CN101161239A - PLGA Gemcitabine sustained-release microsphere and its preparing method - Google Patents
PLGA Gemcitabine sustained-release microsphere and its preparing method Download PDFInfo
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Abstract
A PLGA gemcitabine slow release microball is disclosed in the present invention belonging to pharmaceutical field. The PLGA gemcitabine slow release microball is prepared by supplementary polylactic acid-glycolic acid and raw material medicine gemcitabine with the weight ratio of 95 :5 to 35 :65. The present invention has the advantages that: preparing gemcitabine and polylactic acid-glycolic acid into slow release microball, directly sendint to tumor part by syringe to execute regional slow release chemotherapy, gemcitabine are released slowly, the tumor part has high medicine concentration, long duration, strong action intensity, big killing force to tumor cell, thereby can improve curative effect to tumor; medicine are centralized to act on local, whole body has little medicine amount, adverse response is minimal. After medicine are released, polylactic acid-glycolic acid start to degradate as lactic acid and glycolic acid, further degradate as carbon dioxide and water, no effect to human body.
Description
Technical field
The present invention relates to a kind of injectable PLGA (polylactic acid one glycolic) Gemcitabine sustained-release microsphere and preparation method thereof, more specifically, be to be that adjuvant, gemcitabine are injectable sustained-release micro-spheres of making of active drug and preparation method thereof, belong to the pharmaceutics field with biodegradable polylactic acid one glycolic.
Background technology
Polylactic acid one glycolic is a kind of good biocompatibility, biodegradable, safety good, physicochemical property is excellent pharmaceutical macromolecular material, is used for medical material in the adjuvant of medicament slow release preparation and the human body; This material is degraded to lactic acid and the glycolic that has in the human body earlier after entering human body, further is degraded to carbon dioxide again and water excretes, and is harmless.Polylactic acid-glycollic acid degradation rate in vivo can be regulated by the content and the ordering of glycolic in its molecule.
The gemcitabine chemical name is 2 '-deoxidation-2 ', 2 '-difluoro cytidine hydrochlorate, this product is the cell cycle specific antimetabolitas, mainly act on the DNA tumor cell of synthesis stage, it is S phase cell, under certain condition, can stop the progress of G1 phase to the S phase, thereby inhibition growth of tumour cell, be applicable to nonsmall-cell lung cancer, cancer of pancreas, bladder cancer, the treatment of breast carcinoma and other entity tumors, its active anticancer is relevant with administering mode, and tumor locus gemcitabine concentration is high more, it is long more to keep the active drug concentration time, good more to the effect of oncotherapy.The gemcitabine half-life is short, the whole body administration, and the tumor locus drug level is low, and the time of keeping is short, weak curative effect, and systemic drug concentration height, untoward reaction is serious.
Summary of the invention
The technical problem to be solved in the present invention is: but provide that a kind of toxicity is low, the little direct injection of untoward reaction is in the PLGA Gemcitabine sustained-release microsphere of the capable regional sustained-release chemotherapy of tumor locus.
Another object of the present invention provide this PLGA Gemcitabine sustained-release microsphere preparation method.
For achieving the above object, the present invention is by the following technical solutions:
A kind of injectable PLGA Gemcitabine sustained-release microsphere is made with 95: 5～35: 65 weight ratio by adjuvant polylactic acid-glycollic acid and crude drug gemcitabine.
Described gemcitabine is antitumor drug 2 '-deoxidation-2 ', 2 '-difluoro cytidine hydrochlorate.
Lactic acid and glycolic molecular proportion are 95: 5～10: 90 in the described polylactic acid-glycollic acid molecule.
Described microsphere diameter is 1 μ m～250 μ m.
Test shows that the prepared PLGA Gemcitabine sustained-release microsphere release in vitro degree of the present invention is: 24 hours: 10%～80%; 120 hours: 25%～95%; 360 hours: more than 60%.
The preparation method of PLGA Gemcitabine sustained-release microsphere: pulverize gemcitabine and make the fine powder of diameter less than 20 μ m; Polylactic acid-glycollic acid is dissolved in the solvent, presses formula proportion the gemcitabine fine powder is added in the PLGA solution, mix homogeneously, spray drying under the vacuum, sieve the PLGA Gemcitabine sustained-release microsphere; The weight ratio of polylactic acid-glycollic acid and gemcitabine is 95: 5～35: 65.
Described solvent is selected from one or more in dichloroethanes, dichloromethane, chloroform, oxolane, acetone, ethyl acetate and the carbon dioxide.
Microsphere can also adopt conventional method preparations such as solvent method, emulsion process, supercritical methanol technology.
Using method of the present invention:, behind microsphere and iodized oil (or water, surfactant such as tween 80) mix homogeneously, directly be injected into the capable regional sustained-release chemotherapy of tumor focus by syringe according to the consumption of stipulating clinically.
