CN110393803A - A kind of taxol and polypeptide are total to delivery system, preparation method and application - Google Patents
A kind of taxol and polypeptide are total to delivery system, preparation method and application Download PDFInfo
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- CN110393803A CN110393803A CN201910726966.6A CN201910726966A CN110393803A CN 110393803 A CN110393803 A CN 110393803A CN 201910726966 A CN201910726966 A CN 201910726966A CN 110393803 A CN110393803 A CN 110393803A
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/10—Peptides having 12 to 20 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
- A61K47/6907—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a microemulsion, nanoemulsion or micelle
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
- A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
- A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
- A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
- A61K47/6939—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being a polysaccharide, e.g. starch, chitosan, chitin, cellulose or pectin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
Abstract
The disclosure belongs to anti-tumor drug delivery technique field, and in particular to a kind of taxol and polypeptide are total to delivery system, preparation method and application.Immunization therapy and chemotherapy are united and applied in oncotherapy with good effect in the prior art.Alloferon-1, a kind of basic peptide, some researches show that the polypeptides to promote function of immune system by the NK cell in activation tumor microenvironment in recent years.Inventor thinks Alloferon-1 and taxol combination being expected to have good therapeutic effect, in order to realize the total delivering of two kinds of drugs, present disclose provides a kind of Alloferon-1-Heparin/Protamine nanometers of PTX-DOTAP@total delivery systems, degradation based on -1 pair of heparin of acetic acid heparinase in tumor microenvironment, realization system is whole in the disintegration of tumor locus and the enrichment of drug, it is especially applied to the treatment of melanoma, there is good effect.
Description
Technical field
The disclosure belongs to anti-tumor drug delivery technique field, and in particular to a kind of PTX-DOTAP@Alloferon-1-
Heparin/Protamine is total to delivery system, preparation method and the application in anti-tumor drug preparation.
Background technique
The information for disclosing the background technology part is merely intended to increase the understanding to the general background of the disclosure, without certainty
It is considered as recognizing or implying in any form that information composition has become existing skill well known to persons skilled in the art
Art.
Melanoma originates from melanocyte, is the highest tumor kind of grade malignancy in skin neoplasin.Metastasis melanin tumor
The five year survival rate of patient is reduced to 17% from 98%.In general, tumor microenvironment (tumor microenvironment,
TME the matrix in) is mainly made of structural proteins and glycosaminoglycan, and the main component of the latter is heparan sulfate proteoglycan
(HSPGs).In TME, HSPG stores a large amount of biotic factors (such as b-FGF, VEGF and TGF-β), and these factors are in body
Have height strategic in this war between body and tumour.However, result is usually the heparan in tumor microenvironment
The degradation HSPG of enzyme -1, allows a large amount of releases of these biotic factors, activates subsequent signal transduction path, lead to new blood vessel shape
At epithelial-mesenchymal converts (EMT) and metastases.In addition, to also contribute to tumour thin in the matrix gap generated in tumor microenvironment
The invasion and transfer of born of the same parents.These are studies have shown that pernicious high metastatic tumo(u)r is often expressed as high-caliber heparitinase -1, especially
In melanoma.
For such malignant tumour, scientists propose various countermeasures and therapeutic scheme.Some researchers point out,
The cause of disease of not agnate melanoma is not fully consistent, therefore the corresponding measure taken of early prevention and inspection is not yet
Together.In addition there is research to update and improve melanoma method by stages, be conducive to clinically formulate more rationally effective treatment
Scheme improves curative effect of medication, reduces side effect.5 years melanoma therapeutic schemes are summarized over there are also some researchers
Variation, and be mutated melanoma patients for BRAF and propose reasonable first-line treatment scheme.In short, treating melanoma at present
Method mainly includes chemotherapy, molecular targeted therapy, immunologic test point blocking treatment and tumor vaccine therapy.
A kind of broad-spectrum anti-cancer drug of the taxol as classics, can inhibit the proliferation of tumour cell, is usually used in clinical treatment
Melanoma.Under normal operation, between micro-pipe and tubulin dimer there are dynamic equilibrium, but taxol can destroy it is this
Dynamic equilibrium induces tubulin polymerization and prevents depolymerization, and stablizes micro-pipe, causes cell that cannot be formed during mitosis
Spindle and then inhibition cell division and proliferation, and tumour cell is made to be stuck in the G2/M phase, to play antitumor action.So
And long-time service taxol may cause patient and taxol resistance phenomenon occurs.Fortunately, in recent years, the treatment of immunization therapy
Effect becomes more and more satisfactory in cancer treatment.Researcher attempts to use immunization therapy and chemotherapy drugs in combination, and
It was found that this treatment method substantially increases the curative effect of drug.
Alloferon-1, (HGVSGHGQHGVHG), a kind of basic peptide of 13 amino acid have strong when it is dissolved in water
Positive charge.The peptide is initially used for antiviral and antibacterium field, but in recent years it has also been found that it has good result to treatment of cancer.
Some reports also turn out that the basic peptide can discharge IFN- by natural kill (NK) cell in activation tumor microenvironment, NK cell
γ, TNF-beta and perforin are reversed by the immune system of inhibiting tumour cells, to show antitumor action.It is ground for above-mentioned
Study carefully background, inventor thinks: using free drug to melanoma carry out treatment have the defects that it is obvious, comprising: drug
Whole body distribution and indiscriminate attack can lessen the curative effect and normal bodily tissue is damaged.Meanwhile it being protected without carrier
The drug of shield will be directly by the neutralization of the phagocytosis of macrophage and conditioning molecule, the metabolic function and kidney of liver
Excretory function can also reduce half-life period of the drug in blood circulation.On can significantly being improved using nanoparticle delivery form
Defect is stated, increases medicine stability and drug in the enrichment of tumor locus.
