CN110201161A - A kind of composition, preparation method and its application in killer strain - Google Patents
A kind of composition, preparation method and its application in killer strain Download PDFInfo
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Abstract
The invention belongs to antineoplastic target medicine research and development field more particularly to a kind of composition, preparation method and its applications in killer strain.The present invention provides a kind of composition, raw material includes: black squama, 5-ALA, polyethylene glycol and TfR binding peptide.The present invention also provides a kind of preparation methods of above-mentioned composition, and the present invention provides a kind of application of above-mentioned composition or the product obtained with above-mentioned preparation method in killing SAS, HSC-2 and HSC-3 cell strain.It can be obtained through measuring, compared with positive control drug, product made from technical solution provided by the invention has significant inhibiting effect for the growth and breeding of tumor cell line, while can also further promote the apoptosis of tumour cell.A kind of composition, preparation method and its application in killer strain provided by the invention, solve in the prior art, and there is be difficult to take into account the technological deficiency in body stability, targeting ability of aggregation and vector degradation performance difference for tumor-targeting drug.
Description
Technical field
The invention belongs to a kind of antineoplastic target medicine research and development field more particularly to composition, preparation method and its killing
Application in cell strain.
Background technique
Oral squamous cell carcinoma (Oral squamous cell carcinoma, OSCC) be head-neck malignant tumor it
One, and disease incidence, in trend is risen year by year, the therapeutic modality of the disease is mainly operative treatment at present.But operative treatment is usual
Large area oral cavity tissue organ can be cut off, the postoperative quality of life of patient is seriously affected.Therefore, how efficient non-invasive therapy OSCC
It is that urgent clinical needs one of solve the problems, such as.
Tumour sound motivation therapy (sonodynamic therapy, SDT) is using ultrasonic activation selective aggregation swollen
The sound sensitizing drug of tumor tissue, the sound sensitizing drug for SDT treatment is since its internal stability is undesirable and target tumor is enriched with energy
The problems such as power is limited, mostly also in conceptual phase.It has now been found that SDT is more in glioma, breast cancer and liver cancer etc.
There is specific therapeutic effect in the non-invasive therapy of the kind non-tumor disease such as tumour and atherosclerotic plaque.
Sound sensitiser 5-ALA is the product of heme catabolism process in human body, has good biological safety and outstanding
Acoustodynamic effect, paid close attention to by numerous researchers.Nano material is improved as the hydrophily that carrier can improve sound sensitiser
Its stability in vivo.Meanwhile using the solid tumor high-permeability and retention effect of nano material, sound sensitiser can be increased and existed
Passive target enrichment in tumor tissues.Wherein, it mainly includes liposome and cationic polymer etc. that organic nano carrier is common.
Organic nano carrier carrying drug ratio with higher, in vivo be easy degradation the advantages that, but the higher positive potential in its surface cause its compared with
High cytotoxicity and poor chemical stability；Inorganic nano carrier has stronger chemical stability and controllable physics
Performance, and it is easy to the advantages that being surface modified achievable multifunction, still, inorganic nano vector degradation performance is poor,
Biocompatibility limits its application in nano-carrier.
Therefore, a kind of composition, preparation method and its application in killer strain are developed, for solving existing skill
In art, there is be difficult to take into account the technology in body stability, targeting ability of aggregation and vector degradation performance difference for tumor-targeting drug
Defect becomes those skilled in the art's urgent problem to be solved.
Summary of the invention
In view of this, being used for the present invention provides a kind of composition, preparation method and its application in killer strain
It solves in the prior art, there is be difficult to take into account in body stability, targeting ability of aggregation and vector degradation for tumor-targeting drug
The technological deficiency of energy difference.
The present invention provides a kind of composition, the raw material of the composition includes: black squama (BP), 5-ALA
(5-ALA), polyethylene glycol (PEG) and TfR binding peptide (T7).
Preferably, in terms of mass parts, the raw material of the composition includes: 1~2 part of black squama, 5-ALA part 50
~80 parts, 12~32 parts of polyethylene glycol and 6~16 parts of TfR binding peptide.