Advantage of the present invention is:
1. but novel PLGA Gemcitabine sustained-release microsphere direct injection provided by the invention makes the tumor locus medication amount many in the regional sustained-release chemotherapy of tumor focus position row, the high longer duration of drug level, and action intensity is big, local good effect; The systemic drug amount is few, and blood drug level is low, almost has no adverse reaction; Thereby reach the effect that improves reduction untoward reaction evident in efficacy.
2.PLGA the Gemcitabine sustained-release microsphere direct injection is in the regional sustained-release chemotherapy of tumor focus position row, the systemic drug amount is few, and blood drug level is low, almost has no adverse reaction; The tumor patient (can not bear weak patients, the patients with terminal of multiple dosing) that can not bear traditional chemotherapy can be treated preferably.
3.PLGA Gemcitabine sustained-release microsphere directly injects at tumor locus, medicine directly in the tumor cell effect, has been avoided first pass effects such as liver, the utilization ratio of drug height.
The present invention will be further described below in conjunction with accompanying drawing and better embodiment, so that the public has whole to summary of the invention and understand fully, and is not qualification to protection domain of the present invention.Aforementioned part fully discloses the protection domain that the present invention can implement, and therefore allly any well known in the artly is equal to replacement according to what the disclosure of invention was carried out, all belongs to infringement of the present invention.
Description of drawings
Fig. 1 is a high effective liquid chromatography for measuring gemcitabine canonical plotting.
Fig. 2 is the high-efficient liquid phase chromatogram of gemcitabine and internal standard substance, and G is a gemcitabine, and T is a ftorafur.
Fig. 3 is drug level-time plot in the A group tumor.
Fig. 4 is B group pharmaceutical concentration-time curve figure.
Fig. 5 is tumor growth curve figure.
Fig. 6 is the relative rate of increase figure of tumor.
Fig. 7 is treatment group tumour inhibiting rate figure.
The specific embodiment
Embodiment 1: preparation PLGA Gemcitabine sustained-release microsphere
With ultrafine crusher gemcitabine is made the fine powder of diameter less than 20 μ m; With PLGA[90: 10 (lactic acid and glycolic molecular proportions)] 4g is dissolved in 40mL acetone and makes the PLGA acetone soln, add gemcitabine fine powder 0.4g, mix homogeneously, spray drying under the vacuum, collect microsphere, sieve particle diameter less than the PLGA Gemcitabine sustained-release microsphere (A) of 100 μ m.
Embodiment 2: preparation PLGA Gemcitabine sustained-release microsphere
PLGA (80: 20) 4g is dissolved in the 40mL oxolane makes the PLGA tetrahydrofuran solution, add gemcitabine fine powder 0.8g, mix homogeneously, spray drying under the vacuum is collected microsphere, sieve particle diameter less than the PLGA Gemcitabine sustained-release microsphere (B) of 100 μ m.
Embodiment 3: preparation PLGA Gemcitabine sustained-release microsphere
PLGA 4g is dissolved in the 40mL dichloromethane makes the PLGA dichloromethane solution, add gemcitabine fine powder 1.2g, mix homogeneously, spray drying under the vacuum is collected microsphere, sieve particle diameter less than the PLGA Gemcitabine sustained-release microsphere (C) of 100 μ m.
Embodiment 4: preparation PLGA Gemcitabine sustained-release microsphere
PLGA (75: 25) 4g is dissolved in the 40mL chloroform makes the PLGA chloroform soln, add gemcitabine fine powder 1.8g, mix homogeneously, spray drying under the vacuum is collected microsphere, sieve particle diameter less than the PLGA Gemcitabine sustained-release microsphere (D) of 100 μ m.
Embodiment 5: preparation PLGA Gemcitabine sustained-release microsphere
PLGA (55: 50) 4g is dissolved in the 40mL ethyl acetate makes the PLGA ethyl acetate solution, add gemcitabine fine powder 4.2g, mix homogeneously, spray drying under the vacuum is collected microsphere, sieve particle diameter less than the PLGA Gemcitabine sustained-release microsphere (E) of 100 μ m.
