CN104606134B - A kind of double targeting composite nanoparticles for loading the HCPT of 7 ethyl 10 and its preparation method and application - Google Patents
A kind of double targeting composite nanoparticles for loading the HCPT of 7 ethyl 10 and its preparation method and application Download PDFInfo
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Abstract
The invention provides a kind of double targeting composite nanoparticles and its preparation method and application, further, the invention provides a kind of double targeting composite nanoparticles for loading the HCPT of 7 ethyl 10 and its preparation method and application, the double targeting composite nanoparticles for the HCPT of 7 ethyl of load 10 that the present invention is provided, not only drugloading rate is big, envelop rate is high, and extend the effective acting time of the HCPT of 7 ethyl 10 in vivo, and the combination for being effectively promoted nano-particle and tumour cell is acted on by active targeting, reduce toxic side effect of the medicine to normal cell, the therapeutic effect of medicine can be significantly improved.
Description
Technical field
The invention belongs to nano-medicament carrier field, and in particular to a kind of double targets of load SN38
To composite nanoparticle and its preparation method and application.
Background technology
Stomach cancer is one of most common malignant tumour in the world, is also one of death rate highest tumour, death toll exists
Position is ranked second in world's tumor mortality case.Most of patients is advanced metastatic disease or after surgical discectomy in diagnosis
There is relapse and metastasis, survival rate is less than 10% within 5 years.Irinotecan（CPT-11）It is be widely used in stomach cancer second line treatment hemizygous
Into soluble camptothecin derivative.Similar with other camptothecine mechanisms of action, it suppresses topoisomerase I, makes it
Inactivation, causes DNA single-strand breaks, hinders DNA replication dna to be synthesized with RNA, so as to suppress the division of cell.But, CPT-11 is being controlled
Therapeutic effect and clinical practice also receive a definite limitation, and its reason mainly has two aspects, first, and CPT-11 needs carboxylic in vivo
Active metabolite SN38 is changed into the presence of acid esters enzyme and plays treatment of cancer effect, but research shows that CPT-11 turns in human body
The efficiency for being melted into SN38 is only 2 ~ 8%, and this make it that the bioavilabilities of CPT-11 in vivo are extremely low；Secondly, CPT-11 has marrow
The toxic side effects such as suppression, great pain is brought to patient over the course for the treatment of.Therefore, if by CPT-11 active metabolite
SN38（SN38）It is directly used in curing gastric cancer, it will the defect for overcoming CPT-11 bioavilabilities low,
So as to improve chemotherapy effect.But SN38 strong-hydrophobicity makes its just rapid precipitation after human recycle system is entered, it is difficult to quilt
Lesion tissue absorbs, so as to limit its application clinically.Therefore, the solubility of SN38 in vivo how is improved, is extended
Its circulation time in human body and useful effect concentration of the medicine in lesions position is improved, be in curing gastric cancer using SN38
Difficult point.
The exploitation of targeted nano drug delivery system is the maximally efficient means that solve the above problems.On the one hand, nanometer medicine
Thing transmission system can be contained hydrophobicity chemotherapeutics, improve the solubility of hydrophobic drug in vivo, it is to avoid medicine
Thing is degraded by the various enzymes in blood, extends its internal effective cycle time；On the other hand, carrier is carried out using targeting ligand
Sex modification is targetted, medicine is concentrated in diseased region and discharges, improve drug availability and reduce its toxic side effect.But it is simple
For the design of tumor targets and exploitation nano-medicament carrier, often do not reach best therapeutic effect.On the one hand, single target
To nano-medicament carrier, it can only be combined with a target recipient of tumor cell surface, and this acceptor may be in therapeutic process
In because pathology, physiology, the influence of microenvironment factor lose combination and the mediation between part；On the other hand,
Effect of the expression degree of target site acceptor also to single targeting vector has a huge impact, and the overexpression behavior of acceptor
Only occur in some privileged sites and do not have popularity, and the part and target that these are likely to limit single targeting vector
The specific binding of acceptor, and then reduce its targeting and therapeutic effect.By contrast, double targeting vectors can provide multiple targets
Specific binding is realized to the acceptor of recognition site and target site, the diversity of target site acceptor is also ensure that, weakens very
To eliminating the low defect of specific binding rate that single receptor faced.Therefore, double targeted nanometer medicine carriers are in cancer
There is stronger advantage in treatment.
