CN103933579A - Cationic liposome, and preparation method and application thereof - Google Patents
Cationic liposome, and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a cationic liposome, and a preparation method and application thereof. The cationic liposome is prepared from the following components in parts by weight by adopting a reverse evaporation method: 1 part of DOPE (dioleoyl phosphatidyl ethanolamine), 1 part of DC-Chol and 2.5 parts of OQCMC (octadecyl quaternized carboxymethyl chitosan). A cationic liposome complex adopts the cationic liposome as an encapsulating layer, wherein at least one of a medicinal carrier, a gene carrier or a gene-medicine co-carrier is encapsulated in the encapsulating layer. The liposome component is close to the cytomembrane component, the cationic liposome has better biodegradability, better biocompatibility, low immunity and low cell toxicity; the liposome is an amphoteric matter, so that the liposome can be used for encapsulating polar chemotherapeutic drugs or non-polar chemotherapeutic drugs or simultaneously encapsulating polar and non-polar drugs.
Description
[technical field]
The present invention relates to a kind of cationic-liposome and its preparation method and application.
[background technology]
Non-viral gene vector is one of current most potential carrier in gene therapy, cationic-liposome particularly, have with respect to viral genetic vector and can produce in enormous quantities and production cost is low, can repeat, the high and low immunogenicity of safety, good biocompatibility due to it, can be written into the advantages such as exogenous genetic fragment that size reaches 52kb simultaneously.This carrier has become one of study hotspot of gene therapy at present.
Although cationic-liposome is mainly due to band higher charge at present, can cause cell and occur to shrink, suppress normal cell division, Profilin kinase activity, in kytoplasm, produce the toxicity such as cavity as kytoplasm, but by cationic-liposome being modified or being added the means such as negative high polymer, auxiliary fat, searching optimum N/P can obtain high transfection efficiency, high targeting, the cationic-liposome of low cytotoxicity.It is more and more applied clinically.Wherein take cationic-liposome DMRIE/DOPE as genophore, cystic fibrosis gene (pGT21) is transported to target cell, now in clinicalⅰstage conceptual phase; The DOTAP cationic-liposome that is at present loaded with paclitaxel in Germany has entered the clinical II phase and has tested.
So far cancer remains the disease can not be cured, and no matter in developed country or developing country, all becomes the main killer of life.In the whole process of bringing out, inspire, worsening in cancer, because it is that dilatancy, diffusivity corrode growth model, whole process mechanism is extremely complicated simultaneously, not yet finds at present a kind of effective Therapeutic Method.Tradition cancer treatment method, as surgery, chemotherapy, radiotherapy etc., in most situation, therapeutic effect remains undesirable, because there is very large recurrence probability after cancer surgeries, the toxic and side effects of chemotherapy and the cancer that surpasses half all can produce multidrug resistance, radiotherapy normal tissue has serious lethal effect, and these are all main challenges that traditional treatment of cancer faces, and therefore novel, effective strategy of cancer treatment urgently occurs.The phospholipid (cationiclipids) of cationic-liposome (cationicliposome, CLP) Chang Youhe positive electricity forms jointly with neutral auxiliary phospholipid (co-lipids).Due to the lipid of positively charged and the nucleic acid (DNA of bear electricity, RNA, oligonucleotide (AODNS)) electrostatic interaction forms cationic-liposome-gene composite (lipoplexes), can form cationic-liposome-gene-chemotherapeutics ternary complex with chemotherapeutics again, by subcutaneous injection or lumbar injection, carry out blood circulation and enter target organ, and do with the surface of cell membrane of bear electricity, by endocytosis or cell fusion effect, cross and enter in born of the same parents, through a series of transportations, finally enter nucleus, carry out related gene expression, reticent (knocking out) and Drug therapy effect.Conventional genophore is divided into viral genetic vector and non-viral gene vector two classes at present.Common non-viral gene vector is as cation superpolymer, calcium phosphate, cationic polypeptide and cationic-liposome etc.What wherein development was the most ripe is cationic-liposome.But shortcoming be its transfection efficiency still lower than viral genetic vector, be therefore necessary to develop the safety novel lipide better with transfection activity, employing cationic-liposome carries altogether gene and chemotherapeutics carries out therapeutic alliance, to bring into play better healing effect.