Embodiment 6: assay
With water is solvent, and the gemcitabine crude drug is a reference substance, and ultraviolet method is measured the medicament contg that makes PLGA Gemcitabine sustained-release microsphere A, B, C, D, E among the embodiment 1～5 and is respectively:
A 8.87±0.12；
B 16.39±0.36；
C 22.74±0.41；
D 30.94±0.56；
E 49.85±0.47；
Embodiment 7: the release test
The microsphere that makes among the embodiment 1～5 is respectively got three parts, every part contains gemcitabine 5mg, is respectively charged in the nylon filter bag, seals, put 10mL tool plug in vitro, adding distil water 10mL, close plug is put in 37 ℃ of water-baths and is incubated, take out invisible spectro aqueous solution in different time points, the distilled water that adds synthermal consubstantiality simultaneously, close plug continues to be incubated in 37 ℃ of water-baths.Measure gemcitabine amount in the water with ultraviolet method, calculate release, the result is as follows:
Table 1: the release of embodiment 1-5PLGA Gemcitabine sustained-release microsphere
The acute toxicity test of embodiment 8:PLGA-Gemcitabine sustained-release microsphere
One, experiment material
1. animal and cell strain
The Balb/c-nu nude mice: the SPF level, body weight: 16 ± 2 grams, age is female all around; Beijing dimension tonneau China experimental technique company limited, the quality certification: SCXK (capital) 2007-0001.
Human pancreas cancer transplantation tumor cell strain (JF305): central laboratory of Tumour Inst., Chinese Medical Academy provides.
2. medicine and reagent
The PLGA-Gemcitabine sustained-release microsphere: HeFei University of Technology's controlled release drug institute provides, and particle diameter is less than 100 μ m, content of dispersion 20%, and the design slow-release time is 30 days.Prepare according to embodiment 1 method.
Iodized oil: Guerbet, Roissy, France.06LU003B effect duration: 2009-03
Two, cell culture and tumor strain inoculation
1. cell is taken out from liquid nitrogen, be placed on rapidly to shake in 37 ℃ of water-baths and make its quick dissolving, dropwise put into 10ml serum-free RPMI-1640,1000rmin
-1, centrifugal 10 minutes.Abandoning supernatant, adding contains in the RPMI-1640 of 10% serum.Put into culture bottle, 37 ℃, 5%CO
2Cultivate in the incubator.
2. treat its 90% compound after, wash twice with PBS, add the about 1ml of 0.1% trypsinization liquid, cover cellular layer repeatedly, discard Digestive system, bottleneck is built, be positioned over 37 ℃, 5%CO
2In the incubator 2-5 minute, inverted microscope is observed peptic cell down, and was circular between the cell, adds new culture fluid, with dropper cell blown and beaten into cell suspension, and behind the counting, in cell packing culture bottle, the density of passage cell is not less than 5 * 10
5/ ml.Put into 37 ℃, 5%CO
2Cultivate in the incubator.
3. continue to cultivate to make cell concentration reach 1 * 10
8/ ml.
4. take out tumor cell in liquid nitrogen, after 37 ℃ of recoveries of thawing rapidly, the right side axillary fossa that is inoculated in nude mice is subcutaneous, makes every inoculation inoculation 0.2ml (1 * 10
7/ ml).The back transplantation tumor growth of 3 weeks reaches about the 1.5cm diameter.Go down to posterity according to a conventional method.
5. put to death mice, peel off tumor under aseptic condition, normal saline cleans for several times, reject downright bad tissue, select well-grown tumor tissues, with knife blade tumor tissues is cut into the piece of tissue of 3-4mm size, to organize inoculator that tumor tissues is transplanted to the right side axillary fossa subcutaneous with special.
Three, grouping and medication
1. grouping:
The A group: healthy nude mice, it is subcutaneous that the PLGA-Gemcitabine sustained-release microsphere is injected into nude mice.
The B group: tumor bearing nude mice is injected into the PLGA-Gemcitabine sustained-release microsphere in the tumor.
2. medication:
The tumor bearing nude mice diameter of tumor begins administration when reaching 0.6cm-0.8cm.With iodized oil and PLGA-Gemcitabine sustained-release microsphere mixing, adopt the 1ml injector syringe to connect the 22G injection needle, above-mentioned suspension is injected in the subcutaneous and tumor bearing nude mice tumor of healthy nude mice, every nude mice injection volume is 0.05ml.
Four, experimental technique
Adopt the UDP method to set dosage earlier, the A group is a predose with 270mg, and the B group is a predose with 8.64mg, has or not dead dosage with 1.2 times of differential risings or decline according to animal.
Five, evaluation index: LD
50
Adopt method of maximum likelihood to measure LD
50, formula is:
N=animal subject number
The C=arithmetic average and
Common ratio=1.2
LogLD
50=Log (drug dose of i=0)+C/N * Log1.2 logarithm of negating draws LD
50
Six. the result
1.A group is used 7 of animals altogether, LD when calculating the healthy nude mice subcutaneous administration of PLGA-Gemcitabine sustained-release microsphere
50=256.30mg/Kg (table 2).
Animal subject situation during table 2:PLGA-Gemcitabine sustained-release microsphere health nude mice subcutaneous administration
Annotate: △ represents survival, * representing death, n represents the life or death number of elements of each dosage.
