CN109999001A - A kind of Nano medication and preparation method thereof - Google Patents
A kind of Nano medication and preparation method thereof Download PDFInfo
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- A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
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Abstract
The invention discloses a kind of Nano medications and preparation method thereof；More particularly to the Nano medication of a kind of load hydrophobic drug and hydrophilic medicament simultaneously, the Nano medication is that hydrophobic drug A and hydrophilic medicament B is conjugated by the disulfide bond that can be cut by GSH, nanoparticle obtained by self assembly.Form the hydrophobic prodrug in the hydrophilic one end in an one end, nano liposomes can be self-assembled into water, it can be achieved that drug effective delivering, while that realizes hydrophobic drug (such as: taxol) and hydrophilic medicament (such as: gemcitabine) cooperates with medication.The present invention is made by esterification, has the advantage that the advantages that at low cost, easily prepared, drugloading rate is high.
Description
Technical field
The invention belongs to medicament nano technical field, it is related to a kind of Nano medication and preparation method thereof.
Background technique
Cancer is one of most destructive disease in the world, and the main reason for lead to death.It will be diagnosed to be every year
More than 10,000,000 new cases, it is contemplated that the year two thousand thirty cancer related mortality number is up to 12,000,000.Therefore, treatment of cancer is 21
Century is a huge challenge.The important method that chemotherapy is treatment of cancer is carried out using anticancer drug.
However, its effect is low by drug solubility, non-selective, to the toxicity of healthy organ, efficiency reduces and drug resistance
The limitation of property.Polymer micelle nanoparticle is one of most important pharmaceutical carrier in cancer nanosecond medical science.
Most of in these micellar structures are made of the hydrophilic shell of hydrophobic core and Pegylation for carrying medicine,
To improve colloidal stability and stealthy effect.
Summary of the invention
Purpose: the present invention provides a kind of nano-carrier, the delivery system be can be used for synergistic treatment malignant tumour or other
The therapeutic agent and gene of malignant disease such as liver fibrosis, cirrhosis etc. deliver administration nano-drug administration system altogether, are related to a kind of self assembly
It forms the nanometer of load drug and gene altogether and is total to delivery system, self-assembled nanometer delivery system of the invention can be by drug and gene
It is delivered in target cell simultaneously, discharges gene and drug in target cell cytoplasm.
Technical solution: in order to solve the above technical problems, the technical solution adopted by the present invention are as follows:
A kind of Nano medication, the Nano medication are hydrophobic drug A and hydrophilic medicament B by that can be cut by GSH
Disulfide bond conjugation, nanoparticle A-L-B obtained by self assembly, the chemical structural formula of nanoparticle are as follows:
Wherein, 1,2,3 n.
Preferably, the hydrophobic drug A is hydrophobic anticancer drug, is selected from taxol, camptothecine, Suo Lafei
Buddhist nun；
The hydrophilic medicament B is hydrophily anticancer drug, is selected from gemcitabine, capecitabine, zoledronic acid；
The nanoparticle is spherical morphology, and partial size is 5~200nm.
Further, the Nano medication, the chemical structural formula of the nanoparticle are as follows:
On the other hand, the present invention also provides the preparation methods of above-mentioned Nano medication, comprising:
(1) hydrophobic drug A is connected to form compound A-L by esterification and linker；
(2) again hydrophilic medicament B is reacted to form compound A-L-B by esterification with compound A-L；
(3) compound A-L-B is dissolved in organic solvent and forms organic phase, by organic phase toward water phase by way of dropwise addition
Middle dropwise addition makes it be self-assembled into medicament nano particle.
Preferably, the preparation method of the Nano medication, the linker be 4,4 '-two thio two butyric acid,
Bis- thiodiglycolic acid of 3,3'- dithiodipropionic acid or 2,2'-.
Further, when hydrophobic drug A is taxol, hydrophilic medicament B is gemcitabine, and when n is 3, described is received
The preparation method of rice drug, comprising:
(1) hydrophobic anticancer drug taxol is connected to be formed with 4,4 '-two thio two butyric acid by esterification
Compound 1；
(2) again hydrophilic medicament gemcitabine is reacted to form compound 2 by esterification with compound 1；
(3) compound 2 is dissolved in organic solvent and forms organic phase, organic phase is dripped by way of dropwise addition into water phase
Adding makes it be self-assembled into medicament nano particle.
