CN104606134A - 7-ethyl-10-hydroxycamptothecine supported dual-targeting composite nanoparticle as well as preparation method and application thereof - Google Patents
7-ethyl-10-hydroxycamptothecine supported dual-targeting composite nanoparticle as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a dual-targeting composite nanoparticle as well as a preparation method and an application thereof, and further provides a 7-ethyl-10-hydroxycamptothecine supported dual-targeting composite nanoparticle as well as a preparation method and an application thereof. According to the 7-ethyl-10-hydroxycamptothecine supported dual-targeting composite nanoparticle, the drug loading capacity and the encapsulation efficiency are high, the effective acting time of 7-ethyl-10-hydroxycamptothecine in a body is prolonged, combination of nanoparticles and cancer cell is effectively promoted through the active targeting action, the toxic and side effect of drugs on normal cells are reduced, and the drug treatment effect can be remarkably improved.
Description
Technical field
The invention belongs to nano-medicament carrier field, two targeting composite nanoparticles being specifically related to a kind of load SN38 and its preparation method and application.
Background technology
Gastric cancer is one of modal malignant tumor in the world, and be also one of tumor that mortality rate is the highest, death toll ranked second position in world's tumor mortality case.Most of patients is advanced metastatic disease when diagnosing or occurs relapse and metastasis after surgical discectomy, and within 5 years, survival rate is less than 10%.Irinotecan (CPT-11) is the semi-synthetic solubility camptothecin derivant being widely used in gastric cancer second line treatment.Similar with other camptothecine mechanisms of action, it suppresses typeⅠtopoisomerase, makes its inactivation, causes DNA single chain interruption, hinders DNA replication dna and RNA to synthesize, thus the division of T suppression cell.But, CPT-11 also receives a definite limitation at therapeutic effect and clinical practice, its reason mainly contains two aspects, first, CPT-11 changes into active metabolite SN38 and plays treatment of cancer effect under needing the effect of carboxy-lesterase in vivo, but research shows that the efficiency that CPT-11 changes into SN38 in human body is only 2 ~ 8%, and this makes CPT-11 bioavailability in vivo extremely low; Secondly, CPT-11 has the toxic and side effects such as bone marrow depression, brings great misery over the course for the treatment of to patient.Therefore, if the active metabolite SN38 (SN38) of CPT-11 is directly used in curing gastric cancer, the defect that CPT-11 bioavailability is low will be overcome, thus improve chemotherapy effect.But the strong-hydrophobicity of SN38 makes it just separate out rapidly after entering human recycle system, be difficult to be absorbed by lesion tissue, thus limit its application clinically.Therefore, how improving SN38 dissolubility in vivo, extend its circulation time in human body and improve the useful effect concentration of this medicine at lesions position, is that application SN38 is in the difficult point of curing gastric cancer.
The exploitation of targeted nano drug delivery system is the most effective means that solve the problem.On the one hand, hydrophobicity chemotherapeutics can be carried out bag and carry by Nano medication transmission system, improves hydrophobic drug dissolubility in vivo, avoids medicine by the various enzymatic degradation in blood, extend effective cycle time in its body; On the other hand, utilize targeting part to carry out targeting modification to carrier, make medicine can concentrate release at diseased region, improve drug availability and reduce its toxic and side effects.But simple for a tumor targets design and development nano-medicament carrier, often do not reach best therapeutic effect.On the one hand, single targeted nano pharmaceutical carrier, can only be combined with of a tumor cell surface target recipient, and this receptor may over the course for the treatment of because the impact of pathology, physiology, microenvironment factor loses combination between part and mediation; On the other hand, the expression degree of target site receptor also has a huge impact the effect of single targeting vector, and the overexpression behavior of receptor only occurs in some privileged sites does not have popularity, and these all likely limit the part of single targeting vector and the specific binding of target recipient, and then reduce its targeting and therapeutic effect.By contrast, two targeting vector can provide the receptor of multiple targets identification site and target site to realize specific binding, also ensure that the multiformity of target site receptor, weakens and even eliminates the low defect of specific binding rate that single receptor faces.Therefore, two targeted nanometer medicine carrier has stronger advantage in treatment of cancer.
