CN107028882A - The cancer target nanoscale medicine delivery system and preparation method and application of a kind of physically encapsulation - Google Patents
The cancer target nanoscale medicine delivery system and preparation method and application of a kind of physically encapsulation Download PDFInfo
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Abstract
The invention discloses the cancer target nanoscale medicine delivery system and preparation method and application of a kind of physically encapsulation, its low waist system includes the peptide modified targeting elements of RGERPPR, nano-micelle and antineoplastic, the targeting element are DSPE polyethylene glycol RGERPPR；The micella is the glutamic acid side chain on polyglutamic acid PGG macromolecular scaffolds and skeleton；The antineoplastic is taxol PTX.The glutamic acid branch terminals of polyglutamic acid macromolecular scaffold are bonded with antineoplastic PTX by ester bond, form PGG PTX, then the DSPE in PTX and targeting element passes through hydrophobic interaction, targeting element hydrophobic side DSPE parcel is entered in PGG PTX, targeting anti-tumor nanoscale medicine delivery system is formed.This delivery system can be used as the targeted delivery of anti-tumor medicine, and obtain good curative effect on treatment glioma, extend the median survival interval of BALB/C glioma nude mices.
Description
Technical field
The invention belongs to field of pharmaceutical preparations, specifically realize targeting element modification in nanometre glue with physically encapsulation effect
Preparation method and use in beam medicine.
Background technology
Glioma is one of most common brain tumor, accounts for the 40% of the brain tumor incidence of disease, its deterioration degree and art
High recurrence rate and invasion and attack speed is fast, causes glioma patient's survival period short afterwards：I phase being clinically diagnosed to be or II phase disease
People's time-to-live is 6-8, and III phase patient is 3 years, and IV phase patient typically only has 12-18 months.It is used as intracranial tumors, nerve
Glioma has many unique places, and its tumour formation and development has the various barriers of complexity, such as enzyme barrier, blood-brain barrier
(BBB) and tumour blood-brain barrier (BBTB), hinder medicine or delivery system reaches tumor locus.Brain colloid is treated at present
The method of knurl mainly has operation, radiation and chemotherapy.But because glioma is in infiltrative growth, and more close to central nervous system
System, therefore be difficult to, by complete tumor resection of performing the operation, be easily caused tumor recurrence.It is dull-witted that radiation therapy easily causes radioactivity；It is conventional
Chemotherapeutics makes it to glioma therapeutic effect because often leading to serious toxic side effect to tumor tissues non-selectivity
It is not good, have a strong impact on the quality of life of patient.
Cancer target nanoscale medicine delivery system refers to that nano-carrier contains swollen using tumor tissues special physiological and pathological feature
Knurl is diagnosed or medicine is built-up has the delivery system for targetting positioning function to tumor tissues.Due to clinically conventional
Chemotherapeutics, without targeting, often results in the serious toxic side effect of normal tissue to tumor tissues, to avoid causing wound to patient
Evil, so as to limit the administration consumption of cancer therapy drug.One of primary goal of successful oncotherapy is in reduction pair as far as possible
The medicine of transmission sufficient amount is to tumour while normal structure is injured.Most of chemotherapeutics is due to low to tumor tissues dosage
Imperfect tumor response can be caused in optimal dose, it will cause disease palindromia and the resistance to the action of a drug.But targetted with the addition of polypeptide
The cancer target nanoscale medicine delivery system of molecule then can effectively solve this problem.
To realize effective medicine delivery and reaching tumor locus and the effect accumulated, it is necessary to find a kind of suitable target
To part.Neuropilin-1 (NRP-1) is that have on a kind of I type transmembrane glycoprotein of cell surface, many tumor tissues of the mankind
NRP-1 expression, including prostate cancer, breast cancer, melanoma, cancer of pancreas and glioma, but in corresponding normal group
Knit in epithelial cell and do not express but.Research is shown in human glioma's clinical pathology sample that to have detected NRP-1 notable
Express, and its expression quantity increases with the increase of glioma grade malignancy.There is researcher to pass through polypeptide triage techniques recently
Filtering out a class has the polypeptide of tumor vessel penetrability and tumor tissue penetration, and this kind of polypeptide can be mediated covalently to be connected with it
The drug molecule or nanoscale medicine delivery system connect penetrates tumor vessel wall and penetrates into inside tumor, is distributed to whole tumor tissues
In, this kind of polypeptide is referred to as " tumour penetrating peptide ".Research shows that RGERPPR is NRP-1 ligands specific, shows to tumour
Cell and tumor vascular dual compatibility.Its target tumor vascular wall can be made to tumor vascular compatibility, and tumour is penetrated
Property can make it penetrate tumor vessel wall to penetrate into inside tumor tissues, and the compatibility to tumour cell can make itself and tumour cell
Specific binding, therefore as targeted molecular, this kind of polypeptide has more obvious compared with general tumour cell targeted molecular
Advantage.