Summary of the invention
For the studies above background, inventor thinks to share taxol and Alloferon-1 into the treatment in melanoma
It is expected to receive good therapeutic effect.However, since the target cell of taxol and Alloferon-1 are that tumour cell and NK are thin respectively
Born of the same parents, the total delivering for designing a kind of two kinds of drugs of nanosystems realization have technical difficulty.It is well known that heparitinase -1 can be with
Degradation heparan, further research, which demonstrates heparitinase -1 also, has the ability of degradation heparin, this is found to be offer one
There is kind the new support material of enzyme reaction to provide Research Thinking, be expected to realize the total delivering of two kinds of drugs.
Total delivery system of the disclosure according to the Research Thinking for taxol and Alloferon-1 expands research, obtains
To nanosystems (the PTX-DOTAP@of a kind of taxol cationic micelle and the assembling of Alloferon-1 compound polyelectrolyte
It Alloferon-1-Heparin/Protamine), is a kind of tumor microenvironment responsive nano grain based on heparin, it can
To complete the effect that taxol and Alloferon-1 are carried altogether and substep delivers.By chemotherapy in conjunction with immunization therapy, has and stablize
Property it is good, preparation process is simple, increase drug enrichment at tumour effect.
In order to realize the technical effect, the disclosure the following technical schemes are provided:
For the disclosure in a first aspect, providing a kind of compound polyelectrolyte, the compound polyelectrolyte raw material includes polypeptide
Class drug, protamine sulfate and heparin.
Preferably, the polypeptide drug is anti-tumor drug, is Alloferon-1 further.
Preferably, the heparin is heparin sodium.
Heparin sodium has lasting anti-thrombosis function, uses for a long time as anticoagulation medicine, in addition also has anti-flat
Sliding muscle cell multiplication, anti-inflammatory, antitumor and a variety of physiological activity such as antiviral, the sugar chain knot of area load high density negative electrical charge
Structure.Protamine sulfate is a kind of natural basic protein being mainly made of arginine, carries a large amount of positive charge, by with
Heparin sodium, which forms stable salt, makes heparin lose anticoagulation, is a pair of of antagonistic substance.The two is capable of forming stable compound
Object can improve the unstability of free polypeptide drugs for the transmitting of polypeptide drug to a certain extent.DOTAP is a kind of
It is commonly used for preparing the positively charged phospholipid molecule of cationic-liposome, can be used for capturing hydrophobicity by forming micella
PTX.By optimizing preparation process, the negatively charged pentalyte with appropriate particle size is completely covered positively charged
DOTAP micella is expected to form a kind of long circulating pharmaceutical carrier to form final electronegative nanoparticle.
Disclosure second aspect provides a kind of nanoparticle, and the nanoparticle for delivering taxol and Alloferon- altogether
1；In the nanoparticle, the taxol is taxol cationic micelle form, and the Alloferon-1 is to gather described in first aspect
Polymer electrolyte form.
Taxol has good therapeutic effect as a kind of cytotoxic anti-tumor drug, to solid tumor, but lacks target
Tropism, and it is water-soluble poor.German MediGene company develops a kind of taxol cationic-liposome, with taxol, 1,
2- dioleoyl -3- trimethylamine groups propane (1,2-dioleoyl-3-trimethylammonium-propane, DOTAP), 1,
2- dioleyl phosphatidyl choline (1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC) is raw material system
At being a kind of using tumor neogenetic blood vessels as the drug of target.The taxol that the disclosure provides delivers altogether with Alloferon-1 is
System is assembled by taxol cationic micelle and compound polyelectrolyte, and strong negative electricity is presented in entire nanoparticle surface, facilitates this
Nanoparticle realizes long circulating in blood.
In addition, due to expression heparitinase -1 high in tumor microenvironment, acetic acid heparinase -1 can degrade rapidly nanometer
Heparin in particle outer layer.The ion as caused by the electrolyte in gap spreads the depolymehzation process for having dominated nano particle, adjoint
The nucleoprotamine of concentration gradient release, and the degradation of heparin molecule also leads to the electrostatic attraction between heparin and nucleoprotamine
Power reduces.Finally, this series of factors will form positive feedback loop, accelerate the disintegration of nano particle.Electricity is thought in previous research
The ion diffusion of solution matter induction is an important factor for promoting charged drugs molecule release in compound polyelectrolyte, and the disclosure provides
Nanoparticle promote nano particle depolymerization and alloferon-1 to release by the synergistic effect of ion diffusion and heparitinase -1
It puts, more intelligently.
Nano particle will discharge immune system of the Alloferon-1 to activate NK cell to inhibit with reversing tumor cell.Separately
On the one hand, under the double action that enzymatic hydrolysis and ion are spread, the positively charged DOTAP exposure of nanoparticle core, partial size reduces
And be easier to penetrate into inside tumor in the nano particle of positive charge and be easier by the electronegative tumour cell endocytosis of height, from
And taxol is improved in the enrichment at tumour cell position, improve the therapeutic effect of chemicals.
The disclosure third aspect, provides the preparation method of nanoparticle described in second aspect, and the preparation method includes preparation
The preparation of taxol cationic micelle, Alloferon-1 compound polyelectrolyte and nanoparticle.
Disclosure fourth aspect provides nanoparticle described in compound polyelectrolyte and/or second aspect described in first aspect
Application in preparation of anti-tumor drugs.
Compared with prior art, the beneficial effect of the disclosure includes:
1, present disclose provides the modes of a kind of taxol and Alloferon-1 use in conjunction, by chemotherapy and are immunized
Treatment combines, and provides a kind of therapy that cell toxicity medicament is combined with immunotherapy medicaments, and clinical treatment effect can be improved
Fruit.
2, in order to realize the total delivering of two kinds of drugs, present disclose provides a kind of taxol cationic micelle with
The nano-delivery system of Alloferon-1 pentalyte assembling, the delivery system utilize -1 pair of heparin of heparitinase
Degradation makes nanosystems concentrate on tumor locus disintegration.The Alloferon-1 of exposure is realized to tumor microenvironment after disintegration
The activation of middle NK cell, and taxol is then easier to enter tumour cell under charge effect, reinforces therapeutic effect.It is especially right
In the highly expressed tumour of this heparitinase of melanoma, there is good therapeutic potential.