Found in the research of stomach cancer cell surface receptor, HER2 acceptors have gene in the ﹪ of 15 ﹪~20 stomach cancer
Amplification and the overexpression of albumen, gastric cancer infiltration positive HER2 are strong, and DFS phase is short, poor prognosis；And CD44 high table
It is related up to the transfer to kinds of tumors and high proliferation activity, including stomach cancer.At present, can specific recognition the two target spots part
Respectively AHNP polypeptides and hyaluronic acid（HA）.Wherein, AHNP is a kind of polypeptide for the anti-HER2 of 1.5KDa that recent development comes out
Fragment, has high-affinity with HER2 acceptors, can induce the rapid endocytosis of acceptor；And hyaluronic acid（HA）It is a kind of generally existing
Biodegradable with extraordinary physicochemical property in the natural polysaccharide in human body, good biocompatibility is non-toxic, without immune
Originality, and can be specifically bound with CD44.Therefore, if AHNP polypeptides and hyaluronic acid can be utilized（HA）For HER2 and CD44
Two kinds of acceptor designs are a kind of to load SN38 double targeted nano delivery systems, it will be possible to greatly improve chemotherapy pair
The therapeutic effect of stomach cancer.
The content of the invention
The present invention, can industry for a kind of the not enough there is provided step is simple and easy to apply of existing single targeted nano drug-carrier
Change the preparation method of double targeting composite nanoparticles of expanding production.The double targeting composite nanoparticles of gained can be to 7- ethyls -10-
The hydrophobic drugs such as HCPT have it is stronger contain ability and higher stability, and pair express simultaneously CD44 and
Her2 stomach cancer cell has stronger targeting and therapeutic effect.
The mode that the present invention is connected using chemical bond modifies hexadecylamine and target polypeptide AHNP in hyaluronic acid
（HA）Side chain, becomes the amphipathic lipoid that can be combined with two kinds of stomach cancer cell surface receptor targeted, and utilize the targeting class
Fat is emulsifying agent, by a step emulsion process, is prepared a kind of with degradable high molecular polymer Poly(D,L-lactide-co-glycolide
（PLGA）For core, hyaluronic acid lipoid peptide modified AHNP is double targeting composite Nano drug-carriers of shell, including as follows
Step：
S1. hexadecylamine, AHNP polypeptides and hyaluronic acid are reacted under action of coupling agents, obtains double targeting lipoids, will
Double targeting lipoids are configured to the aqueous solution, produce double targeting lipoid aqueous solution；
S2. Poly(D,L-lactide-co-glycolide is dissolved in dichloromethane/alcohol mixed solution, obtains mixed organic solvents；
S3. gained mixed organic solvents in step S2 are added dropwise in S1 steps in double targeting lipoid aqueous solution of gained,
Obtain double targeting lipid solutions；
S4. by double targeting lipid solutions of gained in S3 steps it is ultrasonic after, obtain it is ultrasonic after double targeting lipid solutions；
S5. double targeting lipid solutions after ultrasound in step S4 are added in ultra-pure water, and is continuously stirring to organic solvent
Volatilization completely；
S6. material obtained by step S5 is centrifuged and collects the nano-particle of solidification, after cleaning-drying, produced described double
Target composite nanoparticle.
Preferably, in the S1 steps coupling agent be 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides and
N-hydroxysuccinimide.
Preferably, the molecular weight of hyaluronic acid is the KDa of 60 KDa ~ 200 in the S1 steps.
Preferably, the amount of weighing of Poly(D,L-lactide-co-glycolide is 10 ~ 20 mg, the dichloro in the S2 steps
The addition of methane/alcohol mixed solution is 0.5 ~ 2 mL, and the volume ratio of the dichloromethane/alcohol mixed solution is 5 ~ 2：1.
Preferably, mixed organic solvents and double volume ratios 1 for targetting the lipoid aqueous solution in the S3 steps:2~5.