Polymer micelle is a kind of nucleocapsid structure body, and it has hydrophily shell and hydrophobic cores.Hydrophobic inner core is general
It is made up of biodegradable polymer, it plays a part of water-insoluble drug bank.Due to require polymer to cell without
Toxicity simultaneously can be by renal metabolism.The distribution of micella in vivo depends primarily on the property of shell, meanwhile, the property of shell is also to glue
The stability and micella of beam and the interaction of plasma protein, cell membrane play a decisive role, the viscosity micella of micella
The release behavior of physical stability and medicine.
The content of the invention
It is an object of the invention to build a kind of act on based on physically encapsulation to realize that RGERPPR-PEG-DSPE targets element
Modify PGG-PTX delivery systems and preparation and application, the targeting drug delivery for realizing tumour.The delivery system of the present invention can lead to
Intravenous injection administration is crossed, the targeting mediated by RGERPPR can penetrate tumor vessel wall in tumor locus, penetrate into
Enter inside tumor tissues.
The object of the present invention is achieved like this：
A kind of cancer target nanoscale medicine delivery system of physically encapsulation, feature is that it is peptide modified that the system includes RGERPPR
Targeting element, nano-micelle and antineoplastic, the targeting element be PEG-DSPE-
RGERPPR(DSPE-PEG-RGERPPR)；The micella is the glutamic acid branch on polyglutamic acid (PGG) macromolecular scaffold and skeleton
Chain；The antineoplastic is taxol (PTX).Polyglutamic acid macromolecular scaffold and glutamic acid side chain are connected by peptide bond,
Glutamic acid branch terminals are bonded with antineoplastic PTX by ester bond, form PGG-PTX, and its structure is as follows：
Then the DSPE in PTX and targeting element is by hydrophobic interaction, by targeting member
Part hydrophobic side DSPE parcel enters in PGG-PTX, so as to form targeting anti-tumor nanoscale medicine delivery system；
In formula, R=Na or taxol.
A kind of preparation method of above-mentioned delivery system, feature is that this method comprises the following steps：
Step 1：Prepare peptide modified targeting element：By polypeptide RGERPPR sulfydryl and distearoylphosphatidyl ethanol
The maleimide base group reaction of amine-polyethylene glycol-maleimide, connection composition targeting element.Reaction equation is as follows：
Step 2:A, PGG-PTX and b, RGERPPR-PEG-DSPE, mol ratio a are weighed respectively:B=98:2.By PGG-PTX
Dissolved with 0.5% sodium cholate solution, RGERPPR-PEG-DSPE dichloromethane:Acetone=3:1 mixing is molten
Liquid dissolves.B solution is added in a solution, is vortexed when being added dropwise；Cell Ultrasonic Cell Disruptor ultrasound 300w, super 2s in ice bath, interval
3s, ultrasonic 2min obtains emulsion；The sodium cholate solution 8ml, magnetic agitation 10min of addition 1% into emulsion.35 DEG C of water-bath rotations
Evaporating organic solvent；4 DEG C, 21000g centrifugation 45min abandon supernatant, precipitation are resuspended to obtain into nanoparticle solution with physiological saline；
Sample is by being connected with the G-50 sephadex columns of protein purification instrument, the separating-purifying under 228nm ultraviolet detection wavelength.