Find in the research of stomach cancer cell surface receptor, HER2 receptor has the amplification of gene and the overexpression of albumen in the gastric cancer of 15 ﹪ ~ 20 ﹪, and the gastric cancer infiltration of the HER2 positive is strong, and the disease free survival phase is short, poor prognosis; And the high expressed of CD44 is active relevant with high proliferation to the transfer of kinds of tumors, comprise gastric cancer.At present, the part of these two target spots of specific recognition AHNP polypeptide and hyaluronic acid (HA) can be respectively.Wherein, AHNP is the polypeptide fragment of the anti-HER2 of a kind of recent development 1.5KDa out, has high-affinity with HER2 receptor, can inducing receptor endocytosis rapidly; And hyaluronic acid (HA) is a kind of natural polysaccharide be prevalent in human body, there is extraordinary physicochemical property, biodegradable, good biocompatibility, avirulence, non-immunogenicity, and can with CD44 specific binding.Therefore, if AHNP polypeptide and hyaluronic acid (HA) can be utilized for two targeted nano delivery systems of HER2 and CD44 two kinds of a kind of energy of receptor design load SN38, the therapeutic effect of chemotherapy to gastric cancer will likely be improved greatly.
Summary of the invention
The present invention is directed to the deficiency of existing single targeted nano drug-carrier, provide a kind of step simple, can the preparation method of two targeting composite nanoparticles of industrialization expanding production.The two targeting composite nanoparticle of gained can have stronger bag loading capability and higher stability to hydrophobic drugs such as SN38s, and has stronger targeting and therapeutic effect to the stomach cancer cell of expressing CD44 and Her2 simultaneously.
Hexadecylamine and target polypeptide AHNP are modified at hyaluronic acid (HA) side chain by the mode that the present invention utilizes chemical bond to connect, become the amphipathic lipoid that can be combined with the two kinds of receptor targeted in stomach cancer cell surface, and utilize this targeting lipoid to be emulsifying agent, by a step emulsion process, prepare one with degradable high molecular polymer Poly(D,L-lactide-co-glycolide (PLGA) for core, the peptide modified hyaluronic acid lipoid of AHNP is two targeting composite Nano drug-carrier of shell, comprises the steps:
S1. hexadecylamine, AHNP polypeptide and hyaluronic acid are reacted under action of coupling agents, obtain two targeting lipoid, described pair of targeting lipoid is mixed with aqueous solution, obtain two 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 is dropped in two targeting lipoid aqueous solutions of gained in S1 step, obtain two targeting lipid solution;
S4. by after ultrasonic for two targeting lipid solution of gained in S3 step, obtain ultrasonic after two targeting lipid solution;
S5. the two targeting lipid solution after ultrasonic in step S4 are added in ultra-pure water, and be continuously stirring to organic solvent and volatilize completely;
S6. by nanoparticle that is centrifugal for step S5 gained material and collection solidification, after cleaning-drying, described pair of targeting composite nanoparticle is obtained.
Preferably, in described S1 step, coupling agent is 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy-succinamide.
Preferably, in described S1 step, hyaluronic molecular weight is 60 KDa ~ 200 KDa.
Preferably, in described S2 step, the amount of taking of Poly(D,L-lactide-co-glycolide is 10 ~ 20 mg, and the addition of described dichloromethane/alcohol mixed solution is 0.5 ~ 2 mL, and the volume ratio of described dichloromethane/alcohol mixed solution is 5 ~ 2:1.
Preferably, volume ratio 1:2 ~ 5 of mixed organic solvents and two targeting lipoid aqueous solution in described S3 step.