3, the preparation method of the disclosure hardly uses organic solvent, preparation method letter almost without chemical synthesis process
Easy row.The nano-particles size uniformity that this method is prepared is good, and can be used for industrial production and clinic with drugization
Purpose.
Detailed description of the invention
The Figure of description for constituting a part of this disclosure is used to provide further understanding of the disclosure, and the disclosure is shown
Meaning property embodiment and its explanation do not constitute the improper restriction to the disclosure for explaining the disclosure.
Fig. 1 is that PTX-DOTAP acts on tumour cell schematic diagram in the disclosure；
Fig. 2 is the preparation method and characterization of PTX-DOTAP@Alloferon-1-Heparin/Protamine in embodiment 1
Figure；
Wherein, Fig. 2 a) be PTX-DOTAP@Alloferon-1-heparin/Protamine preparation flow schematic diagram；
Fig. 2 b) reduce and the charge overturning phase for the granularity of PTX-DOTAP@Alloferon-1-heparin/Protamine
Between, PTX and alloferon-1 are distributed to different target cell schematic diagrames；
Fig. 2 c) it is screened for the optimal proportion of PTX-DOTAP@Alloferon-1-heparin/Protamine；
Fig. 2 d) be PTX-DOTAP@Alloferon-1-heparin/Protamine size distribution；
Fig. 2 e) it is distributed for the TEM photo of PTX-DOTAP@Alloferon-1-heparin/Protamine；
Fig. 2 f) be PTX-DOTAP@Alloferon-1-heparin/Protamine Zeta-potential.
Fig. 3 is the performance characterization result of PTX-DOTAP and Alloferon-1-Heparin/Protamine in embodiment 1
Figure；
Wherein, Fig. 3 a) it is protamine and heparin ratio gradient curve figure in embodiment 1；
Fig. 3 b) be embodiment 1 in Alloferon-1-Heparin/Protamine nanoparticle TEM photo figure；
Fig. 3 c) be embodiment 1 in Alloferon-1-Heparin/Protamine nanoparticle grain size distribution；
Fig. 3 d) be embodiment 1 in Alloferon-1-Heparin/Protamine nanoparticle potential image.
Fig. 3 e) be embodiment 1 in PTX-DOTAP nanoparticle grain size distribution；
Fig. 3 f) be embodiment 1 in PTX-DOTAP nanoparticle potential image.
Fig. 4 is the PTX-DOTAP@alloferon-1- in embodiment 1 when PTX-DOTAP content is slightly higher or excessively high
The partial size and TEM image of heparin/Protamine；
Wherein, Fig. 4 a) be (protamine+heparin): nanoparticle TEM schemes when DOTAP mass ratio is 4.2；
Fig. 4 b) it is (protamine+heparin): nanoparticle grain size distribution when DOTAP mass ratio is 4.2；
Fig. 4 c) be (protamine+heparin): DOTAP mass ratio is 3.0 nanoparticle TEM figure；
Fig. 4 d) be (protamine+heparin): DOTAP mass ratio is 3.0 nanoparticle grain size distributions.
Fig. 5 is PTX-DOTAP@Alloferon-1-heparin/Protamine release performance characterization result in embodiment 2
Figure；
Wherein, Fig. 5 a) it is B16F10 melanoma, the amount for the heparitinase -1 expressed in liver and normal skin tissue；
Fig. 5 b) at 37 DEG C presence or absence of the DTX that dissociates in the case where heparin enzyme, dissociate alloferon-1 and PTX-
The In-vitro release curves of DOTAP@Alloferon-1-heparin/Protamine (as a result n=3 is shown with average value ± SD)；
Fig. 5 c) it is the PTX-DOTAP@Alloferon- in nothing, enzyme, four kinds of buffer environments of electrolyte and enzyme+electrolyte
1-Heparin/Protamine shows obvious phenomenon, TEM photo and signal animation in different time.
Fig. 6 is the cell in vitro ingestion result figure of nano particle in embodiment 3:
Fig. 7 is nanoparticle in vitro toxicity testing result figure in embodiment 4；
Fig. 8 is internal distribution results figure after being administered in embodiment 5；
Wherein, Fig. 8 a) it is that fluorescent image, dotted line indicate mouse tumor stove in Mice Body after intravenous (IV) drug.
Fig. 8 b) it is drug distribution map in isolated viscus；
Fig. 8 c) it is isolated viscus drug content histogram.
Fig. 9 is the immune system result figure of inhibiting tumour cells in tumor microenvironment in embodiment 5；
Wherein, Fig. 9 a) be each group mouse peripheral blood in IFN-γ level variation histogram；
Fig. 9 b) be each group mouse peripheral blood in TNF-α level variation histogram；
Fig. 9 c) it is NKG2D content histogram in each group mice tumor sections；
Fig. 9 d) it is CD94 content histogram in each group mice tumor sections.
Figure 10 is mouse tissue Pathological findings in embodiment 5；
Specific embodiment
It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the disclosure.Unless another
It indicates, all technical and scientific terms used herein has usual with disclosure person of an ordinary skill in the technical field
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to restricted root
According to the illustrative embodiments of the disclosure.As used herein, unless the context clearly indicates otherwise, otherwise singular
Also it is intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet
Include " when, indicate existing characteristics, step, operation, device, component and/or their combination.
As background technique is introduced, immunization therapy and chemotherapy are united and applied in oncotherapy in the prior art
With good effect.Alloferon-1, a kind of basic peptide, some researches show that the polypeptides in recent years can be micro- by activation tumour
NK cell in environment promotes function of immune system.Inventor thinks to be expected to have with taxol combination by Alloferon-1 good
Good therapeutic effect, in order to realize the total delivering of two kinds of drugs, present disclose provides a kind of PTX-DOTAP@Alloferon-1-
Heparin/Protamine nanometers of total delivery systems, based on the degradation of -1 pair of heparin of acetic acid heparinase in tumor microenvironment,
Realization system is whole in the disintegration of tumor locus and the enrichment of drug.