The antitumor delivery system of micelle nano of targeting element is enclosed with to physical action；
Step 3：Determine the molecular weight and purity of the polypeptide；
Step 4：Determine the targeting element DSPE-PEG-RGERPPR nuclear magnetic spectrums；
Step 5：Determine particle diameter, the form of the nanoscale medicine delivery system；
Step 6：Determine the vitro drug release feature of the nanoscale medicine delivery system；
Step 7：Determine the external intake to glioma U87MG tumour cells of the nanoscale medicine delivery system；
Step 8：Determine the external activity rating to glioma U87MG tumour cells of the nanoscale medicine delivery system；
Step 9：Determine the internal anti-glioma active effect of the nanoscale medicine delivery system.
By the missing nanoparticle of fluorescence, examination tumour cell is respectively to being loaded with fluorescent dye cell membrane green fluorescence probe
(DIO) targeting micelle nano medicine and the cellular uptake without targeting micelle nano medicine, as a result confirm that the present invention passes through physics
Package action can realize that the micelle nano delivery system of targeting element RGERPPR-PEG-DSPE modifications enters tumour cell.
The delivery system of the present invention has following characteristics：Particle diameter is spherical in shape in 100nm or so；In phosphate buffer
It is more stable；Drug release characteristics are also improved；Intake of the tumour cell to the delivery system is bright in cellular uptake experiment
Aobvious to increase, the delivery system is preferable to the targeting of tumour；The delivery system is notable in external anti-glioma activity rating
Enhance growth in vitro inhibitory action of the micelle nano medicine to glioma；By being injected intravenously administration, pass through tumour EPR
Effect and RGERPPR mediations are by it targeted to tumor locus, and the targeting that this delivery system can be used as anti-tumor medicine is passed
Send, and good curative effect is obtained on treatment glioma, extend the median survival interval of BALB/C glioma nude mices.
Brief description of the drawings
Fig. 1 is delivery system preparation flow schematic diagram of the present invention；
Fig. 2 is peptide C-RGERPPR efficient liquid phases HPLC collection of illustrative plates of the present invention；
Fig. 3 is peptide C-RGERPPR of the present invention mass M S collection of illustrative plates；
Fig. 4 is MAL-PEG-DSPE and RGE-PEG-DSPE nuclear-magnetisms NMR spectra of the present invention；
Fig. 5 is PGG-PTX and RGE/PGG-PTX grain-size graphs of the present invention；
Fig. 6 is PGG-PTX and RGE/PGG-PTX transmission electron microscope pictures (A) PGG-PTX, (B) RGE/PGG-PTX of the present invention；
Fig. 7 is nanoscale medicine delivery system long-time stability lab diagram of the present invention；
Fig. 8 is nanoscale medicine delivery system drug release patterns figure of the present invention；
Fig. 9 is that U87MG neuroglial cytomas qualitative, quantitative of the present invention absorbs figure；
Figure 10 is U87MG cytoactives curve map of the present invention；
Figure 11 is glioma nude mice survivorship curve figure after present invention administration.
Embodiment
It will be helpful to further understand the present invention by following embodiments, but be not intended to limit present disclosure.
It is the delivery system preparation flow schematic diagram of the present invention refering to Fig. 1.
Macromolecule Prodrug PGG-PTX paclitaxel carried medicines amount is 35%.
Embodiment 1
Peptide C-RGERPPR sign
The polypeptide sterling being commercially available is characterized with HPLC, LC-MS method.
Analytic type HPLC chromatogram method is as follows, chromatographic column：Diamonsil C18 (5 μm, 200 × 4.6mm)；Mobile phase A：
0.1%TFA water, Mobile phase B：0.1%TFA acetonitriles；Elution program：0~2min：5%B, 2~32min：5%~65%B, 32
~33min：65%~90%B, 33~36min：90%B；, 36~37min：90%~5%B, 37~45min：5%B；Stream
Speed：0.7mL/min；Column temperature：40℃；Wavelength：UV 214、280nm.Fig. 2 shows that its purity is more than 98%；Fig. 3 shows polypeptide point
Son amount is 970.4, and mass M S collection of illustrative plates shows that its molecular weight is consistent with theory.
