The nanoparticle pharmaceutical that bevacizumab and dexamethasone for intravitreal injection carry altogetherTechnical field
The present invention discloses the nanoparticle pharmaceutical delivering that a kind of bevacizumab for intravitreal injection and dexamethasone carry altogetherSystem, while the preparation method of the nanoparticle pharmaceutical delivery system is additionally provided, it is related to pharmaceutical technology field.
Background technique
Glucocorticoid medicine is clinical ophthalmology common drug, intraocular to retina choroid new vessels etc. HypertrophicThe entophthamia such as lesion and uveitis, retinal vasculitis have definite curative effect.In view of its systemic toxic side effect and eyeThe distinctive barrier structure of ball, it is considered that intravitreal approach is administered better than oral or intravenous.But word in vitreumDrug metabolism is eliminated very fast after administration, and drug effect is of short duration；Multiple dosing then will increase intraocular hemorrhage, cataract, detachment of retina etc.The incidence of complication.Poly lactic-co-glycolic acid (PLGA) nanoparticle is have biological degradability and biocompatibility novelAtrigel, it is expected to which part reaches long-acting, stable drug concentration within the eye.
Dexamethasone is glucocorticoid medicine, has anti-inflammatory, antiendotoxin, immune, Hemorrhagic shock and enhancing is inhibited to answerSwash the pharmacological actions such as reaction, therefore is widely used in each section and treats a variety of diseases, such as autoimmune disease, allergy, inflammation, asthmaAnd dermatology and ophthalmology disease.Studies have shown that glucocorticoid can by release local noradrenaline vasoconstriction,Reduction or the expression of silencing vascular endothelial growth factor (VEGF), induction of vascular endothelial Apoptosis inhibit blood vessel endothelium proliferation.Glucocorticoid has quick, powerful and nonspecific anti-inflammatory effect simultaneously.It is effective to various inflammation.
Bevacizumab (Avastin®) be recombination Humanized monoclonal antibodies, it comprises the structural area of human antibody andIn combination with the complementary determining region of the source of mouse monoclonal antibody of VEGF, it can be combined with human vascular endothelial growth factor (VEGF) and block its biologyActivity.It is the medicine of the inhibition Tumor Angiongesis of first, the U.S. listing that gets the Green Light.The U.S. has doctor clinical successively in recent yearsIt is found when treatment, since Avastin effective object is anti-Vascular Endothelial outgrowth factor (VEGF), it is abnormal macula area can be neutralizedHigh concentration eye illness VEGF, thus inhibit age-related macular degeneration (AMD) deteriorate, it is common for treating this elderly, the eye disease successful that can lead to severe visual damage is very big.Avastin®Because confirming that there is treatment retinopathy by clinical practiceEffect and cheap, is widely applied by oculist always.
Treated with combined medication is concerned because that may generate the therapeutic effect for being substantially better than drug single therapy.RationallyDrug combination can play the synergistic effect of drug effectively to improve curative effect, can also be dropped by reducing single pharmaceutical quantitiesLow adverse reaction.
Summary of the invention
The present invention discloses the Nano medication that a kind of bevacizumab for intravitreal injection and dexamethasone carry altogether, solvesThe deficiency of dexamethasone half-life short ensure that the high activity of bevacizumab in preparation process.
The present invention provides the nanoparticle pharmaceuticals that a kind of bevacizumab for intravitreal injection and dexamethasone carry altogether, areSurface for intravitreal injection connects bevacizumab, and inside loads the nanoparticle pharmaceutical delivery system of dexamethasone.NanoparticlePLGA in matrix enables to dexamethasone slowly steadily to discharge；Bevacizumab is connected to by covalent bond using PLGA as matrixNanoparticle surface, still ensure that bevacizumab and VEGF effect activity；Two kinds of drug synergisms, drug combination are significantImprove anti-angiogenesis effect.
Invention further provides the nanometer medicines that a kind of bevacizumab for intravitreal injection and dexamethasone carry altogetherThe preparation method of object.
The Nano medication that a kind of bevacizumab and dexamethasone for intravitreal injection of the present invention carries altogether, passes throughFollowing technical scheme is realized:
By the ground plug in the polymer nanoparticle of the core-shell structure prepared using high molecular polymer PLGA as carrier material, nanoparticleMeter Song and the bevacizumab of nanoparticle surface connection are constituted.