More specifically, the preparation method of the Nano medication, specifically includes:
1) taxol is dissolved in DMF, then is charged with 4,4 '-two thio two butyric acid and catalyst EDCI (1-
(3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride) and DMAP (4-dimethylaminopyridine), reaction is stirred at room temperature and obtains
Compound 1；
2) compound 1 is dissolved in DMF, gemcitabine and catalyst EDCI (1- (3- dimethylamino-propyl)-is added
3- ethyl-carbodiimide hydrochloride) and DMAP (4-dimethylaminopyridine), reaction is stirred at room temperature and obtains compound 2；
3) compound 2 is dissolved in acetone and forms organic phase, organic phase is added dropwise to water with the drop rate of 1~5 μ L/s
Xiang Zhong is made compound 2 uniformly and then self assembly medicament nano particle, is freeze-dried by stirring and/or ultrasound to obtain the final product.
Further, the preparation method of the Nano medication, in reaction process, the taxol of addition: 4,4 '-two is thio
Two butyric acid: the molar ratio of gemcitabine is 1:(1~3): (1~9).
Further, the preparation method of the Nano medication, dissolved with DSPE-PEG5000 as steady in the water phase
Determine agent.The addition of stabilizer can make the partial size of the nanoparticle of preparation smaller, be distributed between 10nm~20nm.
As the third aspect of the present invention, the present invention also provides the Nano medications in preparation prevention and/or treatment cancer
Application in the drug of disease.
The utility model has the advantages that Nano medication provided by the invention and preparation method thereof, while load hydrophobic drug and hydrophily
Taxol and gemcitabine are conjugated by the disulfide bond that can be cut by GSH, it is hydrophobic to form the hydrophilic one end in an one end by drug
Prodrug, nano liposomes can be self-assembled into water, it can be achieved that drug effective delivering, while realizing hydrophobic drug (such as: purple
China fir alcohol) with hydrophilic medicament (such as: gemcitabine) cooperate with medication.With high dispersion, stability, size tunable, can be real
Now cooperate with medication；And the bioavilability and stability of drug are enhanced, the sanguimotor time is extended.The present invention passes through ester
Change reaction to be made, has the advantage that the advantages that at low cost, easily prepared, drugloading rate is high.
Detailed description of the invention
Fig. 1 is the nuclear magnetic resonance spectroscopy of compound 1 prepared by embodiment 1；
Fig. 2 is the mass spectrum of compound 1 prepared by embodiment 1；
Fig. 3 is the nuclear magnetic resonance spectroscopy of compound 2 prepared by embodiment 2；
Fig. 4 is the mass spectrum of compound 2 prepared by embodiment 2；
Fig. 5 is the positive ion mass spectrum of medicament nano particle release gemcitabine；
Fig. 6 is the negative ion mass spectrum of medicament nano particle release gemcitabine；
Fig. 7 is the positive corpusc(u)le mass spectrum of medicament nano particle release taxol；
Fig. 8 drug be self-assembly of in water Nano medication TEM figure, intuitively show regular spherical morphology,
One partial size and the dispersibility of height；
Fig. 9 drug is self-assembly of the TEM figure of Nano medication with DSPE-PEG5000 in water, intuitively shows regular
Spherical morphology, the dispersibility of uniform partial size and height；
Figure 10 is inhibitory effect of the medicament nano particle to MCF-7 cell；
Figure 11 is medicament nano particle to the inhibitory effect in 4T1 cell.
Specific embodiment
The present invention is further illustrated below in conjunction with attached drawing and following embodiments, it should be appreciated that attached drawing and following embodiments
It is merely to illustrate the present invention, is not intended to limit the present invention.
The present invention provides a kind of therapeutic agents to deliver new approaches, i.e., prepares height by the self assembly that FDA ratifies drug
Dispersion, size tunable, stability is good, the guaranteed novel nano drug of safety, to solve step existing in the prior art
The problems such as cumbersome, low efficiency, repeatability difference.