Embodiment 2
DSPE-PEG-RGERPPR synthesis and sign
Take C-RGERPPR sterlings 25mg to be dissolved in Ph7.0-7.4 2ml PBS (Ph7.0-7.4,1xPBS) solution, take horse
Carry out acid imide-polyethylene glycol-phosphorus fat complexes DSPE-PEG-Mal (molar equivalent is 0.8 times of RGERPPR) and be dissolved in 0.5ml
DMF, is added in polypeptid solution, and ultrasound removes bubble, and stirring reaction about 1 hour, DSPE-PEG-Mal reactions are complete, dialysis
(molecular cut off 3500Da, dialysis medium is water) removes excessive C-RGERPPR and DMF, and freeze-drying obtains RGERPPR-
PEG-DSPE, H1-NMR.H1-NMR collection of illustrative plates shows that Fig. 4 are RGERPPR-PEG-DSPE and maleimide-polyethylene glycol-phosphorus
The nuclear magnetic spectrum of fat complexes (Mal-PEG-DSPE).RGERPPR-PEG-DSPE nuclear magnetic spectrums are maleimide in 6.7ppm
Characteristic peak is disappeared, and display RGERPPR-PEG-DSPE is synthesized successfully.
Embodiment 3
The preparation of physically encapsulation effect RGERPPR-PEG-DSPE modifications PGG-PTX nanoscale medicine delivery system and sign
A, PGG-PTX and b, RGERPPR-PEG-DSPE, mol ratio a are weighed respectively:B=98:2.PGG-PTX is used
0.5% sodium cholate solution dissolving, RGERPPR-PEG-DSPE dichloromethane:Acetone=3:1 mixed solution dissolving.By b
Solution is added in a solution, is vortexed when being added dropwise.Cell Ultrasonic Cell Disruptor ultrasound 300w, super 2s in ice bath, is spaced 3s, ultrasound
2min obtains emulsion.The sodium cholate solution 8ml, magnetic agitation 10min of addition 1% into emulsion.35 DEG C of water-bath rotary evaporations are removed
Remove organic solvent；4 DEG C, 21000g centrifugation 45min abandon supernatant, precipitation are resuspended to obtain into nanoparticle solution with physiological saline.Sample leads to
The G-50 sephadex columns for being connected with protein purification instrument are crossed, the separating-purifying under 228nm ultraviolet detection wavelength.Obtain physics
Effect is enclosed with the antitumor delivery system of micelle nano of targeting element.
Prepare the micelle nano delivery system (RGE/PGG- that macromolecule carries prodrugs of paclitaxel (PGG-PTX) and modification targeting
PTX) 1mg/ml, crosses 0.22um filter membranes, is determined in its particle size and particle diameter distribution, Fig. 5 and shown with Malvern laser particle analyzer
Nano particle diameter distribution situation, the average grain diameters of two kinds of nanoparticles of PGG-PTX and RGE/PGG-PTX be respectively 25nm and
109nm or so.
PGG-PTX and RGE/PGG-PTX form is observed by transmission electron microscope (TEM), need to 1% phosphorus tungsten
Sour negative staining prepares sample, and concrete operations are as follows：(1) sample solution is dripped on copper mesh；(2) air drying about 10 minutes,
Unnecessary liquid is sucked with filter paper；(3) 1% phosphotungstic acid is dripped again on copper mesh, and drying at room temperature is sucked after 8 minutes with filter paper
Unnecessary dye liquor；(4) it is placed under transmission electron microscope and observes after sample is completely dried.A and B are respectively PGG-PTX and RGE/ in Fig. 6
The transmission electron microscope picture of two kinds of nanoparticles of PGG-PTX, two kinds of equal comparison rules of nanoparticle form are spherical in shape.
Embodiment 4
Two kinds of nanoparticle stability checkings of PGG-PTX and RGE/PGG-PTX
PGG-PTX and RGE/PGG-PTX is configured to 1.0mg/ml with 1xPBS liquid, determined with dynamic light scattering (DLS)
Particle size and monodispersity index (PDI), are determined once for every two days, and METHOD FOR CONTINUOUS DETERMINATION 3 weeks investigates the steady in a long-term of nano-particle
Property.As a result such as Fig. 7, two kinds of nanoparticles have good stability, and particle diameter is without significant change (p after placing 3 weeks>0.05).