The present invention also provides a kind of preparation method including one composition of any of the above, the preparation methods
Are as follows:
Step 1: preparation BP NSs: black squama prepares BP NSs by liquid phase stripping method；
Step 2: preparation T7-PEG-NH2: Mal-PEG-NH2Solution stirs after mixing with T7-Cys solution, dry T7-
PEG-NH2；
Step 3: preparation BP-PEG-T7NSs:BP NSs solution is mixed with MAL-PEG, is stirred after ultrasonic treatment, obtain BP-
PEG-T7NSs；
Step 4: preparation BP-PEG-T7/5-ALA NSs:BP-PEG-T7NSs is incubated for after mixing with 5-ALA solution, obtain
BP-PEG-T7/5-ALA NSs。
Preferably, the preparation method further include: washing, the washing step carry out after step 4；
Crude product BP-PEG-T7/5-ALA NSs obtained by step 4 obtains sterling BP-PEG- after deionized water is washed 6~8 times
T7/5-ALA NSs。
Preferably, in step 1, the liquid phase stripping method are as follows: black squama is dissolved in NMP, with the intensity ultrasonic of 200~300W
After 5~8h of interrupted oscillating, in 0 DEG C of continuation 6~12h of ultrasound, ultrasound removes the black squama crystal being not peeled off after the completion, takes supernatant
Centrifugation, collecting sediment is BP NSs.
Preferably, the method for the ultrasonic interrupted oscillating are as follows: after every 2~5s of interval, 1~4s of ultrasonication；
It is described to remove the method for black squama crystal being not peeled off are as follows: with the revolving speed centrifugation 15 of 6000~8000rpm~
30min；
The revolving speed of the supernatant centrifugation is 8000~12000rpm, and the time of the supernatant centrifugation is 15~30min.
Preferably, in step 2, the Mal-PEG-NH2The solvent of solution is DMSO and/or DMF, the Mal-PEG-
NH2The concentration of solution is 15~25mg/ml；
In step 2, the solvent of the T7-Cys solution is PBS, and the solubility of the T7-Cys solution is 0.2~0.8mg/
ml；
In step 2, the temperature of the stirring is room temperature, and the time of the stirring is 10~15h, the method for the drying
For freeze-drying；
It further include washing after the completion of the stirring in step 2, the method for the washing is deionized water dialysis.
Preferably, in step 3, the time of the ultrasonic treatment is 20~40min, and the time of the stirring is 2~6h.
Preferably, in step 4, the solvent of the 5-ALA solution is DMSO and/or DMF, the concentration of the 5-ALA solution
For 0.1~0.4mg/ml, the time of the incubation is 20~30h.
The present invention provides a kind of including preparation described in composition described in any of the above one or any of the above one
Application of the product that method obtains in killing SAS, HSC-2, HSC-3, Ca922 and Sa-3 cell strain.
In technical solution provided by the invention, using black squama nanometer sheet as carrier, compared with traditional inorganic nano material,
Black squama is made of single P elements, and P elements are the necessary microelements of body, its metabolism will not cause to be immunoreacted, and makes it
The outstanding biodegradability of biological safety with higher；Meanwhile two-dimentional black phosphorus nanometer sheet is waveform layer structure, is had
High specific surface area makes its application in drug and gene delivery show big advantage.Black phosphorus surface has numerous
Active site is easy to modify, and also has wider forbidden band energy gap, and the transition between singlet state and triplet, excitation three may be implemented
The duration of weight state is 100 μ s, is conducive to it and generates with oxygen molecule interaction1O2, this feature of black phosphorus nanometer sheet
Make it under the excitation of ultrasonic wave, has the characteristic that collaboration promotes acoustodynamic effect.Confirm that black phosphorus nanometer sheet can be efficient through research
Immobilized and stable release 5-ALA, has good biocompatibility and degradability.
Meanwhile in technical solution provided by the invention, for the cancer target delivering effect for further improving nanometer sound sensitiser
Fruit also realizes nanometer sound sensitiser active targeting OSCC cell by the endocytosis that tumor surface specific receptor combines, thus
It is efficiently precisely more enriched in tumor locus, is reduced or avoided in the distribution of normal tissue and accumulative.Technology provided by the invention
In scheme, is targeted and combined by a kind of small molecule small peptide (TfR binding peptide, HAIYPRH, T7) specific recognition
There is high expression TfR (Transferrin Receptor, TfR) in OSCC tumor cell surface；Simultaneously as T7
Steric hindrance is small (Kd < 10nm), in conjunction with TfR after do not influence two sites the 7-mer sequence and 12-mer of Tf
For sequence in conjunction with TfR, exogenous T7 enters the competitive inhibition that not will receive endogenous Tf in vivo, thus very big
It improves and is combined to obtain ability with tumour cell targeting.Nanometer sound sensitiser is constructed using T7 modification black phosphorus nanometer sheet, it can active target
It is intracellular to delivering and discharging 5-ALA to OSCC, realize SDT to the accurate targeted therapy of OSCC.