Preferably, ultrasonic power is 7 ~ 14 W, ultrasonic time 30 ~ 180 seconds in the S4 steps.
Preferably, in the S5 steps after ultrasound double targeting lipid solutions and the volume ratio of ultra-pure water are 1:2~5.
The present invention also provides the preparation method that a kind of double targeting composite nanoparticles load hydrophobic drug, including following step
Suddenly：
S1. by the use of 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides and n-hydroxysuccinimide as
Coupling agent, by hexadecylamine and AHNP polypeptides, is connected to side of the molecular weight ranges for the KDa of 60 KDa ~ 200 hyaluronic acid
Chain carboxy-terminal, produces double targeting lipoids, and it is the water-soluble of 0.5 ~ 5 mg/mL that double targeting lipoids are configured into concentration range
Liquid, obtains double targeting lipoid aqueous solution；
S2. 10 ~ 20 mg Poly(D,L-lactide-co-glycolides are weighed, and according to quality than poly lactic-co-glycolic acid copolymerization
Thing：Hydrophobic drug is 5 ~ 20:1 weighs hydrophobic drug, and above two material is dissolved in 0.5 ~ 2 mL dichloromethane/second
Mixed alkoxide solution, the volume ratio of the dichloromethane/alcohol mixed solution is 5 ~ 2：1, obtain mixed organic solvents；
S3. gained mixed organic solvents in S2 steps are added in the double targeting lipoid aqueous solution of gained in S1 steps, obtained
Double targeting lipid solutions；
S4. it is 7 ~ 14 W in ultrasonic power by double targeting lipid solutions of gained in S3 steps, ultrasonic time is 30 ~ 180s
Under the conditions of, obtain it is ultrasonic after double targeting lipid solutions；
S5. double targeting lipid solutions after ultrasound in step S4 are pressed 1:2 ~ 5 volume ratio is added in ultra-pure water, is continuously stirred
5 h are mixed, organic solvent is volatilized completely；
S6. by material obtained by step S5 by the way that the nano-particle of solidification is collected by centrifugation, and clean cold after 2 ~ 4 times with ultra-pure water
It is lyophilized dry, produce double targeting composite nanoparticles of load hydrophobic drug；
Preferably, the hydrophobic drug includes SN38.
The present invention is amphipathic using double targeting lipoids of independent research, by a step emulsion process, prepares one kind to drop
The high molecular polymer PLGA of solution is core, and double targeting lipoids are double targeting composite nanoparticles of shell, and medium particle diameter is distributed in 200
Between ~ 400 nm.On the one hand, the hyaluronic acid in double targeting lipoids can form hydrated sheath on composite nanoparticle surface, hinder
The absorption of opsonin and lipoprotein in internal serum, improves the stability of nano-particle in vivo, when extending its internal circulation
Between；On the other hand, the hyaluronic acid and AHNP target polypeptides in composite nano-granule subshell can effectively facilitate nano-particle and same
When expression CD44 and Her2 stomach cancer cell specific binding, improve local drug concentration, preferably controlled so as to make it have
Therapeutic effect.
Compared with prior art, the invention has the advantages that：
The double targeting composite nanoparticles for the load SN38 that the present invention is provided, not only drugloading rate
Greatly, envelop rate is high, and extends the effective acting time of SN38 in vivo, and passes through active targeting
Effect has been effectively promoted the combination of nano-particle and tumour cell, reduces medicine and the Tu Fu of normal cell is acted on, significantly
Improve the therapeutic effect of medicine.
Brief description of the drawings
Fig. 1 is the hydrogen nuclear magnetic resonance spectrogram and ultraviolet-visible spectrogram for double targeting lipoids that embodiment 1 is synthesized.
Fig. 2 is the stereoscan photograph of double targeting composite nanoparticles prepared by embodiment 2.
Fig. 3 is 3 pairs of targeting composite nanoparticle different time sections in the PBS containing 10% hyclone albumen of embodiment
Grain size distribution.
Fig. 4 is the double targeting composite nanoparticles and the laser after HGC27 cytosiies 4h that embodiment 4 loads coumarin 6
Laser Scanning Confocal Microscope photo.
