CN106994118A

CN106994118A - A kind of preparation method and its usage for integrating photo-thermal and the nano liposomes of chemotherapy

Info

Publication number: CN106994118A
Application number: CN201610888148.2A
Authority: CN
Inventors: 李叶阔; 段婉璐; 吴晓岩; 唐家伟; 何晶玲; 严飞; 郑海荣
Original assignee: General Hospital of Guangzhou Military Command
Current assignee: General Hospital of Guangzhou Military Command
Priority date: 2016-10-11
Filing date: 2016-10-11
Publication date: 2017-08-01

Abstract

The invention discloses a kind of preparation method and its usage for integrating photo-thermal and the nano liposomes of chemotherapy, belong to pharmaceutical technology field.Sensitising agent IR 780 is encapsulated between liposome bilateral lipid by the present invention, while chemotherapeutic drugs Doxorubicin is encapsulated in into liposome center, is prepared into a kind of DITSL for treating malignant tumor.The nano-lipid volume property of the present invention is stable, and particle diameter distribution is uniform, and high temperature can be produced under laser action, kills cancer cell, and high temperature can also dissolve nano liposomes shell membrane, discharges adriamycin, further the cancer cell of inactivation residual.

Description

A kind of preparation method and its usage for integrating photo-thermal and the nano liposomes of chemotherapy

Technical field

The present invention relates to pharmaceutical technology field, more particularly to a kind of system for integrating photo-thermal and the nano liposomes of chemotherapy Preparation Method and application thereof.

Background technology

Malignant tumour remains disease before the row for triggering world population dead, its primary treatment regimen be operation, radiotherapy, Chemotherapy.However, operative treatment and radiotherapy, chemotherapy are larger to patient trauma, and chemotherapy is the problem of be generally faced with multidrug resistance.Cause This, the tumor therapeuticing method that a kind of patient of non-intruding of searching is resistant to is the focus of clinical research.

Thermotherapy is whole body and (or) local organization is heated to temperature range and the maintenance effectively treated by various methods Certain time kills a kind of method of tumour cell.Thermotherapy has direct lethal effect to tumour cell in itself, and thermotherapy can make Mitochondrial membrane, lysosome membrane, the endoplasmic reticulum of cancer cell are destroyed, and because a large amount of of lysosomal acid hydrolase release Put, cause after birth to rupture, endochylema is excessive；Cause the increase of DNA damage and suppress DNA synthesis；Related gene is influenceed to promote swollen Apoptosis of tumor.

Laserthermia has noninvasive or minimally invasive, recovery time is short compared to operation, radiotherapy, chemotherapy, few intercurrent disease it is excellent Point.It is single but because inside tumor heat distribution is uneven, especially in big blood vessel neighboring area, heat dissipates with blood circulation Pure thermotherapy is difficult to kill all tumour cells, and treatment results difference is big.Therefore, it is still necessary to combine other treatment method to improve heat The curative effect for the treatment of, clinical research shows that the therapeutic alliance of thermotherapy-chemotherapy can be remarkably reinforced antitumor curative effect.

Photo-thermal therapy (Photothermal therapy, PTT) is a kind of oncotherapy side of emerging Noninvasive Method.It is the fuel factor using various pyrogens, is heated to effectively treating temperature by tumour under illumination condition and remains certain Therapeutic strategy of the time so that tumour cell to be killed.Wherein light thermit powder has absorption characteristic in near-infrared (NIR) area, can be by luminous energy Heat energy and local heating are converted into, irreversible cellular damage (apoptosis of tumor cells and coagulation necrosis) is ultimately resulted in.It is near red The PTT of outer induced with laser has obvious advantage compared to traditional operative treatment and chemotherapy, including noninvasive, and recovery time is short, Coincidence is low, and therapeutic process can be monitored at any time.However, because intra-tumor heat distribution is uneven, independent photo-thermal therapy is not enough To kill all tumour cells, cause the results such as the incomplete, tumor recurrence of tumour ablation.Therapeutic alliance can improve treatment Curative effect reduces side effect simultaneously, it has also become a kind of important ideas of cancer therapy.