The system for the Nano medication that a kind of bevacizumab and dexamethasone for intravitreal injection of the present invention carries altogetherPreparation Method, comprising the following steps:
1) the PLGA nanoparticle of dexamethasone is contained using emulsion-solvent evaporation method preparation；
2) method for using chemical coupling, sends out the amino of the carboxyl and bevacizumab in the PLGA of nanoparticle using activatorRaw covalent reaction, antibody is connect with nanoparticle.
The system for the Nano medication that a kind of bevacizumab and dexamethasone for intravitreal injection of the present invention carries altogetherPreparation Method, it is characterised in that specifically includes the following steps:
1) 5~20mg dexamethasone is weighed respectively, and 20mg PLGA is dissolved in acetone, ultrasonic dissolution, as organic phase；
2) polyvinyl alcohol (PVA) aqueous solution for taking 5~20mL 0.5% ~ 5%, as water phase；
3) organic phase is added dropwise in water phase under the conditions of magnetic agitation, room temperature continues to stir at low speed, and volatilization eliminates dichloroMethane eccentric cleaning 3 times, is collected dexamethasone-PLGA nanoparticle and is lyophilized；
4) take the above-mentioned nanometer of 10mg in 10ml 1mol/L hydrochloric acid, and be added 5mg n-hydroxysuccinimide (NHS) and20mg 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDC) is centrifuged off after activating nanoparticle 4hClearly；
5) nanoparticle and 5~20mg bevacizumab activated is incubated in jointly in pH7.4 PBS, and connection 3~for 24 hours, eccentric cleaning 3Salt ion in the secondary bevacizumab and buffer not connected with removing, collects and is lyophilized and cut down to get the shellfish for intravitreal injectionThe Nano medication delivery system that monoclonal antibody and dexamethasone carry altogether.
PLGA of the present invention is carboxy blocking, model 7525 1.5A or 5050 2A.
Dexamethasone of the present invention and PLGA mass ratio are (1~4): 4.
Bevacizumab of the present invention and dexamethasone-PLGA nanoparticle mass ratio are 2: (1~4).
The nanoparticle (Bev/Dex-PLGA NPs) that bevacizumab and dexamethasone of the present invention carry altogether is freeze-drying systemAgent, belongs to intravitreal system, and solvent is usually sterile isotonic aqueous solution.
The positive effect of the present invention is:
By combining dexamethasone and bevacizumab using PLGA as the nanoparticle of matrix, it is prepared for bevacizumab and dexamethasoneThe nanoparticle (Bev/Dex-PLGA NPs) carried altogether, forms the Nano medication delivery system with core-shell structure.It solvesThe deficiency of dexamethasone half-life short ensure that the high activity of bevacizumab in preparation process, be acted synergistically by the two effectiveImprove anti-angiogenesis effect.The Nano medication delivery system that the bevacizumab and dexamethasone that the present invention constructs carry altogether, is oneThe excellent delivery system of kind, is expected to the treatment applied to age-related macular degeneration (AMD).
Detailed description of the invention:
Fig. 1 is the grain size distribution of nanoparticle；
Fig. 2 is the potential image of nanoparticle；
Fig. 3 is that the SEM of nanoparticle schemes；
Fig. 4 is the tablets in vitro curve of nanoparticle；
Fig. 5 is the experiment of bevacizumab high performance liquid chromatography；
Fig. 6 is the experiment of bevacizumab circular dichroism spectra；
Fig. 7 is bevacizumab fluorescence spectrum experiments；
Fig. 8 is the cytotoxicity experiment of nanoparticle；
Fig. 9 is that the internal anti-angiogenesis of nanoparticle is tested.
Specific embodiment
Specific implementation method of the invention, by following embodiment for example, but protection scope of the present invention do not limit toIn this.