In conclusion the raw material of the composition includes: black squama (BP), 5- amino the present invention provides a kind of composition
Levulic acid (5-ALA), polyethylene glycol (PEG) and TfR binding peptide (T7).The present invention also provides in one kind
The preparation method for stating composition, the present invention provides a kind of above-mentioned compositions or the product obtained with above-mentioned preparation method to kill
Application in SAS, HSC-2, HSC-3, Ca922 and Sa-3 cell strain.It can be obtained through measuring, compared with positive control drug,
Product made from technical solution provided by the invention has significant inhibiting effect for the growth and breeding of tumor cell line, together
When can also further promote the apoptosis of tumour cell.A kind of composition provided by the invention, preparation method and its in killing cell
Application in strain, solves in the prior art, and there is be difficult to take into account in body stability, targeting ability of aggregation for tumor-targeting drug
And the technological deficiency of vector degradation performance difference.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
The embodiment of invention for those of ordinary skill in the art without creative efforts, can also basis
The attached drawing of offer obtains other attached drawings.
Fig. 1 is the flow diagram of BP-PEG-T7/5-ALA NSs preparation method provided by the invention；
Fig. 2 is in embodiment 4, and BP-PEG-T7/5-ALA NSs shows the result of tumor cell line growth inhibitory effect
It is intended to；
Fig. 3 is to measure the result schematic diagram of BP-PEG-T7/5-ALA biocompatibility in embodiment 5；
Fig. 4 is to measure the result schematic diagram of BP-PEG-T7/5-ALA blood compatibility in embodiment 5；
Fig. 5 is to measure the result schematic diagram of BP-PEG-T7/5-ALA vivo biodistribution safety in embodiment 5.
Specific embodiment
The embodiment of the invention provides a kind of composition, preparation method and its applications in killer strain, for solving
Certainly in the prior art, there is be difficult to take into account in body stability, targeting ability of aggregation and vector degradation performance for tumor-targeting drug
The technological deficiency of difference.
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
In order to which the present invention is described in more detail, below with reference to embodiment to a kind of composition provided by the invention, preparation method
And its application in killer strain, it is specifically described.
Embodiment 1
The present embodiment is the one of embodiment for preparing BP-PEG-T7/5-ALA NSs.
Step 1: preparation BP NSs: black squama prepares BP NSs by liquid phase stripping method；Wherein, liquid phase stripping method are as follows: black squama
It is dissolved in NMP, after the intensity ultrasonic interrupted oscillating 6h of 200W, removes and be not peeled off after the completion of 0 DEG C of continuation ultrasound 8h, ultrasound
Black squama crystal, take supernatant to be centrifuged, collect sediment be BP NSs.In the present embodiment, the method for ultrasonic interrupted oscillating are as follows:
After every interval 5s, ultrasonication 1s；The method for removing the black squama crystal being not peeled off are as follows: be centrifuged with the revolving speed of 6500rpm
20min；The revolving speed of supernatant centrifugation is 10000rpm, and the time of supernatant centrifugation is 20min.
Step 2: preparation T7-PEG-NH2: Mal-PEG-NH2Solution stirs after mixing with T7-Cys solution, dry T7-
PEG-NH2；Mal-PEG-NH2The solvent of solution is DMF, Mal-PEG-NH2The concentration of solution is 15mg/ml；In step 2, T7-
The solvent of Cys solution is PBS, and the solubility of T7-Cys solution is 0.2mg/ml；In step 2, the temperature of stirring is room temperature, stirring
Time be 15h, dry method be freeze-drying；It further include washing after the completion of stirring in step 2, the method for washing is to go
Ionized water dialysis.
Step 3: preparation BP-PEG-T7NSs:BP NSs solution is mixed with MAL-PEG, is stirred after ultrasonic treatment, obtain BP-
PEG-T7NSs；The time of ultrasonic treatment is 20min, and the time of stirring is 6h.
Step 4: preparation BP-PEG-T7/5-ALA NSs:BP-PEG-T7NSs is incubated for after mixing with 5-ALA solution, obtain
BP-PEG-T7/5-ALA NSs crude product；The solvent of 5-ALA solution is DMSO, and the concentration of 5-ALA solution is 0.1mg/ml, is incubated for
Time be 25h.