Embodiment 5
Two kinds of nanoparticle pharmaceutical release performance checkings of PGG-PTX and RGE/PGG-PTX
PGG-PTX and RGE/PGG-PTX Release Performance：It is that 7.4 buffer salt solutions prepare certain density respectively with pH
PGG-PTX and RGE/PGG-PTX, every group of three repetitions, all samples are positioned in 37 DEG C of constant temperature oscillators.Respectively in difference
Time point takes each one group of every kind of Sample Buffer salting liquid, and the taxol discharged in solution is extracted with ethyl acetate, with efficient
The content (calibration curve method) of hplc determination taxol.As shown in figure 8, RGE/PGG-PTX insoluble drug release is certain
Increased in degree, improve PGG-PTX taxol releases slow release high-volume few shortcoming.
Embodiment 6
Nanoscale medicine delivery system vitro in glioma targeting is verified
Using U87MG cells as model, per hole 3 × 104Cells/ml density is inoculated in 6 orifice plates and cultivates 24, per pore volume
2ml, culture plate is moved into CO2gas incubator, 37 DEG C, and 24h is cultivated under the conditions of 5% carbon dioxide and saturated humidity, is made thin
The adherent length of born of the same parents is to 80%.With the DMEM nutrient solutions containing 10% hyclone prepare a series of various concentrations RGE/PGG-PTX and
PGG-PTX solution.Nutrient solution in culture dish is suctioned out, RGE/PGG-PTX and PGG-PTX serial solution is added, sets simultaneously
Put control group, 37 DEG C of incubation 2h.Aspirate supernatant is abandoned, and is gently washed twice with PBS solution, qualitative with laser co-focusing respectively
With flow cytometer quantitative assessment cellular uptake situation.Nucleus dyes blueness with Hochest.The cell of flow cytometry analysis
First washed with PBS 2 times；Then digest, centrifuge, be resuspended, in triplicate；Cell flow cytomery after resuspension.Refer to
A is that free fluorescein DIO cellular uptakes figure, B are that PGG-PTX parcel DIO cellular uptakes figure, C are RGE/PGG-PTX in Fig. 9, figure
DIO cellular uptake figures are wrapped up, as a result the fluorescence micrograph after being acted on 2 hours with tumour cell respectively in 37 DEG C shows RGE/
PGG-PTX intakes illustrate prepared by RGE/PGG-PTX micelle nanos delivery system apparently higher than intake of the cell to PGG-PTX
Success, and there is good targeting to tumour cell.
Embodiment 7
Determine the external activity rating to glioma U87MG tumour cells of the nanoscale medicine delivery system
Vitro cytotoxicity test is using U87MG cells as model, using CCK-8 kit assays IC50Value examines or check PGG-
PTX and RGE/PGG-PTX and free PTX are to the inhibiting rate of cell, specific method：It is 2 × 10 by density3Cells/ml's
U87MG inoculation of suspension liquid is placed on 37 DEG C of incubators under the conditions of 5%CO2 in 96 orifice plates, cultivates 24 hours；To 96 orifice plates
It is middle to add tri- kinds of drug solutions of Taxol, PGG-PTX and RGE/PGG-PTX of various concentrations, and blank control is set, and it is negative
Compare (not dosing), every group at least sets three multiple holes；96 orifice plates are placed on to 37 DEG C of incubators under the conditions of 5%CO2 after dosing
It is middle to continue to be incubated 48 hours；The liquid in hole is removed, 100 μ l fresh cultures and 10 μ l CCK-8 reagents are added again per hole；
96 orifice plates are placed in 37 DEG C of isothermal vibration casees slight concussion and cultivate 4 hours；The OD values at 450nm are determined with ELIASA.
The survival rate calculation formula of cell：
Cell viability (%)=[A (dosing)-A (blank)]/[A (not dosing)-A (blank)] × 100%.
In formula：
A (blank)：With culture medium and CCK-8 solution without the OD values in the hole of U87MG cells；
A (dosing)：The OD values in the hole with U87MG cells, CCK-8 solution and drug solution；
A (not dosing)：With U87MG cells, CCK-8 solution without the OD values in the hole of drug solution.
As shown in Figure 10, after Taxol, PGG-PTX and RGE/PGG-PTX processing U87MG cells 48h, IC50 values are respectively
0.14 μM, 2.39 μM and 1.44 μM,.It will be apparent that RGE/PGG-PTX antitumor cell growth activity is than the PGG- without targeting
PTX is high, and PGG-PTX IC50Value illustrates that macromolecule connects the nanometer medicine of taxol formation by bonding scheme higher than PTX
Thing, cytotoxicity is substantially reduced.