Therapeutic alliance model carries chemotherapeutics and gene altogether, no matter still in experiment in vitro, has all proved that this model can effectively suppress tumor length, breeding, diffusion in vivo.Particularly in multiple medicines resistance to (multidrug-resistant, MDR) cancer, bring into play more obvious effect.Over nearly 3 years, the studied scholar of cationic-liposome is widely used in carries gene and chemotherapeutics carries out the therapeutic alliance of cancer altogether.
At present, this area is existing attempts the report that carries out treatment of cancer by associating polygenes, and experiment obtains certain effect in vitro.But one of bottleneck of gene perturbation technique is the toxicity of gene itself, therefore be necessary gene and chemotherapeutics to be total to year, particularly in MDR tumor model, carry altogether gene and pharmaceutical carrier experiment in vivo and vitro result shows: carry altogether that gene carries gene with chemotherapeutics cationic-liposome than single bag or chemotherapeutics cationic-liposome is treated, curative effect is better, shows synergistic function.
[summary of the invention]
One of the technical problem to be solved in the present invention, is to provide a kind of cationic-liposome, and the transfection efficiency after its stability and rotaring redyeing gene is greatly improved.
The present invention realizes one of above-mentioned technical problem like this:
A cationic-liposome, described cationic-liposome adopts reverse evaporation to be prepared from by the component of following weight portion: 1 weight portion DOPE, 1 weight portion DC-Chol, 2.5 parts by weight O QCMC.
Two of the technical problem to be solved in the present invention, is to provide a kind of preparation side of cationic-liposome, and cationic-liposome stability prepared by the method and the transfection efficiency after rotaring redyeing gene are greatly improved.
The present invention realizes two of above-mentioned technical problem like this:
A preparation method for cationic-liposome, comprises the following steps:
Step 10, by proportioning get 1 weight portion DOPE, 1 weight portion DC-Chol, 2.5 parts by weight O QCMC are dissolved in chloroform solvent;
Step 11, the solution that step 10 is obtained are mixed thoroughly, add ultra-pure water blend, Probe Ultrasonic Searching 10min;
Step 12, step step 11 is obtained to product at Rotary Evaporators, residual solvent is removed, vacuum state, obtains; The evaporating temperature of described Rotary Evaporators is 35 ℃.
Three of the technical problem to be solved in the present invention, is to provide a kind of cationic-liposome complex, and it can greatly reduce the using dosage of chemotherapeutics, reduces patient's administration number of times, greatly increases patient's postponing property.
The present invention realizes three of above-mentioned technical problem like this:
A cationic-liposome complex, is characterized in that: described cationic-liposome complex be take cationic-liposome as encapsulated layer, is encapsulated with pharmaceutical carrier, genophore or gene and medicine at least one in carrier altogether in described encapsulated layer; Described cationic-liposome is prepared from by following parts by weight of component: 1 weight portion DOPE, 1 weight portion DC-Chol, 2.5 parts by weight O QCMC.
Further, described genophore or gene and medicine altogether the contained gene of carrier be at least one in siRNA.
Further, described siRNA is MRP1 or Bcl-2 gene.
Further, the siRNA that average every 1 μ L cationic-liposome can be sealed 0.1～3.0 μ L is 10～30 μ M.
Further, to seal siRNA be 2.0～3.0 μ g to average every 1 μ L cationic-liposome.
Four of the technical problem to be solved in the present invention, is to provide a kind of cationic-liposome compound system Preparation Method, and its convenient operation, is applicable to large-scale continuous production.
The present invention realizes four of above-mentioned technical problem like this:
A preparation method for cationic-liposome complex, comprises the following steps:
In container, add blank cationic-liposome or be loaded with the cationic-liposome of PTX paclitaxel, then mix with gene siRNA, hatching 20～30min, can obtain CLP-siRNA complex or CLP-PTX-siRNA complex.