Preferably, in described S4 step, ultrasonic power is 7 ~ 14 W, ultrasonic time 30 ~ 180 seconds.
Preferably, the two targeting lipid solution after ultrasonic in described S5 step and the volume ratio of ultra-pure water are 1:2 ~ 5.
The present invention also provides the preparation method of a kind of pair of targeting composite nanoparticle load hydrophobic drug, comprises the following steps:
S1. utilize 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy-succinamide as coupling agent, by hexadecylamine and AHNP polypeptide, be connected to the hyaluronic side chain carboxyl group end that molecular weight ranges is 60 KDa ~ 200 KDa, obtain two targeting lipoid, and described pair of targeting lipoid is mixed with the aqueous solution that concentration range is 0.5 ~ 5 mg/mL, obtain two targeting lipoid aqueous solution;
S2. 10 ~ 20 mg Poly(D,L-lactide-co-glycolides are taken, and according to quality than Poly(D,L-lactide-co-glycolide: hydrophobic drug is that 5 ~ 20:1 is to take hydrophobic drug, above-mentioned two kinds of materials are dissolved in the dichloromethane/alcohol mixed solution of 0.5 ~ 2 mL, the volume ratio of described dichloromethane/alcohol mixed solution is 5 ~ 2:1, obtains mixed organic solvents;
S3. gained mixed organic solvents in S2 step is added in the two targeting lipoid aqueous solution of gained in S1 step, obtains two targeting lipid solution;
S4. be 7 ~ 14 W by two targeting lipid solution of gained in S3 step at ultrasonic power, ultrasonic time is under 30 ~ 180s condition, obtain ultrasonic after two targeting lipid solution;
S5. add in ultra-pure water by the two targeting lipid solution after ultrasonic in step S4 by the volume ratio of 1:2 ~ 5, continuous stirring 5 h, makes organic solvent volatilize completely;
S6. by the nanoparticle that step S5 gained material is solidified by collected by centrifugation, and with ultra-pure water cleaning 2 ~ 4 postlyophilizations, two targeting composite nanoparticles of load hydrophobic drug are obtained;
Preferably, described hydrophobic drug comprises SN38.
The present invention utilizes the amphipathic of two targeting lipoids of independent research, and by a step emulsion process, preparation one is with degradable high molecular polymer PLGA for core, and two targeting lipoid is two targeting composite nanoparticles of shell, and medium particle diameter is distributed between 200 ~ 400 nm.On the one hand, the hyaluronic acid in two targeting lipoid can form hydrated sheath in composite nano-granule sub-surface, and the absorption of opsonin and lipoprotein in serum in baffle, improves nanoparticle stability in vivo, extend the circulation time in its body; On the other hand, the hyaluronic acid in composite nano-granule subshell and AHNP target polypeptide effectively can promote nanoparticle and the specific binding of stomach cancer cell of simultaneously to express CD44 and Her2, raising local drug concentration, thus make it have better therapeutic effect.
Compared with prior art, the present invention has following beneficial effect:
Two targeting composite nanoparticles of load SN38 provided by the invention, not only drug loading is large, envelop rate is high, and extend SN38 effective acting time in vivo, and the combination of nanoparticle and tumor cell is effectively facilitated by active targeting effect, reduce Tu Fu's effect of agents on normal cells, significantly improve the therapeutic effect of medicine.
Accompanying drawing explanation
Fig. 1 is hydrogen nuclear magnetic resonance spectrogram and the ultraviolet-visible spectrogram of two targeting lipoids that embodiment 1 is synthesized.
Fig. 2 is the stereoscan photograph of two targeting composite nanoparticles prepared by embodiment 2.
Fig. 3 is the grain size distribution of embodiment 3 pairs of targeting composite nanoparticles different time sections in the PBS containing 10% hyclone albumen.
Fig. 4 is the laser confocal microscope photo after two targeting composite nanoparticle of embodiment 4 load coumarin 6 and HGC27 cytosis 4h.