Embodiment 1
1) 5mg dexamethasone is weighed respectively, and 7525 1.5A of 20mg PLGA is dissolved in acetone, ultrasonic dissolution, as organicPhase；
2) polyvinyl alcohol (PVA) aqueous solution for taking 5mL 0.5%, as water phase；
3) organic phase is added dropwise in water phase under the conditions of magnetic agitation, room temperature continues to stir at low speed, and volatilization eliminates dichloroMethane eccentric cleaning 3 times, is collected dexamethasone-PLGA nanoparticle and is lyophilized；
4) take the above-mentioned nanometer of 10mg in 10ml 1mol/L hydrochloric acid, and be added 5mg n-hydroxysuccinimide (NHS) and20mg 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDC) is centrifuged off after activating nanoparticle 4hClearly；
5) nanoparticle and 5mg bevacizumab activated is incubated in jointly in pH7.4 PBS, connects 12h, eccentric cleaning 3 times to removeRemove salt ion in not connected bevacizumab and buffer, collect and be lyophilized to get for intravitreal injection bevacizumab andThe Nano medication delivery system that dexamethasone carries altogether.
Embodiment 2
1) 10mg dexamethasone is weighed respectively, and 7525 1.5A of 20mg PLGA is dissolved in acetone, ultrasonic dissolution, as organicPhase；
2) polyvinyl alcohol (PVA) aqueous solution for taking 5mL 0.5%, as water phase；
3) organic phase is added dropwise in water phase under the conditions of magnetic agitation, room temperature continues to stir at low speed, and volatilization eliminates dichloroMethane eccentric cleaning 3 times, is collected dexamethasone-PLGA nanoparticle and is lyophilized；
4) take the above-mentioned nanometer of 10mg in 10ml 1mol/L hydrochloric acid, and be added 5mg n-hydroxysuccinimide (NHS) and20mg 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDC) is centrifuged off after activating nanoparticle 4hClearly；
5) nanoparticle and 5mg bevacizumab activated is incubated in jointly in pH7.4 PBS, connects 12h, eccentric cleaning 3 times to removeRemove salt ion in not connected bevacizumab and buffer, collect and be lyophilized to get for intravitreal injection bevacizumab andThe Nano medication delivery system that dexamethasone carries altogether.
Embodiment 3
1) 10mg dexamethasone is weighed respectively, and 7525 1.5A of 20mg PLGA is dissolved in acetone, ultrasonic dissolution, as organicPhase；
2) polyvinyl alcohol (PVA) aqueous solution for taking 5mL 2%, as water phase；
3) organic phase is added dropwise in water phase under the conditions of magnetic agitation, room temperature continues to stir at low speed, and volatilization eliminates dichloroMethane eccentric cleaning 3 times, is collected dexamethasone-PLGA nanoparticle and is lyophilized；
4) take the above-mentioned nanometer of 10mg in 10ml 1mol/L hydrochloric acid, and be added 5mg n-hydroxysuccinimide (NHS) and20mg 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDC) is centrifuged off after activating nanoparticle 4hClearly；
5) nanoparticle and 5mg bevacizumab activated is incubated in jointly in pH7.4 PBS, connects 12h, eccentric cleaning 3 times to removeRemove salt ion in not connected bevacizumab and buffer, collect and be lyophilized to get for intravitreal injection bevacizumab andThe Nano medication delivery system that dexamethasone carries altogether.
Embodiment 4
1) 10mg dexamethasone is weighed respectively, and 7525 1.5A of 20mg PLGA is dissolved in acetone, ultrasonic dissolution, as organicPhase；
2) polyvinyl alcohol (PVA) aqueous solution for taking 10mL 2%, as water phase；
3) organic phase is added dropwise in water phase under the conditions of magnetic agitation, room temperature continues to stir at low speed, and volatilization eliminates dichloroMethane eccentric cleaning 3 times, is collected dexamethasone-PLGA nanoparticle and is lyophilized；
4) take the above-mentioned nanometer of 10mg in 10ml 1mol/L hydrochloric acid, and be added 5mg n-hydroxysuccinimide (NHS) and20mg 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDC) is centrifuged off after activating nanoparticle 4hClearly；
5) nanoparticle and 5mg bevacizumab activated is incubated in jointly in pH7.4 PBS, connects 12h, eccentric cleaning 3 times to removeRemove salt ion in not connected bevacizumab and buffer, collect and be lyophilized to get for intravitreal injection bevacizumab andThe Nano medication delivery system that dexamethasone carries altogether.