Further, described a kind of cationic-liposome complex purposes in gene and chemotherapeutics are carried in preparation altogether.
Five of the technical problem to be solved in the present invention, is to provide a kind of genomic medicine preparation, makes cationic-liposome complex to carry out better administration with various forms.
The present invention realizes five of above-mentioned technical problem like this:
A genomic medicine preparation, described genomic medicine preparation adds the pharmaceutically complementary composition of acceptable by cationic-liposome complex and is prepared from.
Tool of the present invention has the following advantages:
Cationic-liposome formula of the present invention is unique, by add proper proportion OQCMC in traditional DC-Chol-DOPE, has improved well stability and the particle size distribution of cationic-liposome, sphericity, configuration of surface; And compare with traditional DC-Chol-DOPE, the transfection efficiency after its stability and rotaring redyeing gene is improved.
Material to be encapsulated in the present invention be in cation lipid complex by liposome institute electrostatic attraction part and wrapped the part into liposome.Cationic-liposome complex of the present invention is using above-mentioned cationic-liposome as encapsulating material, and material to be encapsulated is sealed to rear formation complex, also can add pharmaceutically acceptable adjuvant simultaneously and prepare drug composite according to formulation method known in this field.
Cationic-liposome of the present invention can be sealed general medicament active composition and make cation lipid body preparation, also can seal related gene and make cationic-liposome transfecting formulations, also can carry altogether gene and chemotherapeutics and make and carry altogether gene drug carriers.And this carries gene altogether and chemotherapeutics carrier is that this area is known, through transfection, enter cell, expression or the short apoptosis of tumor cells that can suppress related neoplasms albumen, carrier altogether of the present invention has overcome traditional chemotherapy shortcoming simultaneously, can greatly reduce the using dosage of chemotherapeutics, reduce patient's administration number of times, greatly increase patient's postponing property.
The preparation method of cationic-liposome of the present invention, has not only improved raw materials used and consumption, and has optimized preparation parameter, goes back convenient operation, is applicable to large-scale continuous production.The method can obtain the cationic-liposome of narrower particle size distribution, and there is good stability, easily with gene and chemotherapeutics formation ternary complex, gene, PTX envelop rate are high, particularly gene is combined firmly with carrier, and can extend the bioavailability that carrier half-life use in vivo improves genomic medicine.
With cationic-liposome of the present invention, carry altogether gene and ternary complex that chemotherapeutics forms, can significantly improve its transfection efficiency to multidrug resistance cancer cell, can also avoid the removing of vivo immuning system to carrier, improve its action effect, fall end clinical medicine using dosage, also reduce the toxic and side effects of cationic-liposome itself simultaneously.Generally speaking, the stability of liposome of the present invention and envelop rate, transfection efficiency is all improved, and has good using value.The present invention is carried gene and chemotherapeutics altogether for oncotherapy, can effectively suppress the growth of tumor, and the using dosage of medicine also reduces greatly, and can be used for intravenous injection with treatment pulmonary carcinoma, ovarian cancer, breast carcinoma, the multidrug resistance cancers such as hepatocarcinoma, are very suitable for clinical use.Preparation method of the present invention is simply efficient, is applicable to large-scale continuous production, has good application scenario.
[accompanying drawing explanation]
The present invention is further illustrated in conjunction with the embodiments with reference to the accompanying drawings.
Fig. 1 is cationic-liposome CLP infrared spectrogram of the present invention.
Fig. 2 is particle diameter and the potential diagram of cationic-liposome CLP-siRNA of the present invention.
Fig. 3 is the TEM figure of the blank CLP of cationic-liposome of the present invention and CLP-siRNA.
Fig. 4 is the AFM figure of the blank CLP of the present invention and CLP-siRNA.
Fig. 5 is CLP-siRNA gel blocking figure of the present invention.