Detailed description of the invention
If no special instructions, the solvent used in this experiment and reagent are all bought from commercial goods, not purified before using.
Embodiment 1
By the hyaluronic acid (HA of 50 mg different molecular weights, 60 KDa ~ 200 KDa) be dissolved in respectively in dry formamide, add a certain amount of EDC, NHS and hexadecylamine (HAD) afterwards, its mole is respectively 15%, 15% and 10% of carboxyl mole in hyaluronic acid.And at room temperature stirring reaction 24 h.After reaction terminates, reaction solution is dropped in excessive dehydrated alcohol, the hyaluronic acid being connected with hexadecylamine is precipitated out and collected by centrifugation, and repeats this step 2 time.Afterwards by lyophilizing after dialysing 2 days in resolution of precipitate to ultra-pure water and in water.
Take 50mg and be connected with the hyaluronic acid dissolves of hexadecylamine in dry formamide, add a certain amount of EDC, NHS and AHNP polypeptide afterwards, its mole is respectively 7.5%, 7.5% and 5% of carboxyl mole in hyaluronic acid.And at room temperature stirring reaction 24h.After reaction terminates by reaction solution as DMSO and ultrapure middle dialysis and lyophilizing, finally obtain two targeting lipoid (AHNP-HA-HDA), and to be configured to concentration range be the two targeting lipoid aqueous solution of 0.5 ~ 5 mg/mL.
As shown in Figure 1, hydrogen nuclear magnetic resonance spectrum analysis shows, HDA is successfully connected on HA; Uv-vis spectra analysis shows, AHNP polypeptide is also successfully connected on HA.
Embodiment 2
10 mg PLGA are dissolved in the mixed solvent of 0.5 mL dichloromethane/ethanol (v/v=3:1), two targeting lipoids embodiment 1 prepared are configured to the aqueous solution of 2mg/mL, mixed solvent is dropped in the two targeting lipoid aqueous solution of 10mL 2mg/mL, ultrasonic emulsification is carried out with ultrasonic cell disruption instrument, power is 14 w, ultrasonic time 60s, at room temperature continuous stirring 5 h removes organic solvent afterwards, make composite nanoparticle curing molding, last collected by centrifugation also cleans three times with ultra-pure water, and lyophilizing obtains two targeting composite nanoparticle.
As shown in Figure 2, laser particle size analysis shows, gained nanoparticle take 387nm as effective diameter is normal distribution, and polydispersity is 0.203.Observe this Nano microsphere under scanning electron microscope and there is regular spherical design.
Embodiment 3
20 mg PLGA are dissolved in the mixed solvent of 0.5 mL dichloromethane/ethanol (v/v=4:1), two targeting lipoids embodiment 1 prepared are configured to the aqueous solution of 2mg/mL, mixed solvent is dropped in the two targeting lipoid aqueous solution of 10mL 2mg/mL, ultrasonic emulsification is carried out with ultrasonic cell disruption instrument, power is 14 w, ultrasonic time 60s, at room temperature continuous stirring 5 h removes organic solvent afterwards, make composite nanoparticle curing molding, last collected by centrifugation also cleans three times with ultra-pure water, and lyophilizing obtains two targeting composite nanoparticle.Be dispersed to afterwards in the PBS containing 10% hyclone albumen, and its stability is evaluated.
As shown in Figure 3, record this pair of targeted nano-particle by laser particle analyzer and can maintain its particle size distribution at 200 ~ 400 nm in containing the PBS of 10% hyclone albumen, polydispersity maintains between 0.1 ~ 0.3, shows that this pair of targeting composite nanoparticle has good vitro stability.