So far, the treatment methods of existing a variety of joint thermotherapies, wherein with PTT combined chemotherapies in each therapeutic alliance most There is prospect.Clinical research shows that the therapeutic alliance of thermotherapy chemotherapy can be remarkably reinforced antitumor curative effect, killed mainly due to thermotherapy While dead cancer cell, chemotherapeutics is contributed to enter the cytotoxicity of tumour cell and increase chemotherapeutics.PTT and chemotherapeutic Thing has different therapy mechanisms.The former mainly kills tumour cell by hyperpyrexia, is a kind of physical therapy modalities.Near-infrared The wave-length coverage of light is 700 between 1100nm, and organism causes near infrared light to body to the absorption very little of near infrared light Penetration capacity is very strong, can effectively penetrate into biological tissue and be selectively heated under conditions of PTT preparations.After laser irradiation Photo-thermal preparation converts light energy into heat energy, local heating so as to cause the irreversible damage of tumour cell.The latter, chemotherapy is a variety of One of the most frequently used treatment method in the treatment of tumour.Thermotherapy and chemotherapy have respective mechanism of action and cytotoxicity, thermotherapy connection Close chemotherapeutics and generally produce cooperative effect.

Currently, the fast development of nanometer technology, nano material is by the intrinsic specific physical properties of its nanoscale Thermotherapy combined chemotherapy provides an effective platform, and the thermotherapy chemotherapy combined Therapy study of novel nano system just turns into tumour The study hotspot for the treatment of.Come in a variety of multi-functional cages, gold nanorods, CuS nanocages, silicon nanoparticle etc..These drug-loading systems The synergistic effects played are proved by external and experiments in vivo, but the shortcoming of these materials is that insoluble drug release delays Slowly, it is and uncontrollable.In the neighboring area of tumour or the region of big blood vessel is closed on, temperature is not enough at 41 DEG C -45 DEG C after thermotherapy To destroy these nano-carriers so as to discharge medicine.

Temperature sensitive liposome pharmaceutical carrier (Temperature-Sensitive-Liposomes, TSL) is a kind of energy Carrying medicaments and the liposome for effectively discharging medicine under the high temperature conditions.When temperature reaches the phase transition temperature of matrix material When, liposome bimolecular film by " gel " state when being converted to " liquid crystal " state structure, the acyl chain turbulence of its phosphatide and activity Degree increase, the mobility increase of film, the medicine encapsulated discharges rapidly.It is not enough to kill all tumour cells in the high temperature of thermotherapy In the case of, the hyperpyrexia that thermotherapy is produced has been enough to cause temperature sensitivity liposome to undergo phase transition, and is released the drug in knurl.

The content of the invention

In order to solve the deficiency that existing treating malignant tumor is present, present invention aims at provide a kind of collection photo-thermal and change Treat in the preparation method and its usage of the nano liposomes of one.

The present invention is adopted the technical scheme that：

The preparation method that integrates photo-thermal and the nano liposomes of chemotherapy of the present invention is comprised the following steps that：

Lipid and IR-780 is miscible in chloroform, and in vortex, nitrogen dries up into uniform film, and vacuum pumps residual chlorine It is imitative, (NH4) 2SO4 solution is added, lipid membrane is dissolved, IR-780 liposomes are made to clarifying in sonic oscillation in a water bath, Established liposome is extruded from polycarbonate membrane repeatedly with mini-extruder extrusion instrument, the liposome of uniform particle sizes is prepared into, by IR- 780 liposomes are loaded in bag filter, are put into PBS solution and are dialysed, the ammonium sulfate of liposome interior are substituted for into PBS, by Ah mould Plain solution is added dropwise in liposome, the vibration of side edged, and adriamycin is loaded into liposome kernel using ammonium sulphate gradient, is made The nano liposomes of the present invention.

Described lipid is by DPPC, MPPC and distearyl acyl group phosphatidyl Monoethanolamine-polyethylene glycol 2000 composition, the mol ratio of three is 86：10： 4.

The mass ratio of IR-780 and lipid is 1~10：100, preferably 5:100.

The time that vacuum takes out chloroform is 3h.

Bath temperature is 65 DEG C.

Described polycarbonate membrane is 200nm (liposomal diameter).

The time dialysed in PBS solution is 4h.

The concentration of described Doxorubicin solution is 1mg/ml, and the mass ratio of adriamycin and lipid is 1:10~30, be preferably 1:20。

Described ammonium sulphate gradient is after blank liposome is prepared, to remove the ammonium ion in the external aqueous phase of lipid, interior Form an amino molecule and a proton in aqueous phase after the ionization of ammonium ion, amino molecule under cross-film diffusion equally by except Go, which forms the pH gradient between inside and outside aqueous phase, medicine is not carried and is got ready, add after Doxorubicin solution, Doxorubicin molecules Under the driving of pH gradients, cross-film enters in intraliposomal aqueous phase, and with sulfate ion into salt, into salt after form colloidal state and sink Form sediment, be gathered in interior aqueous phase, Doxorubicin molecules and proton combine to form its ionic state in addition, so that membrane permeability is lost, without Can reversely it spread out.

