaker to be reduced it was demonstrated that embedding cell membrane camptothecine key compound exists
Stability on cell membrane.
【Embodiment 3】Embedding cell membrane protoporphyrin key compound is to the embedding film of macrophage and its stability to the time
By macrophage with 1 × 105The density inoculation of individual cells/well, cultivates under the conditions of 37 DEG C in 1mL culture medium.24
After hour, embedding cell membrane protoporphyrin key compound is dissolved in the medium, adds 1mL to contain embedding cell membrane in macrophage former
The culture medium of porphyrin key compound (30 micromoles per liter).After culture 2 hours, by the culture containing embedding cell membrane protoporphyrin key compound
Base suctions out, and has PBS buffer solution that three times cell washing is added new culture medium afterwards, uses confocal laser scanning microscope cell
The fluorescence intensity of interior protoporphyrin.
Result is as shown in Fig. 2 embedding cell membrane protoporphyrin key compound can effectively embed in macrophage cell membrane, same
When, incubation time increases, and the fluorescence intensity of surface of cell membrane is only weaker to be reduced it was demonstrated that embedding cell membrane protoporphyrin key compound exists
Stability on cell membrane.
【Embodiment 4】Embedding cell membrane key compound is respectively embedded into macrophage, and stem cell and erythrocyte and embedding cell membrane are glimmering
Light element key compound embeds the stability to the time for the macrophage
By erythrocyte, macrophage, stem cell is respectively with 1 × 105Individual cells/well density inoculation, under the conditions of 37 DEG C
Cultivate in 1mL culture medium.After 24 hours, by embedding cell membrane fluorescein key compound, embedding cell membrane protoporphyrin key compound, embedding cell membrane
Camptothecine key compound dissolves in the medium respectively, respectively to erythrocyte, macrophage, and add 1mL to contain in stem cell embedding thin
After birth fluorescein key compound (30 micromoles per liter), embedding cell membrane protoporphyrin key compound (30 micromoles per liter), embedding cell membrane Fructus seu radix camptothecae acuminatae (Fructus Camptothecae Acuminatae)
The culture medium of alkali key compound (30 micromoles per liter).After culture 2 hours, the culture medium containing embedding cell membrane key compound is suctioned out, has
Cell washing is added new culture medium three times by PBS buffer solution afterwards, with the intracellular fluorescence of confocal laser scanning microscope
The fluorescence intensity of element.
Result is as shown in figure 3, embedding cell membrane key compound can effectively embed erythrocyte, stem cell and Macrophage Cell
It was demonstrated that the versatility of embedding cell membrane key compound in film.Meanwhile, incubation time increases, and the fluorescence intensity of surface of cell membrane only has
Weaker reduce it was demonstrated that stability on cell membrane for the embedding cell membrane fluorescein key compound.
【Embodiment 5】Embedding cell membrane fluorescein key compound and embedding cell membrane protoporphyrin key compound are simultaneously embedding to macrophage
Film and its stability to the time
By macrophage with 1 × 105The density inoculation of individual cells/well, cultivates under the conditions of 37 DEG C in 1mL culture medium.24
After hour, embedding cell membrane fluorescein key compound and embedding cell membrane protoporphyrin key compound are dissolved in the medium, to macrophage
Middle addition 1mL contains embedding cell membrane fluorescein key compound and embedding cell membrane protoporphyrin key compound (respectively 30 micromoles per liter)
Culture medium.After culture 2 hours, the culture medium containing embedding cell membrane fluorescein key compound and embedding cell membrane protoporphyrin key compound is inhaled
Go out, have PBS buffer solution that cell washing is added for three times new culture medium afterwards, intracellular with confocal laser scanning microscope
Fluorescein and the fluorescence intensity of protoporphyrin.
Result is as shown in figure 4, embedding cell membrane fluorescein key compound and embedding cell membrane protoporphyrin key compound can be simultaneously effective
Embedded macrophage cell membrane in.Demonstrate cancer target cell drug carrier of the present invention can tumor be imaged simultaneously
And treatment.