2.B group is used 12 of animals altogether, LD when calculating tumor bearing nude mice tumor vivo medicine-feeding
50=8.91mg/Kg (table 3).
Animal subject situation during administration in the table 3:PLGA-Gemcitabine sustained-release microsphere tumor bearing nude mice tumor
Annotate: △ represents survival, * representing death, n represents the life or death number of elements of each dosage.
The explanation of this experimental result:
1.PLGA-in administration of Gemcitabine sustained-release microsphere whole body and the mesenchyma stroma of tumors during administration reaction of animal have than big difference.
2. tumor by local application PLGA-Gemcitabine sustained-release microsphere carries out the dosage that the chemotherapy of mesenchyma stroma dosage should be lower than conventional chemotherapy.
Embodiment 9:PLGA-Gemcitabine sustained-release microsphere chemotherapy of mesenchyma stroma pharmacokinetic studies
One. experiment material
1. animal and cell strain
Balb/c-nu nude mice: SPF level, body weight: 16 ± 2 grams, age all around, male and female half and half; Beijing dimension
Tonneau China experimental technique company limited, the quality certification: SCXK (capital) 2007-0001.
Human pancreas cancer transplantation cell strain (JF305): central laboratory of Tumour Inst., Chinese Medical Academy provides.
2. medicine and reagent
The PLGA-Gemcitabine sustained-release microsphere: HeFei University of Technology's controlled release drug institute provides, and particle diameter is less than 100 μ m, content of dispersion 20%, and the design slow-release time is 30 days.
Gemcitabine: Hao Sen pharmaceutcal corporation, Ltd in Jiangsu provides
Iodized oil: Guerbet, Roissy, France.06LU003B effect duration: 2009-03
Two. the inoculation of cell culture and tumor strain
With embodiment 8.
Three. grouping and medication
1. grouping:
A group: transplant successful tumor bearing nude mice with 50 and be divided into 10 subgroups at random, 5 every group, give PLGA-Gemcitabine sustained-release microsphere tumor vivo medicine-feeding chemotherapy of mesenchyma stroma.
B group: transplant successful tumor bearing nude mice with 60 and be divided into 12 subgroups at random, 5 every group, give gemcitabine intraperitoneal administration systemic chemotherapy.
2. medication:
The tumor bearing nude mice diameter of tumor begins administration when reaching 0.6cm-0.8cm, A group: with iodized oil and PLGA-Gemcitabine sustained-release microsphere mixing, adopt the 1ml injector syringe to connect the 22G injection needle, above-mentioned suspension is injected in the tumor bearing nude mice tumor, every nude mice injection volume is 0.05ml; The B group: with iodized oil and gemcitabine mixing, adopt the 1ml injector syringe to connect the 22G injection needle, above-mentioned suspension is injected the tumor bearing nude mice intraperitoneal, every nude mice injection volume is 0.05ml.
Four. specimen is extracted and is handled
1. specimen is extracted
A organizes (PLGA-Gemcitabine sustained-release microsphere tumor bearing nude mice tumor vivo medicine-feeding chemotherapy of mesenchyma stroma group) specimen extracting method: 2h, 12h, 24h, 48h, 72h, 120h, 240h, 480h, 720h, 960h time point are randomly drawed a subgroup after administration, peel off tumor after putting to death animal, it is frozen that specimen is positioned over-20 ℃ of refrigerators.
B organizes (gemcitabine tumor bearing nude mice intraperitoneal administration systemic chemotherapy group) specimen extracting method: 0min, 5min, 10min, 30min, 1h, 4h, 8h, 24h, 48h, 72h, 120h, 240h time point are randomly drawed a group after administration, peel off tumor after putting to death animal, it is frozen that specimen is positioned over-20 ℃ of refrigerators.
2. sample disposal
Every part of specimen adds ammonium acetate buffer 2ml, after the use mortar fully grinds, places centrifuge tube interior centrifugal, 3000rmin
-1, centrifugal 10 minutes, extract supernatant ,-20 ℃ of refrigerators are frozen.
Five. sample analysis
1. instrument
The Waters highly effective liquid phase chromatographic system comprises: 515 pumps, 717 automatic samplers help incubator, temperature
The degree controller, 2487 UV-detector, Epower chromatographic work station, Millipore ultra-pure water system.