For the disclosure in a first aspect, providing a kind of compound polyelectrolyte, the compound polyelectrolyte raw material includes polypeptide
Class drug, protamine sulfate and heparin.
Preferably, the polypeptide drug is anti-tumor drug, is Alloferon-1 further.
Preferably, the heparin is heparin sodium.
Disclosure second aspect provides a kind of nanoparticle, and the nanoparticle for delivering taxol and Alloferon- altogether
1；In the nanoparticle, the taxol is taxol cationic micelle form, and the Alloferon-1 is to gather described in first aspect
Polymer electrolyte form.
In some embodiments, the taxol cationic micelle is made of taxol, DOTAP.
In some embodiments, the raw materials and consumption of the nanoparticle is as follows: taxol 1.200%-1.600%, DOTAP
15.000%-19.000%, protamine sulfate 9.000%-13.000%, Alloferon-1 0.150%-0.450%, liver
Plain sodium 60.000%-80.000%.
It further, is taxol 1.300%-1.500%, DOTAP 16.000%-18.000%, sulfuric acid milt egg
White 10.000%-12.000%, alloferon-1 0.200%-0.400%, heparin sodium 65.000%-75.000%.
In the particular embodiment, the dosage of nanoparticle is as follows: taxol 1.401%, DOTAP 16.853%, sulfuric acid fish
Protamine 11.715%, alloferon-1 0.321%, heparin sodium 69.709%.
The disclosure third aspect, provides the preparation method of nanoparticle described in second aspect, and the preparation method includes preparation
The preparation of taxol cationic micelle, Alloferon-1 compound polyelectrolyte and nanoparticle.
In some embodiments, the taxol cationic micelle the preparation method is as follows: taxol and DOTAP are dissolved in
In chloroform, revolving removes solvent, ultrasonic aquation at room temperature；Further, ultrasonic hydration temperature is 20~40 DEG C, ultrasonic time
For 30~60s.
In some embodiments, the Alloferon-1 compound polyelectrolyte the preparation method is as follows: by sulfuric acid milt
Albumen is slowly added dropwise into heparin sodium aqua and is stirred after mixing with Alloferon-1 and both obtained.
In some embodiments, the preparation step of the nanoparticle is as follows: at room temperature, by the taxol prepared cation
It is added dropwise in Alloferon-1 compound polyelectrolyte to stir and both obtain；Further, dialysis removes excessive Alloferon-1-
Heparin/ nucleoprotamine.
Disclosure fourth aspect provides nanoparticle described in compound polyelectrolyte and/or second aspect described in first aspect
Application in preparation of anti-tumor drugs.
In some embodiments, the anti-tumor drug is anti-melanin tumor medicine.
In order to enable those skilled in the art can clearly understand the technical solution of the disclosure, below with reference to tool
The technical solution of the disclosure is described in detail in the embodiment and comparative example of body.
Transmission electron microscope photo is JEM-2100 transmission electron microscope (Jeol Ltd. in following embodiment
JEOL it) shoots.Granularmetric analysis experiment uses Zetasizer Nano ZS partial size potentiometric analyzer (Britain, Malvem company).
The preparation of 1 PTX-DOTAP of embodiment
1. single factor experiment
The present embodiment for PTX-DOTAP preparation condition investigated, investigation factor include medicine rouge ratio, lipid concentration,
Hydration temperature, ultrasonic time, ultrasonic power and revolving six factors of bath temperature.
1.1 medicine rouge ratios
For the determination of mass ratio between PTX and DOTAP, the present embodiment has carried out 1:5,1:10,1:15,1:20,1:25
(PTX:DOTAP) screening of this five ratios, is successively evaluated with encapsulation rate and drugloading rate.
When PTX:DOTAP is after 1:10, encapsulation rate just has been above 90%, and growth then is little, but it carries medicine
Amount but can therefore sharp fall, so 1:12,1:15,1:18 should be selected further to be screened, and subsequent investigation
It all should be based on drugloading rate.
1.2 lipid concentration
For the determination of DOTAP mass concentration, the present embodiment has been carried out to 0.5,1,2,3,4mg/ml this five sieves measured
Choosing, using drugloading rate as evaluation index.When DOTAP lipid concentration has best drugloading rate in 2mg/ml or so, so should select
1.5,2.0,2.5mg/ml is selected further to be screened.
1.3 hydration temperature
The determination of hydration temperature when for ultrasound, the present embodiment select 20,30,40,50,60 DEG C of this five amounts to be sieved
Choosing, using drugloading rate as inspection target.When ultrasound hydration temperature at 30 DEG C or so with best drugloading rate, so should select
25,30,35 DEG C are selected further to be screened.
1.4 ultrasonic time
For the determination of ultrasonic time, the present embodiment has selected 20s, 40s, 1min, 5min, 10min, 15min this five
Amount is screened, using drugloading rate as inspection target.When ultrasonic time is in 40s or so with best drugloading rate, so should
30s is selected, 40s, 50s are further screened.
1.5 ultrasonic power
For the determination of ultrasonic power, the present embodiment has selected 100,80,60,40, and 20% this five amounts are screened, with
Drugloading rate is inspection target, and the liposome drugloading rate of different ultrasonic power preparation does not change substantially, the variation of ultrasonic power
On drugloading rate substantially without influence.
1.6 revolving bath temperatures
For the determination of revolving bath temperature, the present embodiment has selected 20,30,40,50,60 DEG C of this five amounts to be sieved
Choosing, using drugloading rate as inspection target, temperature is changed to 60 DEG C from 20 DEG C, but drugloading rate does not change substantially, it was demonstrated that revolving water
The variation of bath temperature will not be on drugloading rate substantially without influence.
2. orthogonal design optimization formulation
Experiment of single factor the result shows that, to PTX-DOTAP@Alloferon-1-Heparin/protamine nanoparticle shadow
Sound is biggish because being known as: medicine rouge ratio (A), lipid concentration (B), hydration temperature (C), ultrasonic time (D) pass through orthogonal design L9 (3
^4) table, each prescription of parameter evaluating as investigation, following table are the investigation result of factor level table and orthogonal design respectively.