Embodiment
Unless otherwise instructed, the solvent and reagent used in this experiment are bought from commercial goods, using preceding without pure
Change.
Embodiment 1
By the hyaluronic acid of 50 mg different molecular weights（The KDa of HA, 60 KDa ~ 200）It is dissolved in respectively in dry formamide,
A certain amount of EDC, NHS and hexadecylamine are added afterwards（HAD）, its mole is respectively carboxyl mole in hyaluronic acid
15%, 15% and 10%.And 24 h of reaction are stirred at room temperature.After reaction terminates, reaction solution is added dropwise to excessive anhydrous
In ethanol, the hyaluronic acid for being connected with hexadecylamine is precipitated out and is collected by centrifugation, and repeat the step 2 time.Afterwards will be heavy
Shallow lake is dissolved in ultra-pure water and freezed after being dialysed 2 days in water.
Weigh 50mg and be connected with the hyaluronic acid of hexadecylamine and be dissolved in dry formamide, add afterwards a certain amount of
EDC, NHS and AHNP polypeptide, its mole are respectively 7.5%, 7.5% and 5% of carboxyl mole in hyaluronic acid.And in room
The lower stirring reaction 24h of temperature.Reaction terminate after by reaction solution as DMSO and it is ultrapure it is middle dialyse and freeze, finally give double targetings
Lipoid（AHNP-HA-HDA）, and it is the double targeting lipoid aqueous solution of 0.5 ~ 5 mg/mL to be configured to concentration range.
As shown in figure 1, hydrogen nuclear magnetic resonance spectrogram analysis shows, HDA is successfully connected on HA；Uv-vis spectra analytical table
Bright, AHNP polypeptides are also successfully connected on HA.
Embodiment 2
10 mg PLGA are dissolved to 0.5 mL dichloromethane/ethanol（v/v=3:1）In the mixed solvent, by embodiment 1
The double targeting lipoids prepared are configured to the 2mg/mL aqueous solution, and mixed solvent is added dropwise into the double targeting classes of 10mL 2mg/mL
In the fat aqueous solution, ultrasonic emulsification is carried out with ultrasonic cell disruption instrument, power is 14 w, ultrasonic time 60s, afterwards at room temperature
Continuously stir 5 h and remove organic solvent, make composite nanoparticle curing molding, be finally collected by centrifugation and clean three with ultra-pure water
It is secondary, and the double targeting composite nanoparticles of lyophilized acquisition.
As shown in Fig. 2 laser particle size analysis shows, gained nano-particle is in normal distribution by effective diameter of 387nm, many
Dispersiveness is 0.203.The nanoparticle is observed under ESEM has regular spherical design.
Embodiment 3
20 mg PLGA are dissolved to 0.5 mL dichloromethane/ethanol（v/v=4:1）In the mixed solvent, by embodiment 1
The double targeting lipoids prepared are configured to the 2mg/mL aqueous solution, and mixed solvent is added dropwise into the double targeting classes of 10mL 2mg/mL
In the fat aqueous solution, ultrasonic emulsification is carried out with ultrasonic cell disruption instrument, power is 14 w, ultrasonic time 60s, afterwards at room temperature
Continuously stir 5 h and remove organic solvent, make composite nanoparticle curing molding, be finally collected by centrifugation and clean three with ultra-pure water
It is secondary, and the double targeting composite nanoparticles of lyophilized acquisition.It is dispersed to afterwards in the PBS containing 10% hyclone albumen, and it is right
Its stability is evaluated.
As shown in figure 3, measuring this pair of targeted nano-particle containing 10% hyclone albumen by laser particle analyzer
It can maintain its particle diameter distribution in 200 ~ 400 nm in PBS, polydispersity is maintained between 0.1 ~ 0.3, show that this pair targeting is compound
Nano-particle has preferable vitro stability.