2. reagent and medicine
The gemcitabine standard substance: Hao Sen pharmaceutcal corporation, Ltd in Jiangsu provides
Interior mark: ftorafur, purity 99.46%, Jinan pharmaceutical factory provides
Acetonitrile: Fisher company provides
Deionized water: Millipore company provides
3. chromatographic condition
Chromatographic column: Waters Symmetry C18 (4.6mm * 250nm, 5um)
Mobile phase: 50mmolL
-1Ammonium acetate buffer (get ammonium acetate 3.85g and add deionized water 1000ml,
Regulate pH value to 4.2 with glacial acetic acid)
Acetonitrile: (90: 10)
Flow velocity: 1mLmin
-1
Sample size: 20 μ L
Detect wavelength: 268nm
Column temperature: 25 ℃
4. the preparation of reference substance and interior mark storing solution
Precision takes by weighing gemcitabine standard substance 20.0mg in the 100mL measuring bottle, dissolve and be diluted to scale with deionized water (pH6.8), concentration be 200.0 μ gmL
-1The reference substance storing solution is in 4 ℃ of preservations of refrigerator; Precision takes by weighing ftorafur reference substance 20.2mg in the 100mL measuring bottle, dissolve and be diluted to scale with deionized water (pH6.8), concentration be 202.0 μ gmL
-1Interior mark storing solution is in 4 ℃ of preservations of refrigerator.Facing the time spent is diluted to 10 μ gmL
-1Inner mark solution.
5. tissue sample is handled
Get the tissue sample 100 μ L after the processing, add the inner mark solution 150 μ L of 10 μ gmL-1, mixing adds methanol-acetonitrile (1: 9) 3mL, places 5min behind the vortex 1min, in 3500rmin
-1Centrifugal 10min gets supernatant and places in 60 ℃ of water-baths, and nitrogen dries up, and residue dissolves with 1.5mL mobile phase, centrifugal (15000rmin
-1, 10min), get supernatant, sample introduction 20 μ L.
6. the foundation of standard curve
It is an amount of to get gemcitabine standard substance storing solution respectively, adds deionized water (pH6.8) dilution, makes it to become 0.5,1,5,25,50,100,200 μ gmL
-1The standard solution of Concentraton gradient by above-mentioned tissue sample disposal methods, is prepared 0.05,0.1,0.5,2.5,5,10,20 μ gmL respectively
-1Each 7 parts of organizational standard product, sample introduction 20 μ L detect, and are vertical coordinate with the peak area ratio of gemcitabine and internal standard substance, the concentration of gemcitabine is abscissa, carries out linear regression.
Six. date processing
Das2.0 handles with gemcitabine drug level initial data input pharmacokinetic analysis software, obtain pharmacokinetic parameter, data represent that with Mean ± SD the SPSS11.5 software kit carries out statistical analysis, measurement data is checked with t, and P＜0.01 is thought significant difference.
Seven. the result
1. the regression equation of standard curve and linear relationship
The regression equation of gemcitabine standard curve is Y=7.76e-001X+1.23e-001; R=0.999851.The display standard curve is at 0.05～20 μ gmL
-1Has good linear relationship (Fig. 1) in the scope.
2. plant medication tumor vivo medicine concentration-time graph (abbreviation drug-time curve)
A group crest occurred in 72 hours after administration, descend gradually later on and tend towards stability, and curve reaches 40 days in the above persistent period of 0.34 μ g/ml; B group 5 minutes curves after administration reach summit, sharply descend subsequently, and concentration is 0.30 μ g/ml in the time of 30 minutes, is 0.09 μ g/ml (Fig. 3 and Fig. 4) in the time of 10 days.
3. half-life
The administration chemotherapy of mesenchyma stroma group half-life is 97.39 ± 8.93 hours in the PLGA-Gemcitabine sustained-release microsphere tumor, and the gemcitabine intraperitoneal administration systemic chemotherapy group half-life is 2.97 ± 0.25,2 groups compares, and the result has significant difference, and the P value is less than 0.01 (table 4).
Table 4:2 kind administering mode half-life, area under curve, peak concentration
4. area under curve (AUC)
Area is 383.17 ± 0.83ugmL under the interior administration chemotherapy of mesenchyma stroma sets of curves of PLGA-Gemcitabine sustained-release microsphere tumor
-1, area is 37.08 ± 0.22ugmL under the gemcitabine intraperitoneal administration systemic chemotherapy sets of curves
-1, 2 groups are relatively, and the result has significant difference, and the P value is less than 0.01 (table 4).
5. peak concentration (Cmax)
PLGA-Gemcitabine sustained-release microsphere chemotherapy of mesenchyma stroma group peak concentration is 1.33 ± 0.21, and gemcitabine intraperitoneal administration systemic chemotherapy group phase peak concentration is 0.75 ± 0.23,2 groups to be compared, and the result has significant difference, and the P value is less than 0.01 (table 4).
This test explanation:
1.PLGA-the Gemcitabine sustained-release microsphere chemotherapy of mesenchyma stroma can significantly improve the drug level of tumor by local, the action time of prolong drug.
2.PLGA-the Gemcitabine sustained-release microsphere chemotherapy of mesenchyma stroma can reduce the whole body toxic and side effects of chemotherapeutics.