Embodiment 5
1) 10mg dexamethasone is weighed respectively, and 5050 2A of 20mg PLGA is dissolved in acetone, ultrasonic dissolution, as organic phase；
2) polyvinyl alcohol (PVA) aqueous solution for taking 5mL 2%, as water phase；
3) organic phase is added dropwise in water phase under the conditions of magnetic agitation, room temperature continues to stir at low speed, and volatilization eliminates dichloroMethane eccentric cleaning 3 times, is collected dexamethasone-PLGA nanoparticle and is lyophilized；
4) take the above-mentioned nanometer of 10mg in 10ml 1mol/L hydrochloric acid, and be added 5mg n-hydroxysuccinimide (NHS) and20mg 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDC) is centrifuged off after activating nanoparticle 4hClearly；
5) nanoparticle and 5mg bevacizumab activated is incubated in jointly in pH7.4 PBS, connects 12h, eccentric cleaning 3 times to removeRemove salt ion in not connected bevacizumab and buffer, collect and be lyophilized to get for intravitreal injection bevacizumab andThe Nano medication delivery system that dexamethasone carries altogether.
Embodiment 6
1) 10mg dexamethasone is weighed respectively, and 7525 1.5A of 20mg PLGA is dissolved in acetone, ultrasonic dissolution, as organicPhase；
2) polyvinyl alcohol (PVA) aqueous solution for taking 5mL 2%, as water phase；
3) organic phase is added dropwise in water phase under the conditions of magnetic agitation, room temperature continues to stir at low speed, and volatilization eliminates dichloroMethane eccentric cleaning 3 times, is collected dexamethasone-PLGA nanoparticle and is lyophilized；
4) take the above-mentioned nanometer of 10mg in 10ml 1mol/L hydrochloric acid, and be added 5mg n-hydroxysuccinimide (NHS) and20mg 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDC) is centrifuged off after activating nanoparticle 4hClearly；
5) nanoparticle and 10mg bevacizumab activated is incubated in jointly in pH7.4 PBS, connects 12h, eccentric cleaning 3 times to removeRemove salt ion in not connected bevacizumab and buffer, collect and be lyophilized to get for intravitreal injection bevacizumab andThe Nano medication delivery system that dexamethasone carries altogether.
Embodiment 7
1) 10mg dexamethasone is weighed respectively, and 7525 1.5A of 20mg PLGA is dissolved in acetone, ultrasonic dissolution, as organicPhase；
2) polyvinyl alcohol (PVA) aqueous solution for taking 5mL 2%, as water phase；
3) organic phase is added dropwise in water phase under the conditions of magnetic agitation, room temperature continues to stir at low speed, and volatilization eliminates dichloroMethane eccentric cleaning 3 times, is collected dexamethasone-PLGA nanoparticle and is lyophilized；
4) take the above-mentioned nanometer of 10mg in 10ml 1mol/L hydrochloric acid, and be added 5mg n-hydroxysuccinimide (NHS) and20mg 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDC) is centrifuged off after activating nanoparticle 4hClearly；
5) nanoparticle and 10mg bevacizumab activated is incubated in jointly in pH7.4 PBS, connects 6h, eccentric cleaning 3 times to removeRemove salt ion in not connected bevacizumab and buffer, collect and be lyophilized to get for intravitreal injection bevacizumab andThe Nano medication delivery system that dexamethasone carries altogether.
Embodiment 8
1) 10mg dexamethasone is weighed respectively, and 7525 1.5A of 20mg PLGA is dissolved in acetone, ultrasonic dissolution, as organicPhase；
2) polyvinyl alcohol (PVA) aqueous solution for taking 5mL 2%, as water phase；
3) organic phase is added dropwise in water phase under the conditions of magnetic agitation, room temperature continues to stir at low speed, and volatilization eliminates dichloroMethane eccentric cleaning 3 times, is collected dexamethasone-PLGA nanoparticle and is lyophilized；
4) take the above-mentioned nanometer of 10mg in 10ml 1mol/L hydrochloric acid, and be added 5mg n-hydroxysuccinimide (NHS) and20mg 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDC) is centrifuged off after activating nanoparticle 4hClearly；
5) nanoparticle and 10mg bevacizumab activated is incubated in jointly in pH7.4 PBS, connects 18h, eccentric cleaning 3 times to removeRemove salt ion in not connected bevacizumab and buffer, collect and be lyophilized to get for intravitreal injection bevacizumab andThe Nano medication delivery system that dexamethasone carries altogether.