1 orthogonal experiment factor of table and water-glass
2 Orthogonal experiment results of table
Final prescription is determined in conjunction with orthogonal test and experiment of single factor are as follows: PTX:DOTAP 1:12, DOTAP lipid concentration
For 1.5mg/ml, hydration temperature is 35 DEG C, ultrasonic time 50s.When the ratio of drug and lipid is 1:12, PTX-DOTAP
With preferable drugloading rate (7.67% ± 0.01) and encapsulation rate (99.70% ± 0.15), lipid concentration 1.5mg/ml, aquation
Temperature is 35 DEG C, and ultrasonic time is 50 seconds.Partial size is 52.70 ± 1.491nm (PDI:0.163 ± 0.012), zeta potential 44.5
±0.872mV.The electron micrograph of PTX-DOTAP shows round and uniform partial size.
The ratio of 3.Alloferon-1-Heparin/protamine is screened
In the present embodiment, screened for the ratio of Alloferon-1-Heparin/protamine, wherein
The sequence of Alloferon-1 is as shown in SEQ ID NO:1.Under stirring at room temperature, by 0.3mg/ml nucleoprotamine be slowly added dropwise into
Into 0.3mg/ml heparin, the ratio gradient curve of protamine and heparin a series of is designed.With Alloferon-1-
The potential change of Heparin/protamine is evaluation index, as a result as shown in the table:
The ratio of 3 Heparin/protamine of table is screened
As shown in Fig. 2 c), when protamine:heparin mass ratio is 0.125, the ratio of formulation ingredients reaches full
With.
The component ratio of 4.PTX-DOTAP@Alloferon-1-Heparin/protamine nanoparticle is screened
In the present embodiment, under stirring at room temperature, by PTX-DOTAP (0.5ml 1.5mg/ml DOTAP) be slowly added dropwise into
In 0.3mg/ml Alloferon-1-Heparin/protamine, design a series of (protamine+heparin) with
The ratio gradient curve of DOTAP.It is evaluation with the potential change of PTX-DOTAP@Alloferon-1-Heparin/protamine
Index is as a result as follows:
4 heparin bead-DOTAP ratio of table screens table
As shown in Fig. 3 a), as (protamine+heparin): DOTAP mass ratio is at 4.85, the ratio of formulation ingredients
Reach saturation.At this point, the electron micrograph of nano particle shows uniform shape and partial size, it is not extra in the background
heparin/Protamine。
The characterization of preparation when 5.DOTAP ratio is on the high side
The present embodiment has selected (protamine+heparin): DOTAP mass ratio is carries out when 4.2 and 3.0 two ratios
The preparation of PTX-DOTAP@Alloferon-1-Heparin/protamine nanoparticle, and the dialysis band of 1000KD is not used
Remove extra Alloferon-1-Heparin/protamine nanoparticle.
As shown in figure 3, the partial size of nanoparticle can become very big when the ratio of DOTAP in preparation increases, it is more than
200nm, even up to 500nm, and partial size is very inhomogenous, there are many more the Alloferon-1-Heparin/ not being removed
Protamine nanoparticle.
6.Alloferon-1-Heparin/protamine nanoparticle physicochemical
6.1 form
Take appropriate Alloferon-1-Heparin/protamine nanoparticle solution in cillin bottle, observing its appearance is in
Existing light blue opalescence.One to two drop Alloferon-1-Heparin/protamine nanoparticle solution are taken again, it is appropriate with aquae destillata
Dilution is added dropwise on copper sheet, is dyed with 2% Salkowski's solution, dries 30min, microcosmic by transmission electron microscope observation
Form.Alcohol plastid form rounding under TEM, surface is smooth not to be adhered, and particle size distribution is uniform.
6.2 partial sizes and potential
Take the Alloferon-1-Heparin/protamine nanoparticle solution prepared appropriate, after diluting suitable multiple
Use dynamic light scattering measurement with Malvern laser particle analyzer measure partial size for 26.19 ± 1.631nm (PDI:0.146 ±
0.018)；Zeta current potential is stablized in-42.4 ± 0.246-mV.
The preparation of 7.PTX-DOTAP@Alloferon-1-Heparin/Protamine
(1) preparation of PTX-DOTAP
0.550ml 1mg/ml PTX (being dissolved in chloroform) and 6.616ml 1mg/ml DOTAP (are dissolved in chloroform
In) uniformly mixing, then rotary evaporation 15 minutes at room temperature.Then, at a certain temperature under ultrasonic wave by 4.411ml water
It is slowly added in flask, and is ultrasonically treated certain time.
According to the result of study of single factor exploration, in the step, ultrasonic hydration temperature can be selected in 20~40 DEG C,
Ultrasonic time is selected in 30~60s, is able to achieve good load drug effect fruit.
(2) preparation of Alloferon-1-Heparin/ nucleoprotamine compound polyelectrolyte
After 15.33ml 0.3mg/ml protamine sulfate and 0.42ml 0.3mg/ml alloferon-1 are mixed, In
It is stirred at room temperature and lower mixture is slowly dropped in 91.215ml 0.3mg/ml heparin sodium aqua and is stirred 30 minutes
(600rpm)。
(3) PTX-DOTAP@Alloferon-1- heparin/nucleoprotamine is prepared
It is stirred at room temperature down, 4.411ml PTX-DOTAP is slowly dropped to 106.97ml Alloferon-1- liver
In element/nucleoprotamine compound polyelectrolyte, and stir 10 minutes (200rpm).Then it dialyses 10 points in 1000KD bag filter
Clock is to remove Alloferon-1-Heparin/ nucleoprotamine slightly excessive.