Embodiment 10:PLGA-Gemcitabine sustained-release microsphere pharmacodynamic study
One, experiment material
1. animal and cell strain
Balb/c-nu nude mice: SPF level, body weight: 16 ± 2 grams, age all around, male and female half and half; Beijing dimension tonneau China experimental technique company limited, the quality certification: SCXK (capital) 2007-0001.
Human pancreas cancer transplantation cell strain (JF305): central laboratory of Tumour Inst., Chinese Medical Academy provides.
2. medicine and reagent
The PLGA-Gemcitabine sustained-release microsphere: HeFei University of Technology's controlled release drug institute provides, and particle diameter is less than 100 μ m, content of dispersion 20%, 30 days medicament slow release time.
PLGA-5-fluorouracil slow release microsphere: HeFei University of Technology's controlled release drug institute provides, and particle diameter is less than 100 μ m, content of dispersion 20%, 30 days medicament slow release time.
The PLGA microsphere: Wu Hu Zhongren Pharmaceutical Co., Ltd provides, and particle diameter is less than 100 μ m.
Gemcitabine: Hao Sen pharmaceutcal corporation, Ltd in Jiangsu provides
Iodized oil: Guerbet, Roissy, France.06LU003B effect duration: 2009-03
Two. the inoculation of cell culture and tumor strain
With embodiment 8.
Three. the animal grouping
Be divided into 8 groups at random with transplanting successful tumor bearing nude mice:
The A1 group: the tumor body is implanted into PLGA-Gemcitabine sustained-release microsphere gemcitabine 5mg/kg
The A2 group: the tumor body is implanted into PLGA-Gemcitabine sustained-release microsphere gemcitabine 2.5mg/kg
The A3 group: the tumor body is implanted into PLGA-Gemcitabine sustained-release microsphere gemcitabine 1.25mg/kg
B group: injection gemcitabine gemcitabine 5mg/kg in the tumor bearing nude mice tumor body
The C group: the tumor bearing nude mice intraperitoneal injects gemcitabine gemcitabine 5mg/kg
The D group: tumor bearing nude mice tumor body is implanted into PLGA-5-fluorouracil slow release microsphere 5-fluorouracil 5mg/kg
The E group: tumor bearing nude mice tumor body is implanted into PLGA microsphere PLGA5mg/kg
The F group: tumor bearing nude mice does not give any treatment
Four. medicine preparation and medication
1. medicine preparation
PLGA-Gemcitabine sustained-release microsphere, PLGA-5-fluorouracil slow release microsphere, gemcitabine, PLGA microsphere all use the iodized oil dissolving, and every animal injection volume is 0.05ml.
2. medication
Transplanted tumor size begins administration during for 0.6cm-0.8cm.Under aseptic condition,, adopt the 1ml injector syringe to connect the 22G injection needle and inject in the tumor above-mentioned suspension or intraperitoneal medicine and iodized oil mixing.
Five. observation index observing time is 24 days
1. animals administer afterreaction
The observation of animal local response, general reaction and survival state after the administration.
2. the observation of the weight of animals
Before the administration and after the administration 4,8,12,16,20,24 days, with electronics balance measurement tumor-bearing mice body weight.
3. the observation of gross tumor volume
Before the administration and after the administration 4,8,12,16,20,24 days,, draw tumor growth curve with vernier caliper measurement tumor size.
Gross tumor volume computing formula: V=(A * B
2)/2A is a major diameter, and B is a minor axis.
The relative rate of increase computational methods of tumor:
(a certain point of observation tumor average volume-administration pre-neoplastic average external volume)/matched group tumor average volume
4. tumor weight is measured
Measure tumor weight after putting to death animal, calculate tumour inhibiting rate, computational methods:
Tumor control rate=(the average tumor of the average tumor weight/matched group of 1-administration group is heavy) * 100%
5. immunohistochemistry
Experiment finishes the back and puts to death animal, gets tumor tissues, and section back row immunohistochemical staining is measured vegf expression situation in the tumor tissues.
The immunohistochemical experiment material:
One VEGF antibody (mouse-anti human monoclonal antibodies) 0.1ml
The general two step method 3ml of PV-9000
Two anti-reagent 1: polymer adjuvant
Reagent 2: the goat antirabbit of Radix Cochleariae officinalis enzyme labelling/mice IgG polymer
DAB colour reagent box
Above reagent is provided by Beijing Bioisystech Co., Ltd of Golden Bridge
Sodium citrate citric acid dimethylbenzene dehydrated alcohol 95% ethanol 85% ethanol 70% ethanol
50% alcohol hydrochloric acid ethanol differentiation liquid (70% alcoholic solution that contains 0.25%HCL), 1% ammonia
Above reagent is provided by Beijing chemical reagents corporation
6. experimental implementation step:
(1) dewaxing:
Dimethylbenzene (1) 20min → dimethylbenzene (2) 20min → 100% ethanol (1) 20min → 100% ethanol (2) 20min → 95% ethanol 10min → 85% ethanol 10min → 70% ethanol 10min → 50% ethanol 10min → PBS crosses three times.