Fig. 6 is the active figure of CLP of the present invention and complex inhibition tumor cell thereof.
Fig. 7 is CLP complex CLSM anticancer effect figure of the present invention.
Fig. 8 is CLP complex FCM anticancer effect figure of the present invention.
Fig. 9 is that the common carrier of the present invention is schematic diagram.
[specific embodiment]
Refer to shown in Fig. 1～9, embodiments of the invention are described in detail.
The present invention relates to a kind of cationic-liposome, it is characterized in that: described cationic-liposome adopts reverse evaporation to be prepared from by the component of following weight portion: 1 weight portion DOPE, 1 weight portion DC-Chol, 2.5 parts by weight O QCMC.
The preparation method that the invention still further relates to a kind of cationic-liposome, comprises the following steps:
Step 10, by proportioning get 1 weight portion DOPE, 1 weight portion DC-Chol, 2.5 parts by weight O QCMC are dissolved in chloroform solvent;
Step 11, the solution that step 10 is obtained are mixed thoroughly, add ultra-pure water blend, Probe Ultrasonic Searching 10min;
Step 12, step step 11 is obtained to product at Rotary Evaporators, residual solvent is removed, vacuum state, obtains; The evaporating temperature of described Rotary Evaporators is 35 ℃.
The invention still further relates to a kind of cationic-liposome complex, described cationic-liposome complex be take cationic-liposome as encapsulated layer, is encapsulated with pharmaceutical carrier, genophore or gene and medicine at least one in carrier altogether in described encapsulated layer; Described cationic-liposome is prepared from by following parts by weight of component: 1 weight portion DOPE, 1 weight portion DC-Chol, 2.5 parts by weight O QCMC.
Described genophore or gene and the medicine altogether contained gene of carrier are at least one in siRNA.
Preferably, described siRNA is MRP1 or Bcl-2 gene; The siRNA that average every 1 μ L cationic-liposome can be sealed 0.1～3.0 μ L is 10～30 μ M; It is 2.0～3.0 μ g that average every 1 μ L cationic-liposome is sealed siRNA.
A preparation method for cationic-liposome complex, comprises the following steps:
In container, add blank cationic-liposome or be loaded with the cationic-liposome of PTX paclitaxel, then mix with gene siRNA, hatching 20～30min, can obtain CLP-siRNA complex or CLP-PTX-siRNA complex.
Described a kind of cationic-liposome complex is purposes in gene and chemotherapeutics are carried in preparation altogether.
The invention still further relates to a kind of genomic medicine preparation, described genomic medicine preparation is by the cationic-liposome complex described in claim 3～7 any one, to add the pharmaceutically complementary composition of acceptable to be prepared from.
Below in conjunction with specific embodiment, the present invention is further illustrated.
Embodiment mono-cationic-liposome formula of the present invention and preparation technology are preferred
Select cationic-liposome as target liposome type; after primary screening test; select octadecyl quaternary ammonium salt carboxymethyl chitosan (OQCMC), DOPE (DOPE) and 3-β-N-N '-N '-dimethyl aminoethyl-carbamyl cholesterol (DC-Chol); as basis, the optimization of then filling a prescription.
Cation lipid preparation of the present invention and CLP and siRNA consumption carry out preferably by a certain percentage, see as shown the two mass ratio of 1(and cause complex particle diameter and potential change): CLP-siRNA is along with siRNA mass penalty, its size and charged situation change, presenting particle diameter constantly increases, and current potential constantly declines.There is electrostatical binding effect in this side light siRNA and CLP, is adsorbed on CLP.
Table 1 cationic-liposome and siRNA ratio optimization experiment
The preparation of embodiment bis-cationic-liposome CLP of the present invention
Take respectively the OQCMC of 2.5 weight portions, the DC-Chol of the DOPE of 1 weight portion, 1 weight portion is dissolved in chloroform; After sample dissolution, add several milliliters of ultra-pure water blend, Probe Ultrasonic Searching 20min, then removes residual chloroform at Rotary Evaporators, obtains the OQCMC-CLP that concentration is 2.67mg/mL (V/W) (being called for short CLP), CLP-PTX.Sample sealing, lucifuge, 4 ℃ of preservations of low temperature is standby.