Embodiment 4
20 mg PLGA and 0.5mg fluorescent dye coumarin 6 are dissolved to jointly in the mixed solvent of 0.5 mL dichloromethane/ethanol (v/v=4:1), dropped to the aqueous solution of two targeting lipoids (AHNP-HA-HDA) that 5 mL 2mg/mL embodiments 1 are synthesized, ultrasonic emulsification is carried out with ultrasonic cell disruption instrument, power is 14 w, ultrasonic time 60s, at room temperature continuous stirring 5 h removes organic solvent afterwards, make composite nanoparticle curing molding, last collected by centrifugation also cleans three times with ultra-pure water, and lyophilizing obtains two targeting composite nanoparticles of load coumarin 6.After afterwards itself and the HGC27 cell of simultaneously expressing CD44 and Her2 receptor being hatched 4h altogether, laser confocal microscopy is utilized to observe its targeting ability.
As shown in Figure 4, known by the burnt observed result of laser confocal microscopy, this pair of targeting composite nanoparticle compares to the PLGA nanoparticle not having targeting group, has stronger cross-cell membrane turn-over capacity.
Embodiment 5
20 mg PLGA and 2mg SN38 are dissolved to jointly in the mixed solvent of 0.5 mL dichloromethane/ethanol (v/v=4:1), dropped to the aqueous solution of two targeting lipoids (AHNP-HA-HDA) that 5 mL 2mg/mL embodiments 1 are synthesized, ultrasonic emulsification is carried out with ultrasonic cell disruption instrument, power is 14 w, ultrasonic time 60s, at room temperature continuous stirring 5 h removes organic solvent afterwards, make composite nanoparticle curing molding, last collected by centrifugation also cleans three times with ultra-pure water, and lyophilizing obtains two targeting composite nanoparticles of load SN38.
Laser particle size analysis shows, the two targeting composite nanoparticle of gained is with 406 nm for effective diameter is normal distribution, and polydispersity is 0.269, and drug loading is 11.1%, and envelop rate is 66.8%.
Embodiment 6
20 mg PLGA and 2mg SN38 are dissolved to jointly in the mixed solvent of 0.5 mL dichloromethane/ethanol (v/v=4:1), dropped to the aqueous solution of two targeting lipoids (AHNP-HA-HDA) that 5 mL 2mg/mL embodiments 1 are synthesized, ultrasonic emulsification is carried out with ultrasonic cell disruption instrument, power is 14 w, ultrasonic time 60s, at room temperature continuous stirring 5 h removes organic solvent afterwards, make composite nanoparticle curing molding, last collected by centrifugation also cleans three times with ultra-pure water, and lyophilizing obtains two targeting composite nanoparticles of load SN38.After afterwards itself and the HGC27 cell of simultaneously expressing CD44 and Her2 receptor being hatched 48h altogether, MTT experiment is utilized to detect its external treatment efficiency.
Table 1 is the IC after the two targeting composite nanoparticle of embodiment 6 load SN38 and HGC27 cytosis 48h
50value;
Obtained by vitro toxicity test, the IC50 value of direct SN38 to HGC27 cell is 0.329 μM, the IC50 value of two targeting composite nanoparticles to HGC27 cell of this load SN38 is 0.128 μM, the PLGA nanoparticle of load SN38 (preparation method of this PLGA nanoparticle is identical with preparation described pair of targeting composite nano-granule submethod, but not with AHNP-HA-HAD aqueous solution) IC to HGC27 cell
50value is 0.874 μM, as shown in table 1.Can learn from table 1, two targeting composite nanoparticles of this load SN38 have stronger external treatment effect.
Claims (10)
1. a preparation method for two targeting composite nanoparticle, is characterized in that, comprise the following steps:
S1. hexadecylamine, anti-Her2 peptidomimetics (AHNP polypeptide) and hyaluronic acid are reacted under action of coupling agents, obtain two targeting lipoid, described pair of targeting lipoid is mixed with aqueous solution, obtain two 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 is dropped in two targeting lipoid aqueous solutions of gained in S1 step, obtain two targeting lipid solution;
S4. by after ultrasonic for two targeting lipid solution of gained in S3 step, obtain ultrasonic after two targeting lipid solution;
S5. the two targeting lipid solution after ultrasonic in step S4 are added in ultra-pure water, and be continuously stirring to organic solvent and volatilize completely;
S6. by nanoparticle that is centrifugal for step S5 gained material and collection solidification, after cleaning-drying, described pair of targeting composite nanoparticle is obtained.
