One kind having the function of diagnosis and treatment excretion body and preparation method thereof
Technical field
The invention belongs to nanosecond medical science technical fields, in particular to a kind of to have the function of diagnosis and treatment excretion body and its preparation side
Method.
Background technique
Chemotherapy is still one of major way of oncotherapy.But there are poorly water-soluble, metabolism for most of chemotherapeutics fastly,
Poor biocompatibility is distributed the undesirable defects such as low with Premeabilisation of cells ability in vivo, seriously limit chemotherapeutics effect and
Utilization rate.Therefore, a kind of novel pharmaceutical carrier is constructed, to improve drug solubility and stability, penetration into tissue is improved, mentions
High tumor locus drug concentration, while avoiding being captured by immunocyte is current research hot spot.
Unique advantage of the excretion body in medicament transport, becomes one of research hotspot.Excretion body is by mammal
Cell-derived nano vesicle, diameter is probably in 40-200nm or so.Excretion body can be identified as " itself " by host, therefore its
As a kind of active carrier, the substances such as certain cell RNAs, drug molecule can be delivered in mammalian cell.This seed capsules
Blister corpusculum has unique advantage as carrier, and if its immunogenicity is low, stability in blood is high, transports medicine to cell
High-efficient and stronger enhancing infiltration retention effect of object etc..Currently, existing research person utilizes excretion body carrying medicament, as a result table
It is bright compared with free drug, excretion body carrying medicament have better anti-tumor activity.But excretion body carrying medicament efficiency compared with
Low, patent CN108114290A discloses the preparation method of excretion body that is a kind of while loading chemicals and nano material, uses
Curcumin and nano ferriferrous oxide are loaded into excretion body by electric shifting method, but full text does not have table for excretion body encapsulation rate
It states.Excretion physical efficiency is enough in the load of a variety of different type drugs, including nucleic acid molecules (mRNA, siRNA etc.), chemicals
(curcumin, indocyanine green etc.) and nano material (nano silver, Nanoscale Iron etc.).
Curcumin is a kind of fat-soluble phenolic substances extracted from Zingiber curcuma turmeric rhizome, is contained in structure
There is phenolic hydroxyl group, when peroxidatic reaction of lipid occurs for cell membrane, oxidation reaction can occur for phenolic hydroxyl group, can effectively terminate freedom
Base reaction, thus it shows many physiological activity, such as anti-oxidant, antitumor, many effects such as prevent aging.Experiment in vitro
Show that curcumin can significantly inhibit proliferation, migration and the invasion of tumour cell, and promotes apoptosis of tumor cells.Therefore, turmeric
Element has broad application prospects in kinds cancer.Indocyanine green (ICG) is currently the only a kind of by the close of U.S. FDA certification
Infrared (NIR) dyestuff.Studies have shown that ICG has photodynamic properties, can be generated after capable of being excited by the NIR light of strong penetrating power
Active oxygen.And then tumor locus can be cooperated with by photo-thermal therapy and oxyradical, reach and kills tumour cell
Effect.Therefore, ICG is to be presently considered in photo-thermal therapy most promising drug.
Traditional oncotherapy can only be by Drug inhibition tumour growth, and the diagnosis and the state of an illness for inside tumor are evaluated
It is not prompt enough, accurate.We turn parameter and drug quality ratio by inquiring into different electricity, provide a kind of efficiently preparation and have and examine
The excretion body method for treating integrated function, drug is rapidly and efficiently contained inside excretion body, while can be used in a variety of drugs
Combination contains, and realizes the concertedness and consistency of oncotherapy and diagnosis.
Summary of the invention
The primary purpose of the present invention is that overcome presently, there are technological deficiency, providing a kind of has the function of diagnosis and treatment excretion body
Preparation method.This method is easy to operate, repetitive rate is high.Excretion body obtained encapsulation rate with higher and diagnosis and treatment integration function
Can, can be suitable for a variety of single medicines contain and the total load of multiple combinations drug.The excretion body tool that this method is prepared
There are the functions such as chemotherapy, photo-thermal therapy, photodynamic therapy and imaging, the diagnosing and treating for a variety of diseases such as tumour provides one
The new carrier and preparation method thereof of kind.