The cationic-liposome preparing with infrared spectrum (FTIR), Malvern ParticleSizer, TEM, AFM, carry out the Physickemical Properties of blank CLP and CLP complex.As shown in Figure 1, CLP is by DC-Chol for its infrared spectrogram, and tri-kinds of materials of DOPE and OQCMC have successfully been prepared.In DC-Chol-DOP spectrogram, at 1050cm
-1there is a new absworption peak (P=O-yl) in place, this is corresponding with the carboxyl of DOPE, has proved that DOPE is connected on DC-Chol.Meanwhile, at 1250cm
-1also there is a characteristic peak in (P-O key), this has just illustrated that OQCMC is modified on DC-Chol-DOPE.At 721cm
-1the characteristic peak occurring also further illustrates OQCMC and is connected on DC-Chol-DOPE.Particle diameter through recording this kind of cationic-liposome is in 70nm left and right, and Zeta potential, in 60mV left and right, is evenly distributed, and its particle diameter is different with siRNA mass ratio with CLP from Zeta potential, and occurs respective change, as shown in Figure 2.Being evenly distributed of visible this kind of cationic-liposome, is normal distribution, and span is little.For further carrying out the morphology observation of cationic-liposome, we have carried out TEM and AFM observes.As shown in Figure 3, prepared cationic-liposome size distribution is even, and sphericity is good, and cationic-liposome sphericity is good as can be seen from Figure 4, smooth surface, and size is consistent with particle diameter that laser particle analyzer is surveyed; Fig. 3 left side is the TEM figure of the blank CLP of cationic-liposome, the TEM figure that the right is CLP-siRNA; Fig. 4 left side is the AFM figure of blank CLP, the AFM figure that the right is CLP-siRNA.When having carried altogether after siRNA, result shows that this carrier dimensions is suitable as the delivery vehicle of gene and medicine.
Embodiment tri-CLP-siRNA combination rate tests
Gel electrophoresis experiment is for assessing the firm ability of combination of siRNA and CLP.CLP-siRNA complex is to be composited by different mass ratio (CLP/siRNA mass ratio is 0:1-20:1, and note the first swimming lane is Naked siRNA, so CLP quality is 0).Lipid complex applied sample amount is 5 μ L/ swimming lanes, and in 2.0% (w/v), the high-resolution agarose containing 0.5 μ g/mL bromination second shallow lake (EB), carries out 100V at MOPS buffer, 30min electrophoresis.Experimental result is analyzed gained by ultraviolet gel imaging system.It is more different than the gel blocking band that forms lipid complex body that result shows different quality.As can be seen from Figure 5, swimming lane 1 is naked siRNA, and it is the brightest that band is, and the amount of the siRNA in shows slice is maximum.Along with CLP mass penalty, gel blocking is rising.The known CLP of gel retardation assay and siRNA static form complex.The two mass ratio of CLP and siRNA be 3:1 and more than, in gel electrophoresis retardance experiment, find that presenting gel blocking now looks like.In a word, siRNA is easily combined with CLP and in conjunction with firm, wherein in Fig. 5, swimming lane 1 is naked siRNA, and the two mass ratio of 2-8 is respectively 1 ︰ 1,2 ︰ 1,3 ︰ 1,4 ︰ 1,5 ︰ 1,10 ︰ 1,20 ︰ 1.
Fig. 9 is schematic diagram for the present invention is total to carrier.