Nano liposomes prepared by the method for the present invention can be used for the medicine for preparing treatment tumour.

The positive effect of the present invention is as follows：

Sensitising agent IR-780 is encapsulated between liposome bilateral lipid by the present invention, while chemotherapeutic drugs Doxorubicin is encapsulated In liposome center, a kind of DITSL for treating malignant tumor is prepared into.The nano-lipid volume property of the present invention is stable, grain Footpath is evenly distributed, and high temperature can be produced under laser action, kills cancer cell, and high temperature can also dissolve nano liposomes shell membrane, release Adriamycin is put, further the cancer cell of inactivation residual.

Brief description of the drawings

Fig. 1 is the schematic diagram of the preparation method of the liposome of the present invention.

Fig. 2 is the grain size distribution of the liposome of the method preparation of the present invention.

Fig. 3 is that the cytoactive after thermochemotherapy is quantitatively schemed；(A) PBS, blank liposome, DTSL, ITSL and DITSL are added Cell is whetheing there is laser irradiation (0.6W/cm afterwards², 5min) under survival rate.Thermochemotherapy group cells survival rate is minimum.(B) strengthen (0.8 W/cm after laser irradiation intensity², 5min), thermotherapy group and thermochemotherapy group cell survival rate further lower.

Fig. 4 is to show tumour in mouse tumor figure after each treatment group is treated 21 days, circle.

Embodiment

The following examples are that the present invention is described in further detail.

Embodiment 1

First, DITSL preparation and nature examination

1. method

The preparation of 1.1 DOX (adriamycin)/IR-780 temperature sensitivity liposomes

DPPC, MPPC are taken, (mol ratio is 86 to DSPE-PEG2000：10：4), with IR-780 (IR-780：Lipid=5： 100, mass ratio) it is miscible in chloroform, in vortex, nitrogen dries up into uniform film, and vacuum 3h pumps residual chloroform.Add 250mM(NH₄)₂SO₄Solution, lipid membrane is dissolved, and IR-780 liposomes are made to clarifying in sonic oscillation in 65 DEG C of water-baths (ITSL).Established liposome is extruded from 200nm polycarbonate membranes repeatedly with mini-extruder extrusion instrument, the fat of uniform particle sizes is prepared into Plastid.IR-780 liposomes are loaded in bag filter, the 4h that dialyses is put into PBS solution, the ammonium sulfate of liposome interior is replaced Into PBS.DOX solution (1mg/ml) is added dropwise in liposome, the vibration of side edged, and DOX is loaded into fat using ammonium sulphate gradient Plastid kernel.Evacet (DTSL) only adds phosphatide in film forming procedure, and remaining preparation method is as previously described.

1.2 DITSL are characterized

The particle size of each liposome, particle diameter distribution, dispersion index (PDI), surface potential are by Malvern Zeta granularities Instrument is measured at room temperature.DTSL, ITSL and DITSL absorption spectrum are measured by UV-Vis spectrometer, and DOX swashs in 480nm Hair, IR-780 is excited in 780nm.The measuring method of DOX and IR-780 envelop rate is as follows：First obtained with sepectrophotofluorometer Go out DOX and IR-780 fluorescent value-concentration standard curve.Each liposome prepared is put into super filter tube, passes through ultracentrifugation Method (20,000r/min, 30min) comes out liposome and free medical separation, with sepectrophotofluorometer combination DOX with IR-780 fluorescent value-concentration standard curve measures the drug concentration for being encapsulated in liposome interior, then calculates retention volume.Medicine Envelop rate computational methods are shown in formula (1).

Envelop rate (%)=(medication amount of encapsulating)/(initial incremental amount) × 100. (1)

Stability of 1.3 DITSL under biotic environment

Stability of the DITSL under different biotic environments is tested we used different buffer solutions, including PBS, 30% serum, 50% serum and 90% serum buffer.DITSL suspension (1mM lipids) is incubated in above-mentioned buffer solution at 37 DEG C Educate 24h (I_b).Blank control (I is used as using the DITSL without incubation₀), treat what is be completely severed with 1%TritonX-100 DITSL is used as positive control (I₁₀₀).DOX fluorescent value under the conditions of measuring each with XRF, DOX release rates calculating side Method is shown in formula (2).DITSL stability is represented with 100%-DOX release rates.