Another object of the present invention is to provide what is obtained by above-mentioned preparation method to have the function of diagnosis and treatment excretion body.
The purpose of the invention is achieved by the following technical solution:
A kind of preparation method with diagnosis and treatment excretion body, includes the following steps:
1) excretion body is extracted, is purified: being filled in interstital stem cell culture solution and is extracted from C57 mouse using differential supercentrifugation
The excretion body of cell secretion, obtained excretion weight are suspended from 10mM PBS solution, and -80 DEG C save backup;
2) the excretion body of the curcumin prepared or indocyanine green solution and step 1) sample preparation: is mixed in 10mM PBS
In, it is uniformly mixed, obtains sample;
3) electricity turns: the sample of step 2) is placed in electroporation apparatus and carries out electroporation, the sample after obtaining electroporation;
4) it is incubated for: the sample of step 3) electroporation being placed in cell incubator and is protected from light incubation 30min, after being incubated for
Liquid;
5) it purifies: by the liquid after being incubated in step 4) with 100,000-140,000g centrifugal force 60-100min, taking
Sediment obtains having the function of diagnosis and treatment excretion body.
In one of them embodiment, the C57 mouse does not fill interstital stem cell culture solution for no excretion body serum or not
Containing serum.
In one of them embodiment, in step 2), 0-100 μ g is separately added into 10mM PBS solution described in 200 μ L
Excretion body, 0-300 μ g curcumin or 0-300 μ g indocyanine green.
In one of them embodiment, in step 3), the condition of electroporation are as follows: voltage 0-350V, 100 μ s of pulsewidth,
Every 1000 μ s, discharge time: 0-9 times.
One kind having the function of diagnosis and treatment excretion body, is obtained by above-mentioned preparation method.What is be prepared has the function of diagnosis and treatment excretion
Body has medicament slow release performance.
The present invention has the following advantages and effects with respect to the prior art:
(1) preparation method of the invention operation is simple, and preparation time is short, and repetitive rate is high.What is be prepared has diagnosis and treatment function
Energy excretion body has medicament slow release performance, and encapsulation rate is up to 90%;Its solubility that can be improved hydrophobic drug, stability
And permeability, it avoids being swallowed and being removed by immune system in the living body.
(2) what is be prepared has the function of that diagnosis and treatment excretion body is able to maintain complete nano vesicle structure and mobility, together
When remain the physico-chemical property of carrying medicament, such as chemotherapeutic activity, photo-thermal effect, photodynamic effect and fluorescence imaging effect,
Realize diagnosis and treatment integration.
Detailed description of the invention
Fig. 1 is that the excitation of qualitative and quantitative detection near infrared light has the function of the fluorogram that diagnosis and treatment excretion body generates, in which: A
For excretion body, B be curcumin, C is indocyanine green, D is curcumin and indocyanine green;
Fig. 2 is the encapsulation rate result figure with diagnosis and treatment excretion body, in which: A is that excretion body carries curcumin, B is excretion
Body carries that the indoles mountain valley with clumps of trees and bamboo is green, C is that excretion body carries curcumin and the indoles mountain valley with clumps of trees and bamboo is green, D is that excretion body carries curcumin and the indoles mountain valley with clumps of trees and bamboo is green;
Fig. 3 is the testing result figure with the average grain diameter of diagnosis and treatment excretion body, in which: A is excretion body, B is that electricity turns
Excretion body, C are that carry curcumin, D be that excretion body carries that the indoles mountain valley with clumps of trees and bamboo is green, E is that excretion body carries curcumin and the indoles mountain valley with clumps of trees and bamboo is green to excretion body;
Fig. 4 is the testing result figure with the average potential of diagnosis and treatment excretion body, in which: A is excretion body, B is that electricity turns
Excretion body, C are that carry curcumin, D be that excretion body carries that the indoles mountain valley with clumps of trees and bamboo is green, E is that excretion body carries curcumin and the indoles mountain valley with clumps of trees and bamboo is green to excretion body;
Fig. 5 be vitro detection have the function of the testing result figure of the photo-thermal effect of diagnosis and treatment excretion body, in which: A be phosphate,
B is curcumin, C is that the indoles mountain valley with clumps of trees and bamboo is green, D is curcumin and the indoles mountain valley with clumps of trees and bamboo is green, E is excretion body, F is that excretion body carries curcumin, G is excretion
The body load indoles mountain valley with clumps of trees and bamboo is green, H is that excretion body carries curcumin and the indoles mountain valley with clumps of trees and bamboo is green;
Fig. 6 is the testing result figure with diagnosis and treatment excretion body cumulative in vitro drug release, in which: A is curcumin, B
For the indoles mountain valley with clumps of trees and bamboo is green, C is that excretion body carries curcumin, D is that excretion body carries that the indoles mountain valley with clumps of trees and bamboo is green, E is to carry curcumin and the green (turmeric of the indoles mountain valley with clumps of trees and bamboo
Element), F be excretion body carry curcumin and the indoles mountain valley with clumps of trees and bamboo it is green (the indoles mountain valley with clumps of trees and bamboo is green).