Embodiment 4
By 20 mg PLGA and 0.5mg fluorescent dye coumarin 6 co-dissolves to 0.5 mL dichloromethane/ethanol（v/v=
4:1）In the mixed solvent, be added dropwise to double targeting lipoids that 5 mL 2mg/mL embodiments 1 are synthesized（AHNP-HA-HDA）'s
The aqueous solution, carries out ultrasonic emulsification with ultrasonic cell disruption instrument, and power is 14 w, ultrasonic time 60s, continuous at room temperature afterwards
Stir 5 h and remove organic solvent, make composite nanoparticle curing molding, be finally collected by centrifugation and cleaned with ultra-pure water three times, and
The lyophilized double targeting composite nanoparticles for obtaining load coumarin 6.Afterwards by it with expressing CD44 and Her2 acceptors simultaneously
HGC27 cells are incubated after 4h altogether, and its targeting ability is observed using laser confocal microscopy.
As shown in figure 4, by laser confocal microscopy Jiao observation result, this pair targeting composite nanoparticle compared to
The PLGA nano-particles of group are not targetted, with stronger cross-cell membrane turn-over capacity.
Embodiment 5
By 20 mg PLGA and 2mg SN38 co-dissolves to 0.5 mL dichloromethane/ethanol
（v/v=4:1）In the mixed solvent, be added dropwise to double targeting lipoids that 5 mL 2mg/mL embodiments 1 are synthesized（AHNP-HA-
HDA）The aqueous solution, carry out ultrasonic emulsification with ultrasonic cell disruption instrument, power is 14 w, ultrasonic time 60s, afterwards in room temperature
Under continuously stir 5 h remove organic solvent, make composite nanoparticle curing molding, be finally collected by centrifugation and clean three with ultra-pure water
It is secondary, and the lyophilized double targeting composite nanoparticles for obtaining load SN38.
Laser particle size analysis shows that the double targeting composite nanoparticles of gained are in normal distribution by effective diameter of 406 nm,
Polydispersity is 0.269, and drugloading rate is 11.1%, and envelop rate is 66.8%.
Embodiment 6
By 20 mg PLGA and 2mg SN38 co-dissolves to 0.5 mL dichloromethane/ethanol
（v/v=4:1）In the mixed solvent, be added dropwise to double targeting lipoids that 5 mL 2mg/mL embodiments 1 are synthesized（AHNP-HA-
HDA）The aqueous solution, carry out ultrasonic emulsification with ultrasonic cell disruption instrument, power is 14 w, ultrasonic time 60s, afterwards in room temperature
Under continuously stir 5 h remove organic solvent, make composite nanoparticle curing molding, be finally collected by centrifugation and clean three with ultra-pure water
It is secondary, and the lyophilized double targeting composite nanoparticles for obtaining load SN38.Afterwards by it with expressing simultaneously
The HGC27 cells of CD44 and Her2 acceptors are incubated after 48h altogether, and its external treatment efficiency is detected using MTT experiment.
Table 1 is the double targeting composite nanoparticles of the load SN38 of embodiment 6 and HGC27 cytosiies
IC after 48h50Value；
Obtained by vitro toxicity test, be directly to the IC50 values of HGC27 cells with SN38
0.329 μM, double targeting composite nanoparticles of the load SN38 are to the IC50 values of HGC27 cells
0.128 μM, load the PLGA nano-particles of SN38（The preparation method of the PLGA nano-particles and preparation
Double targeting composite nano-granule submethods are identical, but do not mixed with the AHNP-HA-HAD aqueous solution）To the IC of HGC27 cells50
It is worth for 0.874 μM, as shown in table 1.It can be learnt from table 1, double targetings of the load SN38 are compound to be received
Rice corpuscles has stronger external treatment effect.
Claims (10)
1. a kind of preparation method of double targeting composite nanoparticles, it is characterised in that comprise the following steps：
S1. hexadecylamine, AHNP polypeptides and hyaluronic acid are reacted under action of coupling agents, obtains double targeting lipoids, will be described
Double targeting lipoids are configured to the aqueous solution, produce double targeting lipoid aqueous solution；
S2. Poly(D,L-lactide-co-glycolide is dissolved in dichloromethane/alcohol mixed solution, obtains mixed organic solvents；
S3. gained mixed organic solvents in step S2 are added dropwise in S1 steps in double targeting lipoid aqueous solution of gained, obtain double
Target lipid solution；
S4. by double targeting lipid solutions of gained in S3 steps it is ultrasonic after, obtain it is ultrasonic after double targeting lipid solutions；
S5. double targeting lipid solutions after ultrasound in step S4 are added in ultra-pure water, and it is complete to be continuously stirring to organic solvent
Volatilization；
S6. material obtained by step S5 is centrifuged and collects the nano-particle of solidification, after cleaning-drying, produce double targetings
Composite nanoparticle.