【Embodiment 6】Embedding cell membrane camptothecine key compound and embedding cell membrane protoporphyrin key compound to the embedding film of macrophage and
Its stability to the time
By macrophage with 1 × 105The density inoculation of individual cells/well, cultivates under the conditions of 37 DEG C in 1mL culture medium.24
After hour, embedding cell membrane camptothecine key compound and embedding cell membrane protoporphyrin key compound are dissolved in the medium, to macrophage
Middle addition 1mL contains embedding cell membrane camptothecine key compound and embedding cell membrane protoporphyrin key compound (respectively 30 micromoles per liter)
Culture medium.After culture 2 hours, the culture medium containing embedding cell membrane camptothecine key compound and embedding cell membrane protoporphyrin key compound is inhaled
Go out, have PBS buffer solution that cell washing is added for three times new culture medium afterwards, intracellular with confocal laser scanning microscope
Camptothecine and the fluorescence intensity of protoporphyrin.
Result is as shown in figure 5, embedding cell membrane fluorescein key compound and embedding cell membrane protoporphyrin key compound can be simultaneously effective
Embedded macrophage cell membrane in.Demonstrate cancer target cell drug carrier of the present invention can complete to carry out chemotherapy to tumor
Synergistic treatment with photodynamic therapy.
【Embodiment 7】The embedding cell membrane protoporphyrin key compound and protoporphyrin cytotoxicity to macrophage
Macrophage was seeded in 96 orifice plates with the density of 6000 cells/well, with 100 μ L culture medium culturing 24 hours.
Then, the embedding cell membrane protoporphyrin key compound solution prepared with culture medium and protoporphyrin solution 100 μ L are added separately to each hole
In.All cells are cultivated 48 hours for 37 degrees Celsius under the conditions of lucifuge.In each hole, subsequently add the MTT of 20 μ L 5mg/mL
(MTT is dissolved in PBS).After co-culturing 4h, suction out culture medium, add 150 μ L dimethyl sulfoxide (DMSO).Microplate reader is surveyed
Measure the light absorption value of 570 nanometers in each hole, calculate cell survival rate, so obtain embedding cell membrane protoporphyrin key compound solution and
The toxicity to macrophage for the protoporphyrin.
Result, as shown in fig. 6, embedding cell membrane protoporphyrin key compound solution has less toxicity, has preferably compared with protoporphyrin
Biocompatibility.
【Embodiment 8】The relation to macrophage cell membrane embedding film ability and incubation time for the embedding cell membrane protoporphyrin key compound
By macrophage with 1 × 105The density inoculation of individual cells/well, cultivates under the conditions of 37 DEG C in 1mL culture medium.24
After hour, embedding cell membrane protoporphyrin key compound is dissolved in the medium, adds 1mL to contain embedding cell membrane in macrophage former
The culture medium of porphyrin key compound (30 micromoles per liter).Culture 0 hour respectively, 0.5 hour, 1 hour, after 2 hours and 6 hours, will
Culture medium containing embedding cell membrane protoporphyrin key compound suctions out, and has PBS buffer solution that three times cell washing is added new training afterwards
Foster base, with the fluorescence intensity of the intracellular protoporphyrin of confocal laser scanning microscope.
Result as shown in fig. 7, protoporphyrin in macrophage membrane for the embedding cell membrane protoporphyrin key compound fluorescence intensity with
Incubation time increases and increases.Demonstrating carrying drug ratio in macrophage for the embedding cell membrane protoporphyrin key compound can be by embedding thin
After birth protoporphyrin key compound was regulated and controled with the co-cultivation time of macrophage.
【Embodiment 9】Embedding cell membrane protoporphyrin key compound is to macrophage cell membrane embedding film ability and embedding cell membrane protoporphyrin
The relation of key compound Concentraton gradient
By macrophage with 1 × 105The density inoculation of individual cells/well, cultivates under the conditions of 37 DEG C in 1mL culture medium.24
After hour, embedding cell membrane protoporphyrin key compound is dissolved in the medium, add 1mL to contain embedding cell respectively in macrophage
Film protoporphyrin key compound concentration is 0 micromoles per liter, 5 micromoles per liter, 10 micromoles per liter, 15 micromoles per liter and 30 micromoles/
The culture medium rising.After culture 2 hours, the culture medium containing embedding cell membrane protoporphyrin key compound is suctioned out, have PBS buffer solution to incite somebody to action
Cell washing adds new culture medium three times, afterwards with the fluorescence intensity of the intracellular protoporphyrin of confocal laser scanning microscope.