By measuring the growth activity of inhibition tumor cell, show extracorporeal anti-tumor effect 1. experimental techniques of carrier altogether
Anti tumor activity in vitro research
Vitro cytotoxicity experiment adopts Alamarblue to carry out.Total blank group, blank liposome group, CLP-siRNA group, CLP-PTX group, CLP-siRNA-PTX group.Main experimentation is: Bcap-37 cell suspension (100 μ L), density 1.0 * 10
4/ mL carries out bed board in 96 orifice plates.Treat that Growth of Cells degree of converging is about 60-70%, in fresh culture, respectively organize drug treating.Place incubator and cultivate after 3-5h, washing is removed unconjugated CLP and is added complete medium 100 μ L/ holes (n=6).The ultimate density of siRNA is also 100nM.After respectively at the 1st day, the 3rd day, within the 5th day, sample processing (Alamrblue reagent 10 μ L/ holes, complete medium 90 μ L/ holes), after placing incubator 4h and cultivating respectively at 570nm and 590nm detection OD value.
Apoptosis detects analyzes (LCM and FCM)
Bcap-37 cell suspension (500 μ L) is in 1.0 * 10
5/ mL cell density is planted in 24 orifice plates.To be generatedly carry out the processing of Experimental agents group and transfection, rear cultivation altogether 3 days to the regular period.Then with PBS washed cell 3 times collection, carry out AO/EB dyeing in several minutes.With LCM observation of cell apoptosis.In like manner Bcap-37 cell density is 3.0 * 10
5/ mL kind is planted in 6 orifice plates.When Growth of Cells degree of converging reaches 80%, remove old culture medium, carry out respectively blank group, CLP-siRNA, CLP-PTX, carries altogether PTX and siRNA group and processes and hatch 4h.Then remove culture medium, wash 3 times, add complete medium, cultivate altogether 3 days.Last collecting cell is also used FITC-PI dye marker, upper machine to carry out flow cytometer (FCM) and is detected.According to AnnexinV positive cell, account for total cellular score and judge apoptosis situation.
2. experimental result
CLP carries the extracorporeal anti-tumor research of PTX and siRNA altogether
Although carry altogether cytotoxic drug and siRNA, be used for treating MDR tumor and reported, with the CLP that OQCMC modifies, carry MRP1 or PTX and carry out MDR treatment and not yet reported.As shown in Figure 6, while carrying out Bcap-37 experiment in vitro, CLP-siRNA group is found the 1st day, and the 3rd day, the 5th day, the survival rate of its cell approached blank CLP group, and CLP-PTX group cell survival rate is respectively 78.3 ± 8.9%, 60.3 ± 2.4%, 66.0 ± 2.4%.And that CLP carries the survival rate of siRNA and PTX cell is altogether minimum.Carry altogether group and be respectively 72.2 ± 4.0%, 51.3 ± 2.4%, 40.6 ± 0.9%, therapeutic effect is better.
The apoptosis effect of CLP complex
For assessing blank CLP, CLP-PTX and CLP-siRNA-PTX apoptosis effect, Bcap-37 cell is processed with the different complexs that form of CLP, and analyzes with LCM and FCM.As shown in Figure 7, the apoptosis of CLP-siRNA group is lower than CLP-PTX group.Yet, carry altogether group cell survival rate minimum.Wherein, a in Fig. 7, b, c, d are followed successively by the active figure of inhibition tumor cell of blank CLP, CLP-siRNA (MRP1), CLP-PTX, CLP-siRNA-PTX.As shown in Figure 8, the mortality rate of year obvious cell death inducing of group and cell is the highest altogether.CLP-PTX group, large many cells are not caught by V-FITC, illustrate that apoptosis rate is low, and apoptotic cell accounts for 34.67%, and cell mortality is 5.92%.When for carrying altogether group while processing, apoptotic cell accounts for 11.25%, but mortality rate is 32.79%.Compare with CLP-siRNA group with CLP-PTX group, there is obvious anticancer effect.Wherein, in Fig. 8, a, b, c are followed successively by the anticancer effect figure of blank CLP, CLP-PTX, CLP-siRNA-PTX.