Effect experimental examples 1 (characterization of nanoparticle: partial size, current potential, form)
Experimental material:
Bev/Dex-PLGA NPs, silicon wafer, deionized water of embodiment 1 etc..
Experimental procedure:
With the Bev/Dex-PLGA NPs concentration of deionized water dilution embodiment 1 to 1 mg/mL, it is analyzed using laser particle analyzerPartial size and Potential distribution.
With the Bev/Dex-PLGA NPs concentration of deionized water dilution embodiment 1 to 1 mg/mL, it is added dropwise one and drops to silicon wafer loadWater is eliminated in room temperature volatilization after on body.After sample metal spraying processing on silicon wafer, scanning electron microscope is used under 3 kV voltages(SEM) nanoparticle configuration of surface is observed
Experimental result:
Using the partial size and Potential distribution of the Bev/Dex-PLGA NPs of laser particle analyzer analysis embodiment 1, particle diameter distribution is such asShown in Fig. 1, Potential distribution is as shown in Figure 2.Droplet measurement the results show that the average grain diameter of Bev/Dex-PLGA NPs in 150-Between 200nm, current potential is in 0mV or so.
Using the configuration of surface of the Bev/Dex-PLGA NPs in scanning electron microscope (SEM) observation embodiment 6, such as schemeShown in 3.SEM is the results show that Bev/Dex-PLGA NPs is rounded, and surface is smooth, and size is between 150-200nm.
Effect experimental examples 2 (drugloading rate of nanoparticle and release in vitro behavior are investigated)
Experimental material:
Bev/Dex-PLGA NPs, dexamethasone, acetonitrile, 0.22 μm of miillpore filter, BCA kit in Examples 1 to 8 etc..
Experimental procedure:
Using the encapsulating of dexamethasone in the Bev/Dex-PLGA NPs in high performance liquid chromatography (HPLC) measurement Examples 1 to 8Rate:
1) 10mg freeze-drying Bev/Dex-PLGA NPs is weighed, is dissolved in 1mL acetonitrile, spiral dissolves 10 min, is added 9mL's40% acetonitrile solution, 0.22 μm of filtering with microporous membrane, filtered fluid carry out chromatography.
2) HPLC condition: mobile phase acetonitrile: water (40:60, v/v) solution, flow velocity 1mL/min, Detection wavelength 240nm, into20 μ L of sample amount.
3) calculate according to the following formula quality/investment nanoparticle gross mass of drugloading rate (DL%)=measure dexamethasone ×100%, quality/investment dexamethasone quality × 100% of encapsulation rate (EE%)=measure dexamethasone.
Using the connection of bevacizumab in the Bev/Dex-PLGA NPs in BCA kit method measurement Examples 1 to 8Rate:
1) supernatant in nanoparticle antibody connection procedure is collected, the free shellfish in supernatant is detected by BCA kit and is cut downMonoclonal antibody.
2) the free bevacizumab of calculating antibody bonding ratio (CB%)=investment bevacizumab gross mass-according to the following formulaQuality/Bev/Dex-PLGA nanoparticle gross mass.Measuring bonding ratio is 20~40%(table 1)
Using the release in vitro behavior of the Bev/Dex-PLGA NPs in the method analysis embodiment 6 of dialysis:
1) vitro Release Medium: 7.4 PBS(1% Tween-80 of pH), dialysis bag retention molecular weight (MWCO, 8kD).
2) sample point: 0.5h, 1h, 2h, 4h, 6h, 8h, 12h, 1d, 2d, 3d.HPLC detects dexamethasone content, and calculatesThe preparation of dexamethasone.
Experimental result:
Using the encapsulating of dexamethasone in the Bev/Dex-PLGA NPs in high performance liquid chromatography (HPLC) measurement Examples 1 to 8Rate, measuring encapsulation rate is 50% ~ 75%(table 1).