It is disclosed a kind of Nano medication, the Nano medication is by hydrophobic drug and hydrophilic medicament by can quilt
The disulfide bond conjugation of GSH cutting, nanoparticle obtained by self assembly.
Self assembly (self-assembly) generally refers to basic structural unit (molecule, nano material etc.) and has spontaneously formed
A kind of technology of sequence structure.During self assembly, basic structural unit is spontaneous under the interaction based on non-covalent bond
Be formed as a stabilization, the structure with certain regular geometric appearance.Here " self assembly " refers to hydrophobic drug and parent
Aqueous pharmaceutical spontaneously forms the formation preparation process of spherical ordered structure.
Hydrophobic drug is not particularly limited, preferably hydrophobic drug, more preferably hydrophobic small molecules anticancer drug,
Such as taxol.
Nanoparticle can be spherical morphology.The partial size of nanoparticle is adjustable, such as controllable in 5nm~400nm, preferably
30~200nm.Moreover, the nanoparticle can be with high degree of dispersion.
A kind of simple and easy, environmental-friendly method is also disclosed herein to synthesize with high degree of dispersion, size tunable, steady
Qualitative good, a variety of therapeutic modality collaboration, the guaranteed novel nano drug of safety.Hereinafter, as an example, illustrating above-mentioned nanometer
The preparation method of drug.
Disclosed by the invention preparation is simple, pollution-free, yield is high, at low cost, high-efficient, obtained nanometer medicine
Object particle good dispersion, size tunable, stability are good, are advantageously implemented the accurate targeting to tumour, generate fabulous treatment effect
Fruit is one of the cancer immunotherapies of great application prospect.
Enumerate embodiment further below with the present invention will be described in detail.It will similarly be understood that following embodiment is served only for this
Invention is further described, and should not be understood as limiting the scope of the invention, those skilled in the art is according to this hair
Some nonessential modifications and adaptations that bright above content is made all belong to the scope of protection of the present invention.Following examples are specific
Technological parameter etc. is also only an example in OK range, i.e. those skilled in the art can be done properly by the explanation of this paper
In the range of select, and do not really want to be defined in hereafter exemplary specific value.In following embodiments, if to temperature without especially saying
It is bright, refer both to room temperature.
Embodiment 1: the synthesis of compound 1
First 50mg taxol is dissolved among the DMF of 3ml, then is charged with the 4 of 13.95mg, 4 '-two thio two fourths
(two Asia of 1- (3- dimethylamino-propyl) -3- ethyl carbon 24.69mgEDCI is added in acid into reaction solution again after it is completely dissolved
Amine hydrochlorate) and 0.7mgDMAP (4-dimethylaminopyridine), continue to put into after being stirred at room temperature 1 hour 12.35mg EDCI with
And 0.7mg DMAP, it is stirred at room temperature two days, and partly observe reaction process every 1 hour sample point, to the end of raw material total overall reaction
It can stop reacting.
First reactant is dissolved among methylene chloride after reaction, then plus water extracts two to three times, and by water phase
It collects and continues to extract both sides.Resulting organic phase is all put together and is spin-dried for and then sand processed, with methanol: methylene chloride 1:
40 ratio crosses silicagel column, purifies to product, obtains compound 1, and pass through hydrogen nuclear magnetic resonance spectrogram 1 and mass spectrogram 2
To carry out structural identification.