Result as shown in figure 8, protoporphyrin in macrophage membrane for the embedding cell membrane protoporphyrin key compound fluorescence intensity with
Embedding cell membrane protoporphyrin key compound concentration increases and increases.Demonstrate load in macrophage for the embedding cell membrane protoporphyrin key compound
Medicine rate can be regulated and controled by the concentration of macrophage and embedding cell membrane protoporphyrin key compound.
【Embodiment 10】Embedding cell membrane protoporphyrin key compound macrophage carrier tropism aptitude tests
By macrophage, macrophage, the macrophage that embedding cell membrane protoporphyrin is fitted together to, macrophage, embedding cell membrane is former
The macrophage that porphyrin is fitted together to is seeded in the density of 3000 cells/well respectively and penetrates film upper strata.In each inoculating cell lower floor
It is separately added into the new MEM culture medium of 1mL, the MEM culture medium of 4T1 cell culture, new DMEM culture medium and COS7 cultivated
DMEM culture medium, under the conditions of 37 DEG C cultivate.Respectively in culture 12 hours, 24 hours, after 36 hours and 48 hours, aobvious with being inverted
Micro mirror observes the number of penetration cell.
Result is as shown in figure 9, the embedding film macrophage of embedding cell membrane protoporphyrin key compound is trained in the MEM of 4T1 cell culture
Cultivate through penetrating the number of cells of film with macrophage under the MEM culture medium condition of 4T1 cell culture under the conditions of foster base
Culture is suitable through the number of cells penetrating film.Demonstrating embedding cell membrane protoporphyrin key compound affects on the tropism of macrophage
Less.
【Embodiment 11】Mice embeds the targeting ability to tumor for the cell membrane protoporphyrin key compound macrophage carrier
Detection
4～5 weeks white mice (BALB/c) medial leg subcutaneous injection 100 μ L (PBS) contain number of cells be 1 × 106's
4T1 cell suspension makes tumor, when gross tumor volume grows to about 200mm2When.Embedding cell membrane protoporphyrin key compound is fitted together to macrophage tail
Intravenous injection is in Mice.With fluorescence distribution and change in small animal living body imaging observation mice body.
As shown in Figure 10, mouse tumor region material fluorescence increases and increases result in time, thus proving embedding cell membrane
Protoporphyrin key compound is fitted together to the ability that macrophage has cancer target transport.
【Embodiment 12】Embedding cell membrane protoporphyrin is fitted together to the therapeutic effect to tumor for the macrophage carrier
4～5 weeks white mice (BALB/c) medial leg subcutaneous injection 100 μ L (PBS) contain number of cells be 1 × 106's
4T1 cell suspension makes tumor, when gross tumor volume grows to about 200mm2When.Mice is randomly divided into 6 groups.PBS and macrophage, embedding thin
After birth protoporphyrin is fitted together to macrophage, embedding cell membrane protoporphyrin key compound, the chimeric macrophage of embedding cell membrane protoporphyrin and former porphin
Quinoline tail vein injection.After injection 24 hours and 48 hours, irradiate tumor, every each 5min with 660nm He-Ne laser.Daily
Measurement gross tumor volume, gross tumor volume is according to V=W2× L/2 formula calculates, and wherein W is shorter tumor width, and L is longer swelling
Tumor width, by relative tumour volume (V/V0, V is real-time gross tumor volume, V0For treating pre-neoplastic volume) reflecting gross tumor volume
Situation of change.
Result is shown in Figure 11, is suppressed through the mice tumors grew that embedding cell membrane protoporphyrin is fitted together to after macrophage treatment,
And have more superior tumor inhibition effect with respect to through embedding cell membrane protoporphyrin and protoporphyrin, thus prove the former porphin of embedding cell membrane
Quinoline is fitted together to the therapeutic effect to tumor for the macrophage carrier.Meanwhile, mice weights do not have in a substantial change with therapeutic effect, say
Bright do not have strong toxic and side effects (Figure 12) through the chimeric macrophage treatment of embedding cell membrane protoporphyrin to mice.And tumor in tumor picture
Volume (Figure 13) and weight (Figure 14) change further illustrate embedding cell membrane protoporphyrin and are fitted together to the superior tumor suppression of macrophage
Make and use.
Claims (13)
1. a kind of cancer target cell drug carrier is it is characterised in that be made up of embedding cell membrane medicine key compound and carrier cell,
Embedding cell membrane medicine key compound embeds carrier cell cell membrane, realizes drug delivery by carrier cell to the tropism of tumor.