Thereby can draw the following conclusions: altogether carrier system has better external tumor killing effect than single year PTX or siRNA.Visible carrier is altogether better than single bag to oncotherapy successful and carries PTX or siRNA group, this be because the gene carrying altogether can inhibition tumor cell film surface the expression of chlG, thereby reducing PTX is pumped out outside born of the same parents, indirectly increase the chemotherapeutics concentration in tumor cell, carrier is the first-selection that is expected to become following cancer clinical treatment strategy altogether as seen.
To sum up, by gene and the chemotherapeutics carrier of carrying altogether provided by the invention, tested in vitro and can be improved cancerous cell transfection efficiency, improve gene and drug effect effect, reduce dosage, the while is also reduced the toxicity of cationic-liposome own; In experiment, cationic-liposome carries gene and the chemotherapeutics growth of inhibition tumor cell effectively altogether in vitro, and dosage is significantly less than independent bag and carries gene or PTX dosage form; When application, cationic-liposome is total to vector gene and medicine can be for intravenous injection, pulmonary carcinoma, and ovarian cancer, breast carcinoma, the multidrug resistance cancers such as hepatocarcinoma, are very suitable for clinical use.Simultaneously from this area general knowledge, if there is different requirements, the consumption of genomic medicine injection of the present invention can change in a big way at one.Those skilled in the art can be according to some known factors, such as the kind of disease, and the order of severity of the state of an illness, patient's body weight, dosage form, selected route of administration etc. is determined at an easy rate.
In the invention process, preparation is carried altogether gene and chemotherapeutics complex use cationic-liposome and has been overcome high molecular polymer some defects in gene therapy, and there is following advantage: cationic-liposome due to liposome component and cell membrane component more approaching, so there is better biological degradability, biocompatibility, low immunity, lower cytotoxicity.Because lipid is amphiprotic substance, determined that liposome both can wrap year polarity chemotherapeutics or nonpolar chemotherapy medicine or wrap year polarity and a nonpolar medicine simultaneously again.
Cationic-liposome formula of the present invention is unique, by add proper proportion OQCMC in traditional DC-Chol-DOPE, has improved well stability and the particle size distribution of cationic-liposome, sphericity, configuration of surface; And compare with traditional DC-Chol-DOPE, the transfection efficiency after its stability and rotaring redyeing gene is improved.
Material to be encapsulated in the present invention be in cation lipid complex by liposome institute electrostatic attraction part and wrapped the part into liposome.Cationic-liposome complex of the present invention is using above-mentioned cationic-liposome as encapsulating material, and material to be encapsulated is sealed to rear formation complex, also can add pharmaceutically acceptable adjuvant simultaneously and prepare drug composite according to formulation method known in this field.
Cationic-liposome of the present invention can be sealed general medicament active composition and make cation lipid body preparation, also can seal related gene and make cationic-liposome transfecting formulations, also can carry altogether gene and chemotherapeutics and make and carry altogether gene drug carriers.And this carries gene altogether and chemotherapeutics carrier is that this area is known, through transfection, enter cell, expression or the short apoptosis of tumor cells that can suppress related neoplasms albumen, carrier altogether of the present invention has overcome traditional chemotherapy shortcoming simultaneously, can greatly reduce the using dosage of chemotherapeutics, reduce patient's administration number of times, greatly increase patient's postponing property.
The preparation method of cationic-liposome of the present invention, has not only improved raw materials used and consumption, and has optimized preparation parameter, goes back convenient operation, is applicable to large-scale continuous production.The method can obtain the cationic-liposome of narrower particle size distribution, and there is good stability, easily with gene and chemotherapeutics formation ternary complex, gene, PTX envelop rate are high, particularly gene is combined firmly with carrier, and can extend the bioavailability that carrier half-life use in vivo improves genomic medicine.