1H NMR (500MHz, Chloroform-d) δ 9.18 (s, 1H), 8.13 (d, J=7.3Hz, 2H), 7.78 (d, J=
7.4Hz, 2H), 7.61 (t, J=7.4Hz, 1H), 7.55-7.47 (m, 3H), 7.41 (q, J=6.3,5.5Hz, 6H), 7.32
(dq, J=8.7,4.1,3.7Hz, 1H), 7.20 (d, J=9.1Hz, 1H), 6.30 (s, 1H), 6.25-6.18 (m, 1H), 5.95
(dd, J=9.1,4.0Hz, 1H), 5.67 (d, J=7.0Hz, 1H), 5.55 (dd, J=8.8,3.8Hz, 1H), 4.97 (d, J=
9.3Hz, 1H), 4.42 (dd, J=10.8,6.7Hz, 1H), 4.30 (d, J=8.4Hz, 1H), 4.19 (d, J=8.4Hz, 1H),
3.77 (dq, J=14.0,7.3Hz, 3H), 3.27 (qd, J=7.3,5.2Hz, 2H), 2.80-2.58 (m, 11H), 2.56 (s,
5H), 2.54-2.46 (m, 3H), 2.45 (d, J=4.2Hz, 3H), 2.31 (dd, J=15.4,9.4Hz, 2H), 2.21 (s, 3H),
2.15-2.07 (m, 2H), 2.02 (p, J=6.7Hz, 7H), 1.96 (d, J=5.0Hz, 1H), 1.93 (s, 3H), 1.91-1.86
(m, 1H), 1.68 (s, 3H), 1.40-1.23 (m, 5H), 1.22 (s, 3H), 1.21-1.18 (m, 1H), 1.16 (t, J=7.3Hz,
3H),1.13(s,3H),0.91–0.80(m,2H).
Embodiment 2: the synthesis of compound 2
20mg compound 1 is dissolved among the DMF of 3ml, then is charged with the gemcitabine of 9.8mg, it is completely molten to it
Xie Houzai is charged with the DMAP of the EDCI and 0.25mg of 7.85mg, continues to put into 3.93mg's after being stirred at room temperature 1 hour
The DMAP of EDCI and 0.25mg is stirred at room temperature two days, and partly observes reaction process every 1 hour sample point, all anti-to raw material
It should terminate stop reacting.
First reactant is dissolved among methylene chloride after reaction, then plus water extracts two to three times, and by water phase
It collects and continues to extract both sides.Resulting organic phase is all put together and is spin-dried for and then sand processed, with methanol: methylene chloride 1:
20 ratio crosses silicagel column, purifies to product, obtains compound 2, and pass through hydrogen nuclear magnetic resonance spectrogram 3 and mass spectrogram 4
To carry out structural identification.
1H NMR(500MHz,Chloroform-d)δ8.14–8.08(m,2H),7.81–7.70(m,2H),7.70–7.64
(m, 1H), 7.61 (t, J=7.4Hz, 1H), 7.52 (q, J=7.6,6.2Hz, 2H), 7.47-7.33 (m, 7H), 7.24 (d, J=
7.3Hz, 1H), 6.29 (d, J=5.2Hz, 1H), 6.26-6.12 (m, 2H), 5.92 (dd, J=8.9,5.0Hz, 1H), 5.70-
5.62 (m, 2H), 5.61-5.49 (m, 1H), 5.12 (q, J=4.3,3.5Hz, 1H), 4.98-4.89 (m, 1H), 4.51-4.33
(m, 2H), 4.26 (d, J=8.6Hz, 1H), 4.17 (d, J=8.4Hz, 1H), 3.98 (d, J=8.8Hz, 1H), 3.90-3.83
(m, 1H), 3.75 (d, J=6.9Hz, 1H), 2.95 (s, 1H), 2.88 (s, 1H), 2.87-2.81 (m, 1H), 2.70 (dq, J=
17.8,6.6,5.8Hz, 2H), 2.61 (dt, J=23.5,6.8Hz, 4H), 2.51 (td, J=16.9,8.2Hz, 3H), 2.45-
2.39 (m, 1H), 2.34 (s, 2H), 2.29 (s, 1H), 2.19 (s, 3H), 2.17 (s, 1H), 2.13 (d, J=6.5Hz, 1H),
2.05 (d, J=10.3Hz, 3H), 2.02-1.94 (m, 4H), 1.93 (d, J=5.0Hz, 1H), 1.90 (s, 3H), 1.85 (s,
1H), 1.81 (d, J=18.3Hz, 8H), 1.66 (d, J=11.1Hz, 5H), 1.60 (s, 1H), 1.45-1.39 (m, 1H), 1.37
(s, 2H), 1.33 (s, 1H), 1.30 (d, J=7.4Hz, 1H), 1.28 (d, J=4.0Hz, 3H), 1.25 (s, 8H), 1.22 (s,
1H), 1.19 (s, 3H), 1.11 (s, 3H), 1.03-0.91 (m, 1H), 0.88 (t, J=6.9Hz, 2H), 0.86-0.78 (m,
1H).