Specific embodiment
Below with reference to embodiment, the present invention is described in further detail, and embodiments of the present invention are not limited thereto.
In this application, it separates first, purify excretion body:
1) excretion body is extracted, is purified: it collects C57 mouse and fills interstital stem cell supernatant 200mL progress differential centrifugation:
300g, 10min take supernatant;2,000g, 10min take supernatant;10,000g, 30min take supernatant;100,000g, 70min are resuspended heavy
It forms sediment;100,000g, 70min are resuspended in 200 μ L 10mM PBS, and 30 μ L samples is taken to measure protein content for BCA;
2) curcumin solution is prepared: weighing 4mg curcumin powder, 200 μ L DMSO solutions are added, mixes well, uses 10mM
PBS adds to 1mL, is configured to 2mg/mL curcumin solution;
3) indocyanine green solution is prepared: weighing 4mg indocyanine green powder, 200 μ L DMSO solutions are added, mix well, use
10mM PBS adds to 1mL, is configured to 2mg/mL indocyanine green solution;
Then the drugs such as curcumin, indocyanine green are loaded by excretion body by electroporation again:
4) electricity turns: excretion body, curcumin solution, indocyanine green solution is added by a certain percentage in 10mM PBS solution;
It after mixing, is added in 96 porocyte culture plates, is put into electroporation apparatus, carry out electroporation according to certain reaction condition;
5) it is incubated for: 96 orifice plates being protected from light after perforation and are put into 37 DEG C of incubators, be incubated for 30min;
6) purify: twice through 100,000-140,000g ultracentrifugation, each 70-90min is gone the excretion body after perforation
Clearly, sediment is to have the function of diagnosis and treatment excretion body (the excretion body of carrying medicament);
7) fluorescence detection: by step 6) have the function of that diagnosis and treatment excretion body takes 10 μ L to be diluted to 100 μ L with 10mM PBS after
It is added drop-wise on phosphorimager, detects 700nm, 800nm fluorescent absorption value respectively;
8) Uv-vis is detected: step 6) is had the function of that diagnosis and treatment excretion body ultraviolet-visible spectrophotometer is distinguished
In 421nm detection curcumin, 784nm detection indocyanine green absorption value, and and standard curve control, computational envelope rate;
9) partial size and potentiometric analysis: take step 6) has the function of that diagnosis and treatment excretion body carries out partial size and potentiometric analysis, detection
Particle diameter distribution and current potential;
10) photo-thermal effect: step 6) is had the function of that diagnosis and treatment excretion body is added in 3mL silica dish, is swashed with 808nm is infrared
Light device radiates 5min, and every 30s records temperature change;
11) medicament slow release: take step 6) has the function of that diagnosis and treatment excretion body carries out cumulative in vitro release, calculates medicament slow release
Efficiency.
Embodiment 1
80 μ g excretion bodies are added in 10mM PBS solution, is added to after mixing in 96 orifice plates, is put into electroporation apparatus, according to
Following parameter manipulation: voltage 250V, 100 μ s of pulsewidth, 1000 μ s of interval, discharge time 6 times.Suspension can be put after electroporation
Enter and be incubated for 30min in cell incubator, then 100,000g ultracentrifugation twice, 70min/ times, removes supernatant, with 200 μ L PBS
Precipitating, the excretion body after obtaining electroporation is resuspended.Its partial size is~150nm, as shown in Figure 3.Zeta potential is~-8mV, is such as schemed
Shown in 4.4 DEG C are protected from light storage and carry out subsequent detection and experiment.