Step 5: gained crude product BP-PEG-T7/5-ALA NSs obtains sterling BP- after deionized water is washed 6~8 times
PEG-T7/5-ALA NSs。
In the present embodiment, mass parts meter, the feed ratio of each raw material are as follows: 1 part of black squama, gathers at 80 parts of 5-ALA part
20 parts of ethylene glycol and 6 parts of TfR binding peptide.
Embodiment 2
The present embodiment is the one of embodiment for preparing BP-PEG-T7/5-ALA NSs.
Step 1: preparation BP NSs: black squama prepares BP NSs by liquid phase stripping method；Wherein, liquid phase stripping method are as follows: black squama
It is dissolved in NMP, after the intensity ultrasonic interrupted oscillating 5h of 300W, removes after the completion of 0 DEG C of continuation ultrasound 12h, ultrasound and do not shelled
From black squama crystal, take supernatant to be centrifuged, collect sediment be BP NSs.In the present embodiment, the method for ultrasonic interrupted oscillating
Are as follows: after every interval 2s, ultrasonication 2s；The method for removing the black squama crystal being not peeled off are as follows: be centrifuged with the revolving speed of 6000rpm
15min；The revolving speed of supernatant centrifugation is 12000rpm, and the time of supernatant centrifugation is 15min.
Step 2: preparation T7-PEG-NH2: Mal-PEG-NH2Solution stirs after mixing with T7-Cys solution, dry T7-
PEG-NH2；Mal-PEG-NH2The solvent of solution is DMSO, Mal-PEG-NH2The concentration of solution is 22mg/ml；In step 2,
The solvent of T7-Cys solution is PBS, and the solubility of T7-Cys solution is 0.4mg/ml；In step 2, the temperature of stirring is room temperature, is stirred
The time mixed is 12h, and dry method is freeze-drying；It further include washing after the completion of stirring in step 2, the method for washing is
Deionized water dialysis.
Step 3: preparation BP-PEG-T7NSs:BP NSs solution is mixed with MAL-PEG, is stirred after ultrasonic treatment, obtain BP-
PEG-T7NSs；The time of ultrasonic treatment is 25min, and the time of stirring is 2h.
Step 4: preparation BP-PEG-T7/5-ALA NSs:BP-PEG-T7NSs is incubated for after mixing with 5-ALA solution, obtain
BP-PEG-T7/5-ALA NSs crude product；The solvent of 5-ALA solution is DMS, and the concentration of 5-ALA solution is 0.4mg/ml, incubation
Time is 30h.
Step 5: gained crude product BP-PEG-T7/5-ALA NSs obtains sterling BP- after deionized water is washed 6~8 times
PEG-T7/5-ALA NSs。
In the present embodiment, mass parts meter, the feed ratio of each raw material are as follows: 1.3 parts of black squama, 70 parts of 5-ALA part,
12 parts of polyethylene glycol and 6 parts of TfR peptide 1.
Embodiment 3
The present embodiment is the one of embodiment for preparing BP-PEG-T7/5-ALA NSs.
Step 1: preparation BP NSs: black squama prepares BP NSs by liquid phase stripping method；Wherein, liquid phase stripping method are as follows: black squama
It is dissolved in NMP, after the intensity ultrasonic interrupted oscillating 8h of 260W, removes and be not peeled off after the completion of 0 DEG C of continuation ultrasound 6h, ultrasound
Black squama crystal, take supernatant to be centrifuged, collect sediment be BP NSs.In the present embodiment, the method for ultrasonic interrupted oscillating are as follows:
After every interval 4s, ultrasonication 4s；The method for removing the black squama crystal being not peeled off are as follows: be centrifuged with the revolving speed of 8000rpm
30min；The revolving speed of supernatant centrifugation is 8000rpm, and the time of supernatant centrifugation is 30min.
Step 2: preparation T7-PEG-NH2: Mal-PEG-NH2Solution stirs after mixing with T7-Cys solution, dry T7-
PEG-NH2；Mal-PEG-NH2The solvent of solution is DMSO, Mal-PEG-NH2The concentration of solution is 25mg/ml；In step 2,
The solvent of T7-Cys solution is PBS, and the solubility of T7-Cys solution is 0.8mg/ml；In step 2, the temperature of stirring is room temperature, is stirred
The time mixed is 10h, and dry method is freeze-drying；It further include washing after the completion of stirring in step 2, the method for washing is
Deionized water dialysis.