Embodiment 3: the self assembly of the nanoparticle without stabilizer
The compound 2 of 5mg is dissolved among the acetone of 1ml, is drawn with syringe, and at leisure toward being placed in ultrasonic device
Among 38ml pure water, by rate of addition control in 5 μ L/s, continues ultrasound 1h after organic phase is added dropwise, clarified at this time
Opalescent colloidal.By colloid by the method for freeze-drying, medicament nano particle is obtained.Pass through transmission electron microscope observing to nanometer
Particle is as shown in figure 8, uniform particle diameter, and the partial size of most of nanoparticles is between 150nm~200nm.
Example 4: the self-assembly of stabilizer is done using DSPE-PEG5000
First DSPE-PEG5000 is added among the pure water of 38ml, heating makes it completely dissolved, then by the compound of 5mg
2 are dissolved among the acetone of 1ml, are drawn with syringe, and at leisure toward being placed among the 38ml pure water of ultrasonic device, by rate of addition
Control continues ultrasound 1h after organic phase is added dropwise, obtains clear opalescent colloidal at this time in 5 μ L/s.Colloid is passed through
The method of freeze-drying obtains medicament nano particle.By transmission electron microscope observing to nanoparticle as shown in figure 9, uniform particle diameter,
The partial size of most of nanoparticles is between 10nm~20nm.
Embodiment 5: the drug release of medicament nano particle
Medicament nano particle is dissolved in pure water, GSH is being added thereto, sampling mass spectrum inspection after stirring at normal temperature is reacted 48 hours
Whether the connecting key surveyed between each substance confirmation hydrophilic medicament and hydrophobic drug can be cut off in vivo is released drug
It releases.After reaction 48 hours, the gemcitabine known to Fig. 5, Fig. 6 among Nano medication has been released, and
And compound 2 has disappeared.The taxol among Nano medication has been released as shown in Figure 7, and compound 2 is also
Through disappearing.Glutathione GSH is antioxidant and free radical scavenger important in organism, it can thus be seen that of the invention
The medicament nano particle of preparation can be after reaching lesions position, under GSH effect in vivo, by among Nano medication two
Sulfide linkage cuts off to form mercapto, more active mercapto and then spontaneous attack ester bond and amido bond, and anti-tumor drug is complete
It releases entirely.
Embodiment 6: Inhibition test of the medicament nano particle to MCF-7 cell
2x10 is made with the DMEM culture medium containing 10%FBS in MCF-7 cell in good condition5The cell of cells/well is outstanding
Liquid is added in 96 orifice plates, and every 100 μ L of hole is placed in 5%CO2, 37 DEG C of incubator cultures for 24 hours, then trained with the DMEM of 10%FBS
It is respectively 0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM of this seven concentration ladders in hole that feeding base, which adds drug to,
Degree, every hole is cleaned three times with PBS (pH=7.4) after being incubated for for 24 hours, and after the MTT solution of 20 μ L of addition is incubated for 4h again, removal contains MTT
Culture medium, every hole is added the DMSO of 150 μ L, rocks sufficiently lysigenous crystallization after 15min at room temperature, survey in microplate reader
Fixed absorbance of every hole at 490nm, the results are shown in Figure 10, it can be seen that medicament nano particle has MCF-7 cell good
Good inhibitory effect.