8.PTX-DOTAP@Alloferon-1-Heparin/protamine nanoparticle physicochemical
8.1 form
It takes appropriate PTX-DOTAP@Alloferon-1-Heparin/protamine nanoparticle solution in cillin bottle, sees
It examines its appearance and light blue opalescence is presented.One to two drop PTX-DOTAP@Alloferon-1-Heparin/protamine are taken to receive again
Grain of rice solution is suitably diluted with aquae destillata, is added dropwise on copper sheet, is dyed with 2% Salkowski's solution, 30min is dried, by saturating
Penetrate electron microscope observation micromorphology.Alcohol plastid form rounding under TEM, surface is smooth not to be adhered, and particle size distribution is equal
It is even.
8.2 partial sizes and potential
It takes the PTX-DOTAP@Alloferon-1-Heparin/protamine nanoparticle solution prepared appropriate, dilutes
Used after suitable multiple dynamic light scattering measurement with Malvern laser particle analyzer measure partial size for 106.1 ± 1.113nm (PDI:
0.147±0.005)；Zeta current potential is stablized in -45.1 ± 0.455mV.
The release in vitro behavior of 2 PTX-DOTAP@alloferon-1-heparin/Protamine of embodiment
In the present embodiment, for the release in vitro behavior of PTX-DOTAP@alloferon-1-heparin/Protamine
It is studied.Tumor microenvironment is simulated using 0.01nM Tris-HCl (pH=6.5), uses 20nM Ca2+Simulate tumour micro-loop
Ca in border2+Concentration simultaneously enhances -1 activity of heparitinase.It is shown according to Fig. 5 b), dissociate alloferon-1's and free PTX
Burst size is respectively 85.22 ± 0.82% and 90.01 ± 1.41% in 6 hours respectively, and above-mentioned two groups have been rapidly completed release.
The PTX-DOTAP@alloferon-1-heparin/Protamine group of 50 μ l0.0833IU/ml heparinases was discharged from 0 hour
Alloferon-1, rate of release significant reduction at 10 hours.PTX group discharges rapidly after 2 hours, because of nano particle
PTX in core is not discharged, release speed suddenly by alloferon-1-heparin/Protamine layers of protection
Rate significant reduction at 16 hours.Finally, the burst size of PTX and alloferon-1 is respectively 83.82 ± 1.12% and 81.67
± 0.76%.Not plus in the PTX-DOTAP@alloferon-1-heparin/Protamine group of heparinase, entirely discharging
The alloferon-1 that 12.77 ± 1.58% PTX and 8.43 ± 1.79% is only discharged in experiment, shows PTX-DOTAP@
Alloferon-1-heparin/Protamine nanoparticle can provide outstanding protection for internal drug.As Fig. 5 c) is shown
Every group of the apparent phenomenon and electron micrograph during release, wherein the presence of nano particle makes solution show pale blue
The opalescence of color.When 5min, enzyme group and enzyme+electrolyte group are become cloudy, and generate insoluble White Flocculus, show alloferon-
1-heparin/Protamine (outermost layer of PTX-DOTAP@alloferon-1-heparin/Protamine nano particle)
Enzymatic hydrolysis, and heparin is gradually discharged, expose a part of positively charged nucleoprotamine.After 6 hours, aggregates of nanoparticles
Color become whiter, show the alloferon-1- of PTX-DOTAP@alloferon-1-heparin/Protamine preparation
Heparin/Protamine layers by heparinase it is significant degradation and pass through ion diffusive separation.At 6 hours, electrolyte group became
It is very muddy, and solution contains a large amount of white cotton fibers.This is because ion caused by electrolyte is spread, therefore PTX-DOTAP@
The alloferon-1-heparin/Protamine layer of alloferon-1-heparin/Protamine also slowly dissociates, surface
The nanoparticle aggregate of discharge is together.However, without the participation of heparin enzyme, the significant reduction of the progress of this physical phenomenon.
At 12 hours, enzyme+electrolyte group became very limpid, and the white flocculate in solution almost disappears.Electron microscopic shines
Piece also shows that the solution only contains the individual nanocrystals particle that a small amount of partial size is about 50nm.This is heparin enzyme and ion diffusion phase
In conjunction with as a result, leading to the alloferon-1- of PTX-DOTAP@alloferon-1-heparin/Protamine preparation
Heparin/Protamine layers are almost detached from, exposed inner DOTAP core.Measurement result shows nanometer in solution at this time
The granularity and current potential of particle are respectively 59.30 ± 0.783nm (PDI:0.234 ± 0.032) and 25.4 ± 0.257mV.According to releasing
The burst size of PTX and alloferon-1 that experiment measures are put, the ion diffusion of independent electrolyte cannot promote PTX-DOTAP@
The depolymerization of alloferon-1-heparin/Protamine.Only heparinase and the synergistic effect of ion diffusion causes preparation several
It will be completely dissociated, realize that the granularity of nano particle reduces and charge is converted.Therefore, when nano particle reaches tumor locus,
Particle size, which reduces process, changes the particle size of nano particle to 50nm from about 100nm, enters receiving for tumour to increase
The amount of rice grain.
The cellular uptake of 3 PTX-DOTAP@alloferon-1-heparin/Protamine of embodiment is assessed
Using B16F10 cell it is model evaluation tumour cell to the intake effect of nanoparticle in the present embodiment, passes through cumarin
6 indicator cells absorb situation.Fluorescence intensity is measured by fluorescence microscope.As shown in fig. 6, free C6 group, that is, blank group fluorescence is strong
It spends minimum.The fluorescence intensity of C6-DOTAP group is most strong, shows in tumor microenvironment, PTX-DOTAP@alloferon-1-
Heparin/Protamine can expose positively charged DOTAP under enzymatic degradation effect, increase the cell of nano particle
Ingestion efficiency.
In addition, coumarin 6-HEP NP and heparin preincubate+coumarin 6-HEP NP are much higher than the intake of nano particle
Free coumarin 6.Cellular uptake coumarin 6-HEP NP is not influenced with heparin pre-incubation cell, shows that heparin molecule does not have tumour
Targeting.Finally, carrying out the intake of coumarin 6-CS NP and coumarin 6-HA NP.Compared with coumarin 6-HEP NP, as a result show
Show that hyaluronic acid and chondroitin sulfate have certain targeting to tumour, and enhances tumour cell and nano particle is taken the photograph
It takes.