2. the preparation method of double targeting composite nanoparticles according to claim 1, it is characterised in that in the S1 steps
Coupling agent is 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides and n-hydroxysuccinimide.
3. the preparation method of double targeting composite nanoparticles according to claim 1, it is characterised in that in the S1 steps
The molecular weight of hyaluronic acid is the KDa of 60 KDa ~ 200.
4. the preparation method of double targeting composite nanoparticles according to claim 1, it is characterised in that in the S2 steps
The amount of weighing of Poly(D,L-lactide-co-glycolide is 10 ~ 20 mg, and the addition of the dichloromethane/alcohol mixed solution is
0.5 ~ 2 mL, the volume ratio of the dichloromethane/alcohol mixed solution is 5 ~ 2：1.
5. the preparation method of double targeting composite nanoparticles according to claim 1, it is characterised in that in the S3 steps
The volume ratio 1 of mixed organic solvents and double targeting lipoid aqueous solution:2~5.
6. the preparation method of double targeting composite nanoparticles according to claim 1, it is characterised in that in the S4 steps
Ultrasonic power is 7 ~ 14 W, ultrasonic time 30 ~ 180 seconds.
7. the preparation method of double targeting composite nanoparticles according to claim 1, it is characterised in that in the S5 steps
The volume ratio of double targeting lipid solutions and ultra-pure water after ultrasound is 1:2~5.
8. double targets that a kind of preparation method of double targeting composite nanoparticles described in any one of claim 1 to 7 is prepared from
To composite nanoparticle.
9. application of double targeting composite nanoparticles in load hydrophobic drug is prepared described in claim 8, its feature exists
In the hydrophobic drug is SN38.
10. a kind of double targeting composite nanoparticles load the preparation method of hydrophobic drug, it is characterised in that including following step
Suddenly：
S1. it is used as coupling by the use of 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides and n-hydroxysuccinimide
Agent, by hexadecylamine and AHNP polypeptides, is connected to side chain carboxylic of the molecular weight ranges for the KDa of 60 KDa ~ 200 hyaluronic acid
Cardinal extremity, produces double targeting lipoids, and double targeting lipoids are configured into the aqueous solution that concentration range is 0.5 ~ 5 mg/mL, obtains
Double targeting lipoid aqueous solution；
S2. 10 ~ 20 mg Poly(D,L-lactide-co-glycolides are weighed, and compare Poly(D,L-lactide-co-glycolide according to quality：
Hydrophobic drug is 5 ~ 20:1 weighs hydrophobic drug, and above two material is dissolved in 0.5 ~ 2 mL dichloromethane/ethanol
Mixed solution, the volume ratio of the dichloromethane/alcohol mixed solution is 5 ~ 2：1, obtain mixed organic solvents；
S3. gained mixed organic solvents in S2 steps are added in the double targeting lipoid aqueous solution of gained in S1 steps, obtain double targets
To lipid solution；
S4. it is 7 ~ 14 W in ultrasonic power by double targeting lipid solutions of gained in S3 steps, ultrasonic time is 30 ~ 180s conditions
Under, obtain it is ultrasonic after double targeting lipid solutions；
S5. double targeting lipid solutions after ultrasound in step S4 are pressed 1:2 ~ 5 volume ratio is added in ultra-pure water, continuously stirs 5
H, makes organic solvent volatilize completely；
S6. material obtained by step S5 is done by the way that the nano-particle of solidification is collected by centrifugation, and cleans freezing after 2 ~ 4 times with ultra-pure water
It is dry, produce double targeting composite nanoparticles of load hydrophobic drug；
The hydrophobic drug is SN38.
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