Embodiment 2
80 μ g excretion bodies and 80 μ L curcumin solution (concentration 2mg/mL) are added in 10mM PBS solution, add after mixing
Enter into 96 orifice plates, be put into electroporation apparatus, according to following parameter manipulation: voltage 250V, 100 μ s of pulsewidth are spaced 1000 μ s, electric discharge
Number 6 times.Suspension can be put into cell incubator after electroporation and be incubated for 30min, then 100,000g ultracentrifugation two
It is secondary, 70min/ times, supernatant is removed, it is the excretion body for loading curcumin that precipitating, which is resuspended, with 200 μ L PBS.Its encapsulation rate be~
86.64%, as shown in Figure 2;Its partial size is~170nm, as shown in Figure 3;Zeta potential is~-17mV, as shown in Figure 4;Preparation
The excretion body of obtained load curcumin has medicament slow release performance, as shown in Figure 6.4 DEG C be protected from light storage carry out subsequent detection and
Experiment.
Embodiment 3
80 μ g excretion bodies and 40 μ L indocyanine green solution (concentration 2mg/mL) are added in 10mM PBS solution, after mixing
It is added in 96 orifice plates, is put into electroporation apparatus, according to following parameter manipulation: voltage 250V, 100 μ s of pulsewidth are spaced 1000 μ s, put
Electric number 6 times.Suspension can be put into cell incubator after electroporation and be incubated for 30min, then 100,000g ultracentrifugation
Twice, 70min/ times, remove supernatant, with 200 μ L PBS be resuspended precipitating be load indocyanine green excretion body, encapsulation rate be~
97.79%, as shown in Figure 2;Partial size is~180nm, as shown in Figure 3;Zeta potential is~-6mV, as shown in Figure 4.It is prepared
The excretion body of load indocyanine green have fluorescence imaging, photo-thermal effect and medicament slow release performance, such as Fig. 1, shown in 5,6.4 DEG C are kept away
Light storage carries out subsequent detection and experiment.
Embodiment 4
80 μ g excretion bodies and 40 μ L curcumin solution (concentration 2mg/mL) and 40 μ L are added in 10mM PBS solution
Indocyanine green solution (concentration 2mg/mL) (curcumin: indocyanine green: the mass ratio of excretion body is 1:1:1), is added after mixing
Into 96 orifice plates, it is put into electroporation apparatus, according to following parameter manipulation: voltage 250V, 100 μ s of pulsewidth are spaced 1000 μ s, electric discharge time
Number 6 times.Suspension can be put into cell incubator after electroporation and be incubated for 30min, then 100,000g ultracentrifugation twice,
70min/ times, supernatant is removed, precipitating is resuspended with 200 μ L PBS and as carries curcumin/indocyanine green excretion body, curcumin packet altogether
Envelope rate is~87.87%, and indocyanine green encapsulation rate is~96.97%, as shown in Figure 2;Partial size is~200nm, as shown in Figure 3;
Zeta potential is~-15mV, as shown in Figure 4.The excretion body for the total load curcumin/indocyanine green being prepared have fluorescence at
Picture, photo-thermal effect and medicament slow release performance, such as Fig. 1, shown in 5,6.4 DEG C are protected from light storage and carry out subsequent detection and experiment.
Embodiment 5
80 μ g excretion bodies and 80 μ L curcumin solution (concentration 2mg/mL) and 40 μ L are added in 10mM PBS solution
Indocyanine green solution (concentration 2mg/mL) (curcumin: indocyanine green: the mass ratio of excretion body is 2:1:1), is added after mixing
Into 96 orifice plates, it is put into electroporation apparatus, according to following parameter manipulation: voltage 250V, 100 μ s of pulsewidth are spaced 1000 μ s, electric discharge time
Number 6 times.Suspension can be put into cell incubator after electroporation and be incubated for 30min, then 100,000g ultracentrifugation twice,
70min/ times, supernatant is removed, precipitating is resuspended with 200 μ L PBS and as carries curcumin/indocyanine green excretion body altogether.4 DEG C are protected from light storage
Deposit into row subsequent detection and experiment.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.