Step 3: preparation BP-PEG-T7NSs:BP NSs solution is mixed with MAL-PEG, is stirred after ultrasonic treatment, obtain BP-
PEG-T7NSs；The time of ultrasonic treatment is 40min, and the time of stirring is 5h.
Step 4: preparation BP-PEG-T7/5-ALA NSs:BP-PEG-T7NSs is incubated for after mixing with 5-ALA solution, obtain
BP-PEG-T7/5-ALA NSs crude product；The solvent of 5-ALA solution is DMF, and the concentration of 5-ALA solution is 0.3mg/ml, incubation
Time is 20h.
Step 5: gained crude product BP-PEG-T7/5-ALA NSs obtains sterling BP- after deionized water is washed 6~8 times
PEG-T7/5-ALA NSs。
In the present embodiment, mass parts meter, the feed ratio of each raw material are as follows: 2 parts of black squama, gathers at 50 parts of 5-ALA part
32 parts of ethylene glycol and 4 parts of TfR peptide 1.
Embodiment 4
The present embodiment is the specific implementation for verifying BP-PEG-T7/5-ALA NSs for tumor cell line growth inhibitory effect
Example.
In the present embodiment, experimental group are as follows: negative control group (C group), positive controls application 5-Fu (5-Fu group) 5-ALA
Group, BP-PEG-T7/5-ALA group.Each group is included into (SAS, HSC-2, HSC-3, Ca922, Sa-3) with OSCC tumour cell respectively
It co-cultures, gives the ultrasound (1.0MHz of same dose；1W/cm2；20% duty ratio) treatment 5min, it is thin to establish SDT treatment OSCC
Born of the same parents' model: cell proliferative conditions are detected using CCK-8, calculate cell survival rate.
For experimental results further referring to Fig. 2, result is mean ± standard deviation (n=6), p < 0.05 *, with C group phase
Than with statistical significance.
Embodiment 5
The present embodiment is the measurement BP-PEG-T7/5-ALA NSs specific embodiment of compatibility and metabolic condition in vivo.
5.1 biocompatibility
It is fibroblast (3T3) that cell is selected in measurement, and experiment is divided into three groups: every group is separately added into PBS (control group), two
Silicon oxide sio2And BP-PEG-T7/5-ALA, 6h is co-cultured, cell-proliferation activity is detected using CCK-8, calculates cell survival
Rate.
Experimental results are referring to Fig. 3, as can be drawn from Figure 3, BP-PEG-T7/5-ALA NSs is good.As a result it is
Mean ± standard deviation (n=6), p < 0.05 * have statistical significance compared with control group (C group).
5.2 blood compatibility
By nano-Ag particles (Ag) solution and BP-PEG-T7/5-ALA solution of 2.5 μ g/mL concentration, it is separately added into 15mL
In centrifuge tube, every pipe 10mL, each concentration respectively prepares 3 pipes.10mL physiological saline is added in centrifuge tube as negative control, centrifugation
10mL distilled water is added as positive control, negative control and positive control in pipe and respectively prepares 3 pipes.
Heart takes rabbit blood 4mL (2% Potassium Oxalate Solution 1:9 is anticoagulant), mixes with 5mL normal saline dilution.By sample
Quality control, negative control pipe and positive control pipe are put into 37 DEG C of water-bath 30min in water bath, and it is dilute to be separately added into 0.2mL into each pipe
Anticoagulant rabbit blood is released, continues 37 DEG C of water-bath 60min in constant water bath box, takes out test tube after the water bath is over, be put into centrifuge
800g is centrifuged 5min, observes the color change of supernatant in test tube, Aspirate supernatant, with ultraviolet specrophotometer at 545nm
Read absorbance value.
Wherein, A, A1And A2Respectively indicate experimental group light absorption value, negative control group light absorption value and positive controls light absorption value.
Acquired results are referring to Fig. 4, as can be drawn from Figure 4, BP-PEG-T7/5-ALA has good blood compatibility.
5.3 vivo biodistribution safeties
Nude mice OSCC lotus knurl model is established, control group tail vein injection saline, experimental group tail vein injection concentration are
The BP-PEG-T7/5-ALA solution of 2.5 μ g/mL puts to death mouse and is rapidly separated liver, spleen, heart, kidney and lung device after 15 days
Official.
Acquired results observe main organs histopathology referring to Fig. 5, dyeing by HE, have no apparent damage and disease
Reason changes.Prove BP-PEG-T7/5-AL vivo biodistribution good security.