(2) antigen retrieval (microwave method):
High fiery 3-4min heating citrate buffer to boil → from PBS, take out section, immerse citrate buffer, high fiery 0.5-1min to the fiery 4min that boils → hang down → in be cooled to room temperature → PBS under low fiery 4min (being total to 12-14min) → room temperature and cross three times.
(3) 3%H2O2 incubated at room 5-10 minute, to eliminate the activity of endogenous peroxydase, PBS flushing 2 minutes 3 times.
(4) one anti-(the diluting by 1: 5 according to description) that adds the proper proportion dilution spent the night for 4 ℃.
(5) PBS 2min * 3 drip reagent I (ploymer helper), and 37oC was hatched 30 minutes.
(6) PBS 2min * 3 drip reagent II (ploy peroxidase-anti-mouse/rabbit IgG), hatch 30 minutes for 37 ℃.
(7) PBS 3min * 3, the DAB chromogenic reagent. mirror observes down decision termination time → washes color development stopping → haematoxylin from the beginning to redye 1-5min → 10-30sec of washing → hydrochloride alcohol color separation from the beginning.Mirror is observed decision color separation time → 1% ammonia down and is returned blue 5sec → washing from the beginning.
(8) haematoxylin is redyed, and it is transparent to dewater, mounting.
Place in (9) 50% ethanol 5min → 70% ethanol 5min → 85% ethanol 5min → 95% ethanol 5min → dehydrated alcohol 5min → air and read sheet after 3min → dimethylbenzene (1) 10min → dimethylbenzene (2) 10min → neutral gum mounting → section sealing is good.
7. the result judges:
The positive cell of brown particle appears with endochylema, 10 high power fields of random observation, keep the score by staining power earlier: non-coloring is 0 minute, and light brown is 1 minute, and brown is 2 minutes, dark-brown is 3 minutes, keep the score according to the shared percentage rate of positive cell: positive cell＜10% is 0 minute again, 10%～be 1 minute, 30%～be 2 minutes, 50%～70% is 3 minutes, and＞70% is 4 minutes.Both sums 〉=2 minute can be judged to positive and classification: 2～3 are divided into the weak positive (+), 4～5 is divided into the medium positive (++), 6～7 and is divided into strong positive (+++).
Six. statistical analysis
This experimental data represents that with mean ± SD the spss11.5 software kit carries out statistical analysis, the enumeration data X 2 test, and measurement data is checked with t, and the p value thinks that less than 0.05 significant difference is arranged.
Seven. the result
1. animals administer afterreaction:
(1) part and general reaction
Asthenia occurred on the 3rd day after A1 group, the administration of A2 group, do not take food, weight loss, skin color is rubescent, and this phenomenon continued about 2 weeks.The 2nd day animal activity reduces after A3 group, B group, C group, the administration of D group, and diet descends, and takes a turn for the better the no above-mentioned reaction of E group F group after 2 days.
(2) viewing duration animals survived situation
Dead 1 of A1 treated animal, B are organized dead 2, and C organizes dead 1.Animal all shows as movable the minimizing before death, do not take food, and diarrhoea, skin color is rubescent, sialorrhea.
(3) variation of viewing duration the weight of animals
The treatment group compares with matched group before administration, zero difference between the body weight, and the P value is all greater than 0.05.Administration (B group) and intraperitoneal administration group (C group) body weight are all on a declining curve in PLGA-Gemcitabine sustained-release microsphere treatment group after the administration (A1 group, A2 group, A3 group), the gemcitabine tumor, wherein comparatively obvious with the body weight decline in the 4th day after administration of A1 group, A2 group, B group, C group, all the other are respectively organized body weight and all present ascendant trend.A1, A2 group after administration the 4th day, A3 group after administration the 8th day, B, C organize after after the administration the 12nd day, and body weight is in rising trend.Treatment group body weight is put and comparison in 0 day, matched group each observing time after administration
Put and comparison in 0 day, the equal no difference of science of statistics of result, P＞0.05 (table 5) each observing time after 0 day.
Table 5: the variation of treatment group and control animals body weight
Annotate:
*Represent that each, point was compared with 0 day observing time.
2. the variation of gross tumor volume
A1, A2 group gross tumor volume was compared with matched group after administration in 16,20,24 days, and the result has significant difference, and the P value is all less than 0.05.A3 group, B group, D group gross tumor volume were compared with matched group after administration in 16 days, and the result has significant difference, and the P value is all less than 0.05.C group, E group gross tumor volume are put in above-mentioned observing time and are compared no difference of science of statistics (table 6) with matched group.