With cationic-liposome of the present invention, carry altogether gene and ternary complex that chemotherapeutics forms, can significantly improve its transfection efficiency to multidrug resistance cancer cell, can also avoid the removing of vivo immuning system to carrier, improve its action effect, fall end clinical medicine using dosage, also reduce the toxic and side effects of cationic-liposome itself simultaneously.Generally speaking, the stability of liposome of the present invention and envelop rate, transfection efficiency is all improved, and has good using value.The present invention is carried gene and chemotherapeutics altogether for oncotherapy, can effectively suppress the growth of tumor, and the using dosage of medicine also reduces greatly, and can be used for intravenous injection with treatment pulmonary carcinoma, ovarian cancer, breast carcinoma, the multidrug resistance cancers such as hepatocarcinoma, are very suitable for clinical use.Preparation method of the present invention is simply efficient, is applicable to large-scale continuous production, has good application scenario.
Although more than described the specific embodiment of the present invention; but being familiar with those skilled in the art is to be understood that; our described specific embodiment is illustrative; rather than for the restriction to scope of the present invention; those of ordinary skill in the art are in equivalent modification and the variation done according to spirit of the present invention, all should be encompassed in the scope that claim of the present invention protects.
Claims (10)
1. a cationic-liposome, is characterized in that: described cationic-liposome adopts reverse evaporation to be prepared from by the component of following weight portion: 1 weight portion DOPE, 1 weight portion DC-Chol, 2.5 parts by weight O QCMC.
2. a preparation method for cationic-liposome, is characterized in that: comprise the following steps:
Step 10, by proportioning get 1 weight portion DOPE, 1 weight portion DC-Chol, 2.5 parts by weight O QCMC are dissolved in chloroform solvent;
Step 11, the solution that step 10 is obtained are mixed thoroughly, add ultra-pure water blend, Probe Ultrasonic Searching 10min;
Step 12, step step 11 is obtained to product at Rotary Evaporators, residual solvent is removed, vacuum state, obtains; The evaporating temperature of described Rotary Evaporators is 35 ℃.
3. a cationic-liposome complex, is characterized in that: described cationic-liposome complex be take cationic-liposome as encapsulated layer, is encapsulated with pharmaceutical carrier, genophore or gene and medicine at least one in carrier altogether in described encapsulated layer; Described cationic-liposome is prepared from by following parts by weight of component: 1 weight portion DOPE, 1 weight portion DC-Chol, 2.5 parts by weight O QCMC.
4. a kind of cationic-liposome complex according to claim 3, is characterized in that: described genophore or gene and the medicine altogether contained gene of carrier are at least one in siRNA.
5. a kind of cationic-liposome complex according to claim 4, is characterized in that: described siRNA is MRP1 or Bcl-2 gene.
6. a kind of cationic-liposome complex according to claim 5, is characterized in that: the siRNA that average every 1 μ L cationic-liposome can be sealed 0.1～3.0 μ L is 10～30 μ M.
7. a kind of cationic-liposome complex according to claim 6, is characterized in that: it is 2.0～3.0 μ g that average every 1 μ L cationic-liposome is sealed siRNA.
8. a preparation method for cationic-liposome complex, is characterized in that: described cationic-liposome complex is the cationic-liposome complex described in claim 3～7 any one;
Comprise the following steps:
In container, add blank cationic-liposome or be loaded with the cationic-liposome of PTX paclitaxel, then mix with gene siRNA, hatching 20～30min, can obtain CLP-siRNA complex or CLP-PTX-siRNA complex.
9. according to the purposes in gene and chemotherapeutics are carried in preparation altogether of a kind of cationic-liposome complex described in claim 3～7 any one.
10. a genomic medicine preparation, is characterized in that: described genomic medicine preparation is by the cationic-liposome complex described in claim 3～7 any one, to add the pharmaceutically complementary composition of acceptable to be prepared from.
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| CN108883068A (en) * | 2015-12-30 | 2018-11-23 | 杏国新药股份有限公司 | Use the method for the cationic liposomes preparation of taxane, the non-micro- rouge body preparation and other active agents combined treatment breast cancers of taxane |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108883068A (en) * | 2015-12-30 | 2018-11-23 | 杏国新药股份有限公司 | Use the method for the cationic liposomes preparation of taxane, the non-micro- rouge body preparation and other active agents combined treatment breast cancers of taxane |
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Application publication date: 20140723 |