Embodiment 7: Inhibition test of the medicament nano particle to 4T1 cell
2x10 is made with the DMEM culture medium containing 10%FBS in 4T-1 cell in good condition5The cell of cells/well is outstanding
Liquid is added in 96 orifice plates, and every 100 μ L of hole is placed in 5%CO2, 37 DEG C of incubator cultures for 24 hours, then trained with the DMEM of 10%FBS
It is respectively 0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM of this seven concentration ladders in hole that feeding base, which adds drug to,
Degree, every hole is cleaned three times with PBS (pH=7.4) after being incubated for for 24 hours, and after the MTT solution of 20 μ L of addition is incubated for 4h again, removal contains MTT
Culture medium, every hole is added the DMSO of 150 μ L, rocks sufficiently lysigenous crystallization after 15min at room temperature, survey in microplate reader
Fixed absorbance of every hole at 490nm, as a result as shown in figure 11, it can be seen that medicament nano particle has 4T1 cell good
Inhibitory effect.
The above is only a preferred embodiment of the present invention, it should be pointed out that: for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of Nano medication, which is characterized in that the Nano medication is hydrophobic drug A and hydrophilic medicament B by can quilt
The disulfide bond conjugation of GSH cutting, the chemical structural formula of nanoparticle obtained by self assembly, nanoparticle are as follows:
Wherein, 1,2,3 n.
2. Nano medication according to claim 1, which is characterized in that the hydrophobic drug A is hydrophobic anticancer drug,
Selected from taxol, camptothecine, Sorafenib；
And/or the hydrophilic medicament B is hydrophily anticancer drug, is selected from gemcitabine, capecitabine, zoledronic acid；
And/or the nanoparticle is spherical morphology, partial size is 5~200nm.
3. Nano medication according to claim 1, which is characterized in that the chemical structural formula of the nanoparticle are as follows:
4. the preparation method of Nano medication according to claim 1-3 characterized by comprising
(1) hydrophobic drug A is connected to form compound A-L by esterification and linker；
(2) again hydrophilic medicament B is reacted to form compound A-L-B by esterification with compound A-L；
(3) compound A-L-B is dissolved in organic solvent and forms organic phase, organic phase is dripped by way of dropwise addition into water phase
Adding makes it be self-assembled into medicament nano particle.
5. the preparation method of Nano medication according to claim 4, which is characterized in that the linker is 4,4 '-two sulphur
Generation two butyric acid, bis- thiodiglycolic acid of 3,3'- dithiodipropionic acid or 2,2'-.
6. the preparation method of Nano medication according to claim 3 characterized by comprising
(1) hydrophobic anticancer drug taxol is connected to form chemical combination by esterification and 4,4 '-two thio two butyric acid
Object 1；
(2) again hydrophilic medicament gemcitabine is reacted to form compound 2 by esterification with compound 1；
(3) compound 2 is dissolved in organic solvent and forms organic phase, organic phase, which is added dropwise by way of dropwise addition into water phase, to be made
It is self-assembled into medicament nano particle.
7. the preparation method of Nano medication according to claim 6, which is characterized in that specifically include:
1) taxol is dissolved in DMF, then is charged with 4,4 '-two thio two butyric acid and catalyst EDCI (1- (3- bis-
Methylaminopropyl) -3- ethyl-carbodiimide hydrochloride) and DMAP (4-dimethylaminopyridine), reaction is stirred at room temperature and obtains chemical combination
Object 1；
2) compound 1 is dissolved in DMF, gemcitabine and catalyst EDCI (1- (3- dimethylamino-propyl) -3- second is added
Base carbodiimide hydrochloride) and DMAP (4-dimethylaminopyridine), reaction is stirred at room temperature and obtains compound 2；
3) compound 2 is dissolved in acetone and forms organic phase, organic phase is added dropwise in water phase with the drop rate of 1~5 μ L/s,
By stirring and/or ultrasound, make compound 2 uniformly and then self assembly medicament nano particle, be freeze-dried to obtain the final product.
8. the preparation method of Nano medication according to claim 7, which is characterized in that in reaction process, the Japanese yew of addition
Alcohol: 4,4 '-two thio two butyric acid: the molar ratio of gemcitabine is 1:(1~3): (1~9).
9. according to the preparation method of Nano medication described in claim 4,6 or 7, which is characterized in that be dissolved in the water phase
DSPE-PEG5000 is as stabilizer.
10. application of the described in any item Nano medications of claim 1-3 in the drug of preparation prevention and/or treating cancer.
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