A1, A2 group tumor propagation is slow, and curve is mild, and is wherein obvious with the A1 group.A3, D, B, C group after administration before 12 days curve comparatively mild, become after 12 days precipitous, the most obvious with the C group.Secondly matched group curve steepest is E group (Fig. 5).
Table 6: the variation of treatment group and matched group gross tumor volume
Annotate: the treatment group is compared with matched group
3.PLGA-administration chemotherapy of mesenchyma stroma group in the Gemcitabine sustained-release microsphere tumor (A1 group, A2 group, A3 group) the relative rate of increase of tumor
A1 group tumor propagation speed is the slowest, secondly is the A2 group, and the A3 group is the fastest, and the PLGA-Gemcitabine sustained-release microsphere is dose-effect relationship to the inhibitory action of tumor.(Fig. 6)
4. treatment group tumour inhibiting rate
The A1 group is the strongest to the inhibitory action of tumor, and secondly inhibitory rate for the A2 group, is 40% to 66%; The A3 group is 23%, organizes (24%) near (Fig. 7) with D, and the tumour inhibiting rate of B group, C group, E group is respectively 30%, 20%, 5%.
5.VEGF expression
A1 group, A2 group, A3 organize, F group VEGF all has positive expression, and wherein A1 group vegf expression is minimum, and secondly the A2 group compares with matched group, the result has significant difference, P＜0.05, and A3 group vegf expression is the strongest, compare there was no significant difference as a result, P＞0.05 (table 7) with matched group.
Table 7A1 group, A2 group, A3 group, F group vegf expression situation
Annotate: A1 group, A2 group, A3 group are compared with the F group respectively.
This test explanation:
1.PLGA-the Gemcitabine sustained-release microsphere chemotherapy of mesenchyma stroma can significantly suppress growth of tumor.
2.PLGA-the Gemcitabine sustained-release microsphere chemotherapy of mesenchyma stroma is dose-effect relationship to the inhibitory action of tumor.
3.PLGA-the Gemcitabine sustained-release microsphere chemotherapy of mesenchyma stroma can significantly suppress the expression of tumor tissues VEGF, and inhibitory action is strengthened with the increase of dosage.
4.PLGA-the Gemcitabine sustained-release microsphere chemotherapy of mesenchyma stroma is safe.
To sum up, gemcitabine and polylactic acid-glycollic acid are made sustained-release micro-spheres, directly deliver to the capable regional sustained-release chemotherapy of tumor locus with syringe, gemcitabine is slowly disengaged, tumor locus drug level height, long action time, action intensity is big, lethality to tumor cell is big, thereby can improve the curative effect of tumor; Medicine is concentrated and to be acted on the part, the systemic drug amount seldom, untoward reaction is minimum.After medicine was released and finished, polylactic acid-glycollic acid began to be degraded to lactic acid and glycolic, further is degraded to carbon dioxide and water, and animal body is not had influence.
Claims (7)
1. a PLGA Gemcitabine sustained-release microsphere is characterized in that: made with 95: 5～35: 65 weight ratio by adjuvant polylactic acid-glycollic acid and crude drug gemcitabine.
2. PLGA Gemcitabine sustained-release microsphere according to claim 1 is characterized in that: described gemcitabine is antitumor drug 2 '-deoxidation-2 ', 2 '-difluoro cytidine hydrochlorate.
3. PLGA Gemcitabine sustained-release microsphere according to claim 1 is characterized in that: lactic acid and glycolic molecular proportion are 95: 5～10: 90 in the described polylactic acid-glycollic acid molecule.
4. PLGA Gemcitabine sustained-release microsphere according to claim 1 is characterized in that: described microsphere diameter is 1 μ m～250 μ m.
5. the preparation method of a PLGA Gemcitabine sustained-release microsphere is characterized in that: pulverize gemcitabine and make the fine powder of diameter less than 20 μ m; Polylactic acid-glycollic acid is dissolved in the solvent, presses formula proportion the gemcitabine fine powder is added in the PLGA solution, mix homogeneously, spray drying under the vacuum, sieve the PLGA Gemcitabine sustained-release microsphere; The weight ratio of polylactic acid-glycollic acid and gemcitabine is 95: 5～35: 65.
6. the preparation method of a kind of PLGA Gemcitabine sustained-release microsphere according to claim 5 is characterized in that: described solvent is selected from one or more in dichloroethanes, dichloromethane, chloroform, oxolane, acetone, ethyl acetate and the carbon dioxide.
7. the preparation method of a kind of PLGA Gemcitabine sustained-release microsphere according to claim 5 is characterized in that: described microsphere can also adopt conventional method preparations such as solvent method, emulsion process, supercritical methanol technology.
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