Using the connection of bevacizumab in the Bev/Dex-PLGA NPs in BCA kit method measurement Examples 1 to 8Rate, measuring bonding ratio is 20~40%(table 1)
Using the release in vitro behavior of the Bev/Dex-PLGA NPs in the method analysis embodiment 6 of dialysis, as a result as shown in Figure 4There is slow release behavior after burst release in Bev/Dex-PLGA NPs, adds up release rate in 72 h up to 81.3%.Release behavior tableIt is now sustained again to be first released, and slow release effect is obvious.This is attributed to the fact that the presence of the core-shell structure and surface antibody of nanoparticle,After the degradation characteristic of PLGA has delayed the release of dexamethasone, surface to be coupled monoclonal antibody, releasing for dexamethasone is further delayedIt puts, maintains the treatment concentration of dexamethasone within a certain period of time.
Nanoparticle dexamethasone drugloading rate, encapsulation rate and bevacizumab bonding ratio obtained by 1 Examples 1 to 8 of table
| DL(%) | EE(%) | CB(%) |
| Embodiment 1 | 10.64±0.56 | 53.24±0.77 | 22.47±0.13 |
| Embodiment 2 | 19.08±0.06 | 57.83±0.46 | 25.72±0.09 |
| Embodiment 3 | 22.07±0.47 | 66.88±0.03 | 36.67±0.08 |
| Embodiment 4 | 20.89±0.54 | 63.32±0.67 | 29.87±0.04 |
| Embodiment 5 | 18.56±0.61 | 56.23±0.08 | 24.32±0.37 |
| Embodiment 6 | 23.21±0.35 | 70.33±0.09 | 32.74±0.08 |
| Embodiment 7 | 23.61±0.42 | 71.56±0.78 | 33.33±0.52 |
| Embodiment 8 | 24.71±0.66 | 74.87±0.33 | 37.64±0.12 |
* DL is dexamethasone drugloading rate；EE is dexamethasone encapsulation rate；CB is bevacizumab bonding ratio.
Effect experimental examples 3 (bevacizumab stability study)
Experimental material:
Apply free bevacizumab, acetonitrile, the BCA kit, phosphorus in the nanoparticle Incubating Solution of the Bev/Dex-PLGA NPs in example 6Acid dihydride sodium, disodium hydrogen phosphate etc..
Experimental procedure:
To investigate influence of the Bev/Dex-PLGA NPs preparation process to bevacizumab stability, we take the Bev/ applied in example 6Free bevacizumab in the nanoparticle Incubating Solution of Dex-PLGA NPs is investigated by HPLC, circular dichroism spectra, fluorescence spectrum respectivelyIts level-one, second level, the stability of tertiary structure.
With the stability of HPLC detection bevacizumab primary structure, Herceptin solution is diluted or is concentrated before detectionTo 0.5mg/mL, HPLC condition: mobile phase acetonitrile: water (40:60, v/v) solution, flow velocity 1mL/min, Detection wavelength 240nm, into20 μ L of sample amount.
Using the stability of circular dichroism spectrometer detection bevacizumab secondary structure, Herceptin solution is diluted before detectionOr it is concentrated to 10-5Mol/L, circular dichroism spectra scanning wavelength 190 ~ 260nm of range detect 25 DEG C of temperature.Using Jasco softwareBackground correction solution signal, is converted to mean residue ellipticity for result.
Using the stability of Fluorescence Spectrometer detection bevacizumab tertiary structure, Herceptin solution is diluted before detectionOr it is concentrated to 1mol/L, fluorescence spectrum excitation wavelength 280nm, 305 ~ 550nm of launch wavelength.Background correction solution letter before testingNumber, record fluorescence spectra.
Experimental result:
The stability result of bevacizumab primary structure such as Fig. 5 in nanoparticle Incubating Solution, with natural antibody appearance time almost oneIt causes, illustrates that the antibody in Incubating Solution remains complete antibody structure, there is no denaturation to assemble.
Secondary structure is the basis for guaranteeing its bioactivity, and the change of secondary structure may influence proteinActivity.The bevacizumab secondary structure variation such as bevacizumab in Fig. 6, PBS(pH 7.4) presents apparent negative at 212nmPeak, and the antibody negative peak in Incubating Solution is still obvious, illustrates that secondary structure does not almost change.