The vitro cytotoxicity of 4 PTX-DOTAP@alloferon-1-heparin/Protamine of embodiment measures
In the present embodiment, using Cell counting Kit (CCK) -8 (Cell Counting Kit-8, BestBio,
Shanghai, China), measure the vitro cytotoxicity of different nano particles.
By B16-F10 cell inoculation in 96 orifice plates, 37 DEG C, 6000 cells/wells are trained in 100 μ L complete mediums
It supports overnight.By blank NP (DOTAP@heparin/Protamine), dissociate PTX, and dissociate alloferon-1, and dissociate combination, PTX
NP (PTX-DOTAP@heparin/Protamine), alloferon-1 NP (DOTAP@alloferon-1-heparin/
Protamine), PTX/alloferon-1 NP (PTX-DOTAP@alloferon-1-heparin/Protamine) and control
Group (isometric blank complete medium is added) is added with the paclitaxel concentration of 0.003,0.03,0.3,3,30 and 300 μ g/mL
(concentration of alloferon-1 is 1/8th of taxol) and 0.14,1.4,14,140 and 1400 μ g/mL blank are received in each hole
Rice grain, and in 3 DEG C of incubation 5%CO2Continue 24 hours or 48 hours.Before analysis, CCK-8 solution (10 hole μ L/) is added,
Then it is further incubated for 1 hour.Maximum absorbance is set as 450nm, passes through microplate reader (BioTek Synergy H1, BioTek
Instruments, Inc., Winooski, VT, USA) each hole of scanning optical density (OD).
Versus cell survival rate (RCV) (%) is calculated as RCV (%)=OD test/OD control × 100%, and wherein OD is tested
The OD of the cell with test group and control group processing is respectively represented with OD control.Will be the independent repetition of experiment six times, and use
The half maximum suppression concentration (IC50) of 7.0 software of GraphPad Prism calculating test group.
In order to explore the characteristic induced cell apoptosis in vitro, by B16-F10 cell with 2 × 105The density of a cells/well
It is seeded in 12 orifice plates.After co-culturing from different groups of free drug or nano particle, pass through flow cytometry
(3.0 software of CytExpert) detects in 30 minutes.
B16-F10 is inoculated in 6 orifice plates, inoculum density is 5 × 105Different groups of free drug is added in a cells/well
Or nano particle processing.After incubating 24 hours, collects cell and pass through flow cytometry (FACSCalibur, BD
Biosciences, Franklin Lakes, NJ, USA) analysis propidium iodide intensity and distribution.Use 3.1 software of ModFit
Analysis and processing data.Result of study shows the dosage of comparable sodium, and PTX/alloferon-1 NP group has strongest
Cytotoxicity can significantly reduce tumor cell activity, and as the raising inhibiting effect of Drug level also enhances therewith.Respectively
The influence of group drug cell cycle is as shown in fig. 7, PTX has blocked the cell generation cycle of G2/M phase, and PTX/
Alloferon-1 NP then further increases intake of the cell to drug.
The measurement of embodiment 5 tumor xenograft mouse model and mouse bio distribution
All internal mouse experiments are ratified through Shandong University's animal protection and using the committee.With 1 × 106Quantity exists
Veutro inoculates B16-F10 cell on the right side of C57BL/6 mouse (6-8 week old).When gross tumor volume reaches 200mm3When, by B16-
F10 tumor-bearing mice is grouped (n=3) at random, is injected intravenously free DiR, DiR-HEP/DOTAP (DiR-) DOTAP@heparin/
Protamine), DiR-DOTAP and DiR-HA/DOTAP (DiR-DOTAP@hyaluronic acid/Protamine), dosage are 100 μ g/
kg。
1 hour after administration, 4 hours, 12 hours and 24 hours, anesthetized mice.Pass through Xenogen IVIS Lumina system
System (Caliper Life Sciences, Waltham, MA, USA) obtains realtime graphic.Mouse is put to death after 24 hours, harvests the heart
Dirty, lung, liver, spleen, kidney and tumour for being imaged in vitro.In 4.1 software of Living Image (Caliper Life
Sciences, Waltham, MA, USA) in analyze image.As a result as shown in figure 8, DiR-HEP/DOTAP and DiR-HA/DOTAP
Certain accumulation of the group at 24 hours with good long circulating ability and at tumour.As a result it is also shown that free DiR, DiR-
DOTAP and DiR-HA/DOTAP is accumulated with more major organs, and there is DiR-HEP/DOTAP less liver to accumulate
(Fig. 8 b)).
(1) antitumor research in vivo
In order to assess antitumor efficacy and the safety of nano particle, by 1 × 106B16-F10 cell subcutaneous injection is to often
In C57BL/6 mouse.When gross tumor volume reaches 100mm3When, mouse is randomly divided into 8 groups (n=8), 100 μ are injected intravenously
LPBS, blank NP (DOTAP@heparin/Protamine), free PTX, free alloferon-1, free combination, PTX NP
(PTX-DOTAP@heparin/Protamine), alloferon-1 NP (DOTAP@alloferon-1-heparin/
) and PTX/alloferon-1 NP (PTX-DOTAP@alloferon-1-heparin/Protamine) taxol Protamine
Dosage is 10mg/kg, and alloferon-1 dosage is 1.25mg/kg.Every 3 days measurement gross tumor volumes and weight are anti-in vivo to observe
Tumor efficacy and toxicity.After administration, collects tumour and organ (heart, liver, spleen, lung and kidney) is fixed for
TUNEL, Ki67 and heparanese-1 dyeing.Using GraphPad Prism 7.0, after Kaplan-Meier CNN surviving fraction
Analyze the time-to-live of mouse.Result of study shows that PTX-DOTAP@Alloferon-1-Heparin/Protamine can enhance
Tumor suppression simultaneously delays tumour growth.