Embodiment 8
Determine the activity rating of the external interior anti-glioma of the nanoscale medicine delivery system
Take the logarithm the U87MG cells in growth period, centrifuged after digestion, single cell suspension is separated into phosphate buffer PBS,
1 × 106 cell (being scattered in 10ul PBS) is inoculated with 5ul in corpus straitum on the right side of nude mice with stereotaxic instrument.It is inoculated with position
Put coordinate setting as follows：0.6mm before nude mice bregma, right side 1.8mm, deep 3mm.
Nude mice is randomly divided into 4 groups every group 7,100ul physiology salts are injected within the 11st, 15,18 and 21 day after inoculation respectively
Water, PTX, PGG-PTX, RGE/PGG-PTX, accumulation Taxol injection dosage are 40mg/kg, during the existence of record cast nude mice
Between.Refering to Figure 11, result is shown in figure, physiological saline N.S, free paclitaxel PTX, the PGG-PTX without targeting, has targeting element
RGE/PGG-PTX each groups median survival interval be respectively 22,29,36,47 days.It follows that RGE/PGG-PTX group lotus knurls are naked
The life span of mouse is longer than PGG-PTX groups, illustrates CRNGRGPDC modification and can significantly increase the antitumor of Evacet
Effect.
Claims (4)
1. the cancer target nanoscale medicine delivery system of a kind of physically encapsulation, it is characterised in that it is peptide modified that the system includes RGERPPR
Targeting element, nano-micelle and antineoplastic, the targeting element be PEG-DSPE-
RGERPPR；The micella is the glutamic acid side chain on polyglutamic acid PGG macromolecular scaffolds and skeleton；The antineoplastic is
Taxol PTX；Polyglutamic acid macromolecular scaffold and glutamic acid side chain are connected by peptide bond, glutamic acid branch terminals with it is antitumor
Medicine PTX is bonded by ester bond, forms PGG-PTX, and its structure is as follows：
Then the DSPE in PTX and targeting element is dredged targeting element by hydrophobic interaction
Water end (W.E.) DSPE parcel enters in PGG-PTX, and the cancer target nanometer for forming the physically encapsulation passs medicine
System；In formula, R=Na or taxol.
2. delivery system according to claim 1, it is characterised in that the DSPE-poly- second two
Molecular weight polyethylene glycol is 400~8000 in alcohol-RGERPPR.
3. the preparation method of delivery system described in a kind of claim 1, it is characterised in that this method comprises the following steps：
Step 1：Prepare peptide modified targeting element：By polypeptide RGERPPR sulfydryl and DSPE-poly-
The maleimide base group reaction of ethylene glycol-maleimide, connection composition targeting element；
Step 2:A, PGG-PTX and b, RGERPPR-PEG-DSPE, mol ratio a are weighed respectively:B=98:2；PGG-PTX is used
0.5% sodium cholate solution dissolving, RGERPPR-PEG-DSPE dichloromethane:Acetone=3:1 mixed solution dissolving；By b
Solution is added in a solution, is vortexed when being added dropwise；Cell Ultrasonic Cell Disruptor ultrasound 300w, super 2s in ice bath, is spaced 3s, ultrasound
2min obtains emulsion；The sodium cholate solution 8ml, magnetic agitation 10min of addition 1% into emulsion；35 DEG C of water-bath rotary evaporations are removed
Remove organic solvent；4 DEG C, 21000g centrifugation 45min abandon supernatant, precipitation are resuspended to obtain into nanoparticle solution with physiological saline；Sample leads to
The G-50 sephadex columns for being connected with protein purification instrument are crossed, the separating-purifying under 228nm ultraviolet detection wavelength；Obtain physics
The cancer target nanoscale medicine delivery system of parcel.
4. the application of delivery system described in a kind of claim 1, it is characterised in that the delivery system is used for glioma treatment medicine
The targeted delivery of thing.
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| CN110028674A (en) * | 2018-01-12 | 2019-07-19 | 中国科学院苏州纳米技术与纳米仿生研究所 | A kind of amphipathy macromolecule material system, preparation method and application |
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