The activity of protein keeps relying on its distinctive tertiary structure, and when the tertiary structure of protein changes, activity mustIt will be destroyed.There is fluorescence at 330nm in the bevacizumab tertiary structure variation such as bevacizumab in Fig. 7, PBS(pH 7.4)Spectrum peak, and the antibody fluorescence spectrum peak in Incubating Solution and PBS coincide substantially, illustrate that tertiary structure does not almost change.
Above results proved that Bev/Dex-PLGA NPs of the invention ensure that the high activity of wherein bevacizumab.
Effect experimental examples 4 (cytotoxicity experiment)
Experimental material:
Human RPE Cells in Vitro (ARPE-19 cell), DMEM/F12 culture medium, fetal calf serum, Pen .- Strep,96 microwell plates, dexamethasone, bevacizumab, Bev/Dex-PLGA NPs of embodiment 6, MTT, DMSO etc..
Experimental procedure:
In DMEM/F12 culture medium (10% fetal calf serum, 1% Pen .- Strep), culture bottle is set for ARPE-19 cell cultureIn 37 DEG C, 5% CO2 incubator.The cell of logarithmic growth phase is tested.With 1 × 104The density in a/hole will in pairThe cell inoculation of number phase is in 96 microwell plates, and 37 DEG C of cultures make its adherent for 24 hours, and second day removal culture solution is separately added into and containsAfter 37 DEG C are incubated for for 24 hours, MTT examination is added in medicine (dexamethasone, bevacizumab, the Bev/Dex-PLGA NPs of embodiment 6) culture mediumAgent detects and calculates cell survival rate.
Experimental result:
As a result as shown in figure 8, dexamethasone, bevacizumab, Bev/Dex-PLGA NPs show as almost without toxicity, it was demonstrated thatIts ophthalmic administration safe and feasible, it is free of toxic effects to ocular cell ARPE-19.
Effect experimental examples 5 (anti-angiogenesis experiment)
Experimental material:
Fertilized eggs, methylcellulose, dexamethasone, bevacizumab, Bev/Dex-PLGA NPs of embodiment 6, physiological salineDeng.
Experimental procedure:
Influence using chick chorioallantoic membrane (CAM) experimental evaluation Bev/Dex-PLGA NPs to anti-angiogenesis.In embryonic developmentThe 4th day, the gas chamber of each egg opens one small window (diameter: ~ 10mm).It is with about 2 millimeters of carboxymethyl cellulose films of diameterPharmaceutical carrier.At embryonic period, embryonic phase the 6th day, drug containing dexamethasone, bevacizumab, the Bev/Dex-PLGA NPs of embodiment 6) carrier is lightGently it is implanted into each CAM.It is incubated for the 8th day, 3.7% formaldehyde is fixed and taken pictures.Calculate the blood vessel number in each CAM.For blood vessel amountChange, negative control group (physiological saline) value is set as 100%.
Experimental result:
CAM experimental result Avastin as shown in Figure 9 and dexamethasone can inhibit the growth of new blood vessel, to promoteThe significant decrease of vessel density.The blood vessel number of bevacizumab group and Dexamethasone group is reduced to 65.2% and 83.4%, and Bev/Dex-PLGA NPs group is reduced to 42.7%.These statistics indicate that, Bev/Dex-PLGA NPs anti-angiogenic rebirth effect of the inventionMost preferably, two kinds of drug synergisms, drug combination significantly improve anti-angiogenesis effect.
Conclusion:
The Nano medication delivery system Bev/ that a kind of bevacizumab and dexamethasone for intravitreal injection of the invention carries altogetherThe surface Dex-PLGA NPs is in smooth spheroidal, and average grain diameter is between 150-200nm, and current potential is in 0mV or so.Stall off a meter bale breakingEnvelope rate is in 50% to 75% range, and bevacizumab bonding ratio is in 20% to 40% range.Release behavior shows as first being released to be delayed againIt releases, and slow release effect is obvious.This is attributed to the fact that the presence of the core-shell structure and surface antibody of nanoparticle, and the degradation characteristic of PLGA prolongsDelay the release of dexamethasone further to have delayed the release of dexamethasone after surface is coupled monoclonal antibody, tieed up within a certain period of timeThe treatment concentration of dexamethasone is held.Bev/Dex-PLGA NPs anti-angiogenic rebirth excellent, two kinds of drug synergisms, connectionIt shares medicine and significantly improves anti-angiogenesis effect.