In conclusion the raw material of the composition includes: black squama (BP), 5- amino the present invention provides a kind of composition
Levulic acid (5-ALA), polyethylene glycol (PEG) and TfR binding peptide (T7).The present invention also provides in one kind
The preparation method for stating composition, the present invention provides a kind of above-mentioned compositions or the product obtained with above-mentioned preparation method to kill
Application in SAS, HSC-2, HSC-3, Ca922 and Sa-3 cell strain.It can be obtained through measuring, compared with positive control drug,
Product made from technical solution provided by the invention has significant inhibiting effect for the growth and breeding of tumor cell line, together
When can also further promote the apoptosis of tumour cell.A kind of composition provided by the invention, preparation method and its in killing cell
Application in strain, solves in the prior art, and there is be difficult to take into account in body stability, targeting ability of aggregation for tumor-targeting drug
And the technological deficiency of vector degradation performance difference.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of composition, which is characterized in that the raw material of the composition includes: black squama, 5-ALA, polyethylene glycol
And TfR binding peptide.
2. composition according to claim 1, which is characterized in that in terms of mass parts, the raw material of the composition includes: black
1~2 part of squama, 50~80 parts of 5-ALA part, 12~32 parts of polyethylene glycol and TfR binding peptide 6~16
Part.
3. a kind of preparation method including composition described in claim 1 to 2 any one, which is characterized in that the preparation side
Method are as follows:
Step 1: preparation BP NSs: black squama prepares BP NSs by liquid phase stripping method；
Step 2: preparation T7-PEG-NH2: Mal-PEG-NH2Solution stirs after mixing with T7-Cys solution, dry T7-PEG-
NH2；
Step 3: preparation BP-PEG-T7 NSs:BP NSs solution is mixed with MAL-PEG, is stirred after ultrasonic treatment, obtain BP-PEG-
T7 NSs；
Step 4: preparation BP-PEG-T7/5-ALA NSs:BP-PEG-T7 NSs is incubated for after mixing with 5-ALA solution, BP- is obtained
PEG-T7/5-ALA NSs。
4. preparation method according to claim 3, which is characterized in that the preparation method further include: washing, the washing
Step carries out after step 4；
Crude product BP-PEG-T7/5-ALA NSs obtained by step 4 obtains sterling BP-PEG-T7/ after deionized water is washed 6~8 times
5-ALA NSs。
5. preparation method according to claim 3, which is characterized in that in step 1, the liquid phase stripping method are as follows: black squama is molten
In NMP, after 5~8h of intensity ultrasonic interrupted oscillating of 200~300W, removed after the completion of 0 DEG C of continuation 6~12h of ultrasound, ultrasound
The black squama crystal being not peeled off is removed, supernatant is taken to be centrifuged, collecting sediment is BP NSs.
6. preparation method according to claim 5, which is characterized in that the method for the ultrasound interrupted oscillating are as follows: every interval 2
After~5s, 1~4s of ultrasonication；
The method for removing the black squama crystal being not peeled off are as follows: 15~30min is centrifuged with the revolving speed of 6000~8000rpm；
The revolving speed of the supernatant centrifugation is 8000~12000rpm, and the time of the supernatant centrifugation is 15~30min.
7. preparation method according to claim 3, which is characterized in that in step 2, the Mal-PEG-NH2Solution it is molten
Agent is DMSO and/or DMF, the Mal-PEG-NH2The concentration of solution is 15~25mg/ml；
In step 2, the solvent of the T7-Cys solution is PBS, and the solubility of the T7-Cys solution is 0.2~0.8mg/ml；
In step 2, the temperature of the stirring is room temperature, and the time of the stirring is 10~15h, and the method for the drying is cold
It is lyophilized dry；
It further include washing after the completion of the stirring in step 2, the method for the washing is deionized water dialysis.
8. preparation method according to claim 3, which is characterized in that in step 3, the time of the ultrasonic treatment is 20
~40min, the time of the stirring are 2~6h.
9. preparation method according to claim 3, which is characterized in that in step 4, the solvent of the 5-ALA solution is
DMSO and/or DMF, the concentration of the 5-ALA solution are 0.1~0.4mg/ml, and the time of the incubation is 20~30h.
10. a kind of including system described in composition described in claim 1 to 2 any one or claim 3 to 9 any one
Application of the product that Preparation Method obtains in killing SAS, HSC-2, HSC-3, Ca922 and Sa-3 cell strain.
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