2. the preparation method of according to claim 1 pair of targeting composite nanoparticle, is characterized in that, in described S1 step, coupling agent is 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy-succinamide.
3. the preparation method of according to claim 1 pair of targeting composite nanoparticle, is characterized in that, in described S1 step, hyaluronic molecular weight is 60 KDa ~ 200 KDa.
4. the preparation method of according to claim 1 pair of targeting composite nanoparticle, it is characterized in that, in described S2 step, the amount of taking of Poly(D,L-lactide-co-glycolide is 10 ~ 20 mg, the addition of described dichloromethane/alcohol mixed solution is 0.5 ~ 2 mL, and the volume ratio of described dichloromethane/alcohol mixed solution is 5 ~ 2:1.
5. the preparation method of according to claim 1 pair of targeting composite nanoparticle, is characterized in that, volume ratio 1:2 ~ 5 of mixed organic solvents and two targeting lipoid aqueous solution in described S3 step.
6. the preparation method of according to claim 1 pair of targeting composite nanoparticle, is characterized in that, in described S4 step, ultrasonic power is 7 ~ 14 W, ultrasonic time 30 ~ 180 seconds.
7. the preparation method of according to claim 1 pair of targeting composite nanoparticle, is characterized in that, the two targeting lipid solution after ultrasonic in described S5 step and the volume ratio of ultra-pure water are 1:2 ~ 5.
8. two targeting composite nanoparticles that the preparation method of the two targeting composite nanoparticles described in a claim 1 to 7 is prepared from.
9. the according to claim 8 pair of application of targeting composite nanoparticle in load hydrophobic drug, it is characterized in that, described hydrophobic drug comprises SN38.
10. a preparation method for two targeting composite nanoparticle load hydrophobic drug, is characterized in that, comprise the following steps:
S1. utilize 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy-succinamide as coupling agent, by hexadecylamine and AHNP polypeptide, be connected to the hyaluronic side chain carboxyl group end that molecular weight ranges is 60 KDa ~ 200 KDa, obtain two targeting lipoid, and described pair of targeting lipoid is mixed with the aqueous solution that concentration range is 0.5 ~ 5 mg/mL, obtain two targeting lipoid aqueous solution;
S2. 10 ~ 20 mg Poly(D,L-lactide-co-glycolides are taken, and according to quality than Poly(D,L-lactide-co-glycolide: hydrophobic drug is that 5 ~ 20:1 is to take hydrophobic drug, above-mentioned two kinds of materials are dissolved in the dichloromethane/alcohol mixed solution of 0.5 ~ 2 mL, the volume ratio of described dichloromethane/alcohol mixed solution is 5 ~ 2:1, obtains mixed organic solvents;
S3. gained mixed organic solvents in S2 step is added in the two targeting lipoid aqueous solution of gained in S1 step, obtains two targeting lipid solution;
S4. be 7 ~ 14 W by two targeting lipid solution of gained in S3 step at ultrasonic power, ultrasonic time is under 30 ~ 180s condition, obtain ultrasonic after two targeting lipid solution;
S5. add in ultra-pure water by the two targeting lipid solution after ultrasonic in step S4 by the volume ratio of 1:2 ~ 5, continuous stirring 5 h, makes organic solvent volatilize completely;
S6. by the nanoparticle that step S5 gained material is solidified by collected by centrifugation, and with ultra-pure water cleaning 2 ~ 4 postlyophilizations, two targeting composite nanoparticles of load hydrophobic drug are obtained;
Described hydrophobic drug comprises SN38.
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