2. cancer target cell drug carrier according to claim 1 is it is characterised in that described embedding cell membrane medicine key
Compound, is made up of hydrophilic poly arginine, alkyl chain and bonding medicine, its chemical structural formula formula is as shown in formula I：
Wherein m=5-8；N=1-6；R is bonding medicine, is photodynamic therapy photosensitizer, chemotherapeutics or fluorescence molecule
Any one.
3. cancer target cell drug carrier according to claim 2 is it is characterised in that described embedding cell membrane medicine key
Compound is Palmic acid-lysine (camptothecine)-Arg-Arg-Arg-Arg, Palmic acid-lysine (former porphin
Quinoline)-Arg-Arg-Arg-Arg and Palmic acid-lysine (fluorescein)-Arg-Arg-arginine-
Any one in arginine.
4. the cancer target cell drug carrier according to claim 1-3 is it is characterised in that described carrier cell is to follow
Garland cellss or stem cell.
5. cancer target cell drug carrier according to claim 4 is it is characterised in that described circulating cells are red thin
Born of the same parents, macrophage, mononuclear cell, leukocyte.
6. the cancer target cell drug carrier according to claim 1-3 or 5 any one is it is characterised in that described
Cell drug carrier is made up of two kinds of embedding cell membrane medicine key compounds and carrier cell, and two kinds of embedding cell membrane medicine key compounds are simultaneously
Embedded carrier cell cell membrane, the bonding medicine of two kinds of described embedding cell membrane medicine key compounds is that photodynamic therapy is photosensitive
In agent, chemotherapeutics or fluorescence molecule any two kinds.
7. cancer target cell drug carrier according to claim 4 it is characterised in that described cell drug carrier by
Two kinds of embedding cell membrane medicine key compounds and carrier cell composition, it is thin that two kinds of embedding cell membrane medicine key compounds are simultaneously embedded in carrier cell
After birth, the bonding medicine of described embedding cell membrane medicine key compound is photodynamic therapy photosensitizer, chemotherapeutics or fluorescence
In molecule any two kinds.
8. application in the reagent preparing tumor imaging for the cancer target cell drug carrier described in claim 1.
9. application in preparing chemotherapeutics for the cancer target cell drug carrier described in claim 1.
10. application in preparing photodynamic therapy photosensitizer for the cancer target cell drug carrier described in claim 1.
Cancer target cell drug carrier described in 11. claim 1 can complete tumor imaging and chemotherapy in preparation simultaneously
Application in medicine.
Cancer target cell drug carrier described in 12. claim 1 can complete photodynamic therapy for cancer in preparation simultaneously
With the application in the medicine of chemotherapy.
Cancer target cell drug carrier described in 13. claim 1 can complete photodynamic therapy for cancer in preparation simultaneously
With the application in the medicine of tumor imaging.
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| CN108815520A (en) * | 2018-06-19 | 2018-11-16 | 暨南大学 | A kind of bionical binary synergic nano carrier and the preparation method and application thereof |
| CN110507826A (en) * | 2018-05-21 | 2019-11-29 | 中国科学院上海药物研究所 | A kind of living cells drug-loading system, preparation method and application based on macrophage |
| WO2021010698A1 (en) * | 2019-07-12 | 2021-01-21 | 고려대학교 산학협력단 | Cancer cell-targeted drug delivery carrier and composition for promoting photo-thermal treatment effects, both of which contain m1 macrophages as active ingredient |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110507826A (en) * | 2018-05-21 | 2019-11-29 | 中国科学院上海药物研究所 | A kind of living cells drug-loading system, preparation method and application based on macrophage |
| CN110507826B (en) * | 2018-05-21 | 2021-10-01 | 中国科学院上海药物研究所 | Live cell drug delivery system based on macrophages, and preparation method and application thereof |
| CN108815520A (en) * | 2018-06-19 | 2018-11-16 | 暨南大学 | A kind of bionical binary synergic nano carrier and the preparation method and application thereof |
| CN108815520B (en) * | 2018-06-19 | 2021-04-02 | 暨南大学 | Bionic binary synergistic nano-carrier and preparation method and application thereof |
| WO2021010698A1 (en) * | 2019-07-12 | 2021-01-21 | 고려대학교 산학협력단 | Cancer cell-targeted drug delivery carrier and composition for promoting photo-thermal treatment effects, both of which contain m1 macrophages as active ingredient |
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