(2) immune-related In vivo study
In order to explore NK cell and internal CD8+T cell activation, the present embodiment passes through granzyme B, NKG2D, CD94, IFN-
γ and TNF-α assess the anti-tumor immune response of alloferon-1 induction.To carry the C57BL/6 mouse of B16-F10 as mould
Type puts to death mouse after being injected intravenously different preparations, obtains tumor tissues and carries out immunohistochemical staining, and using corresponding
ELISA kit test Peripheral Blood immune factor.Anti-mouse mAb is used in this study.Particle-resistant enzyme B, anti-CD8+,
Anti- NKG2D, anti-CD94, anti-IFN-γ, anti-tnf-alpha, IFN-γ ELISA and TNF-α ELISA purchased from Abcam (Cambridge,
) and HuaBio (Hangzhou China) UK.Use laser scanning co-focusing microscope (LSM 780, Carl Zeiss, Oberkochen
Germany image) is obtained.Wherein, in mouse peripheral blood IFN-γ and TNF-α level variation such as Fig. 9 a) and b) shown in, tumour
The immunofluorescence results of NKG2D and CD94 such as Fig. 9 c in slice) and it is d) shown.
(3) toxicity research of the PTX-DOTAP@alloferon-1-heparin/Protamine to major organs
In order to check the safety of nano particle in vivo, by fixed vitals (heart, liver, spleen, lung and kidney
It is dirty) it is embedded in paraffin, it is sliced, hematoxylin and eosin dyeing.Use VS120 virtual slide microscope (Olympus
Corp., Tokyo, Japan) it observes the slice of dyeing and takes pictures.Using alanine aminotransferase (ALT) ELISA kit,
Aspartate aminotransferase (AST) ELISA kit and blood urea nitrogen (BUN) ELISA kit test Peripheral Blood index,
Testing result is as shown in table 5 and Figure 10.
AST, ALT, and BUN content (n=3) in the different medication group serum of table 5
ALT, alanine aminotransferase；AST, aspartate aminotransferase；BUN, blood urea nitrogen
(4) it statisticallys analyze
Data are expressed as average value ± standard deviation.Single factor test and two-way ANOVA are used for Multiple range test.Comparing
It tests after carrying out Bonferroni when having group, and is examined when comparing two groups using double tail t.It is repeated in two independent experiments
In-vivo tumour Therapy study is to ensure enough sample sizes and reproducibility.All statistical analysis use GraphPad Prism
It is carried out with 19.0 software of SPSS.It is as follows to count significant property: p < 0.01 p < 0.05, * * * and p < 0.001 * * *.
The foregoing is merely preferred embodiment of the present disclosure, are not limited to the disclosure, for the skill of this field
For art personnel, the disclosure can have various modifications and variations.It is all within the spirit and principle of the disclosure, it is made any to repair
Change, equivalent replacement, improvement etc., should be included within the protection scope of the disclosure.
Claims (10)
1. a kind of compound polyelectrolyte, which is characterized in that the compound polyelectrolyte raw material includes polypeptide drug, sulfuric acid
Nucleoprotamine and heparin.
2. compound polyelectrolyte as described in claim 1, which is characterized in that the polypeptide drug is anti-tumor drug,
It preferably, is Alloferon-1；Or the heparin is heparin sodium.
3. a kind of nanoparticle, which is characterized in that the nanoparticle for delivering taxol and Alloferon-1 altogether；The nanoparticle
In, the taxol is taxol cationic micelle form, and the Alloferon-1 is polymer electrolytic described in claim 2
Matter form.
4. nanoparticle as claimed in claim 3, which is characterized in that the taxol cationic micelle is by taxol, DOTAP system
At.
5. nanoparticle as claimed in claim 3, which is characterized in that the raw materials and consumption of the nanoparticle is as follows: taxol
1.200%-1.600%, DOTAP 15.000%-19.000%, protamine sulfate 9.000%-13.000%,
Alloferon-1 0.150%-0.450%, heparin sodium 60.000%-80.000%；It preferably, is taxol 1.300%-
1.500%, DOTAP 16.000%-18.000%, protamine sulfate 10.000%-12.000%, alloferon-1
0.200%-0.400%, heparin sodium 65.000%-75.000%.
6. the preparation method of any one of the claim 3-5 nanoparticle, which is characterized in that the preparation method includes that preparation is purple
The preparation of China fir alcohol cationic micelle, Alloferon-1 compound polyelectrolyte and nanoparticle.
7. the preparation method of nanoparticle as claimed in claim 6, which is characterized in that the preparation side of the taxol cationic micelle
Method is as follows: taxol and DOTAP being dissolved in chloroform, revolving removes solvent, ultrasonic aquation at room temperature；Preferably, ultrasonic water
Changing temperature is 20~40 DEG C, and ultrasonic time is 30~60s.
8. the preparation method of nanoparticle as claimed in claim 6, which is characterized in that the Alloferon-1 polyelectrolyte is compound
Object the preparation method is as follows: being slowly added dropwise after protamine sulfate is mixed with Alloferon-1 into heparin sodium aqua and being stirred
It mixes and both obtained.
9. the preparation method of nanoparticle as claimed in claim 6, which is characterized in that at room temperature, by the taxol sun prepared from
Sub- micella is added dropwise in Alloferon-1 compound polyelectrolyte to stir and both obtain；Further, dialysis removes excessive
Alloferon-1-Heparin/ nucleoprotamine.
10. providing any one of the compound polyelectrolyte and/or claim 3-5 as claimed in claim 1 or 2 nanoparticle making
Application in standby anti-tumor drug；Preferably, the anti-tumor drug is anti-melanin tumor medicine.
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| WO2021072349A1 (en) * | 2019-10-11 | 2021-04-15 | Tobebio Novel Drug Co., Ltd. | Synergistic combination of chemotherapy and peptide for treating cancer |
| CN116983431A (en) * | 2023-08-30 | 2023-11-03 | 山东大学 | Lipid nanoparticle for encapsulating H-mRNA and preparation method and application thereof |
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