DOX release rates (%)=(I_b-I₀)/(I₁₀₀-I₀) (2)

The external heating of 1.4 induced with laser

PBS,DTSL(DOX:15μg/ml),ITSL(IR-780:15 μ g/ml), and DITSL (IR-780:15μg/ml, DOX:15 μ g/ml) each 1ml added in small test tube.With near-infrared laser (808nm, 0.8W/cm²) each sample is respectively irradiated 5min.While laser irradiation is carried out to sample, every 30 seconds, the temperature of each liposome solutions is entered with thermal infrared imager Row is measured and recorded.

1.5 DITSL temperature sensitive drugs release

In the PBS that DITSL is added to 90% serum, respectively at 37 DEG C, metal bath is carried out under the conditions of 42 DEG C and 50 DEG C.Respectively In timely supplement equal amount after 20s, 40s, 1min, 2min, 3min, 4min, 5min, 10min and 30min sampling, every sub-sampling , 90% blood-serum P BS at identical temperature.Blank control (I is used as using the fluorescent value of the DITSL without incubation simultaneously₀), DITSL determines the fluorescent value of DOX in liposome as total amount (I after being demulsified with 1%TritonX-100₁₀₀), per sub-sampling sample Fluorescent value be I_t, add up successively, calculate accumulative release rate of the medicine in Each point in time at a temperature of each.

The DITSL insoluble drug releases of 1.6 laser controllings

The drug release experiment method of laser controlling is as follows：In the PBS that DITSL and DTSL are inserted to 90% serum, near Infrared laser (808nm, 0.8W/cm²) 20s, 40s, 1min, 2min, 3min, 4min, and 5min are irradiated respectively, measure irradiation The DOX release conditions of two kinds of liposomes of different time, in addition, surveying DOX releases when same time point DITSL is irradiated without laser Amount.

2. testing result

The making of 2.1 liposomes and sign

The manufacturing process of each liposome is then terraced through ammonium persulfate as shown in figure 1, first film aquation method prepares ITSL (green) Degree method is loaded into DOX (red), and DITSL is in dark-brown.Because IR-780 is different from DOX hydrophily, IR-780 is dissolved in liposome In double-layer of lipoid, DOX is encapsulated in the hydrophilic core of liposome.The particle size of each drug-loaded liposome, dispersion index (PDI), Surface potential and envelop rate are shown in Table 1.DITSL particle diameters are in 138.98nm or so, PDI about 0.32.DTSL, ITSL and blank liposomes The particle diameter of body is slightly smaller, respectively about 94.05nm, 84.55 1nm and 77.53nm.All liposomes slightly negative potential, Zeta potential About -10 or so.DOX DITSL envelop rate about 92.52%, IR-780 DITSL envelop rate about 94.47%. DTSL IR-780 envelop rate about 99.98% in middle DOX envelop rate about 97.39%, ITSL.

The blank liposome of table 1., ITSL, DTSL and DITSL sign

Data are means standard deviation, n=3,；Medicine fat is than the mass ratio for medicine and lipid；PDI is polydispersity index.

The liposome optical characteristics measured from ultraviolet-visible spectrophotometer.ITSL has maximum suction at 793nm Peak is received, meets IR-780 Absorption Characteristics, DTSL has absorption maximum at 492nm, it is similar to DOX absworption peaks.DITSL exists simultaneously 793nm and 492nm have notable absworption peak, and strong absorption characteristics of the DITSL near infrared region also demonstrates IR-780 and be suitable as For thermotherapy reagent.

Stability of each liposome under different biotic factors, is being incubated within 12h, DITSL insoluble drug releases are slower. 90% 37 DEG C of serum buffer is incubated after 24h, and the DOX in DITSL all discharges, in 30% serum and 50% serum is wrapped, DITSL stability is slightly good, and about 57% and 53% DOX is discharged respectively.And seldom (about 23%) discharged in PBS.As a result Show that TSL stability in PBS is preferable, but the less stable in high concentration serum.The external heating of 2.2 induced with laser

In order to assess DITSL photo-thermal conversion efficiency, we irradiate lower DITSL heating with thermal infrared imager exploring laser light Situation.In 0.8W/cm²Laser was irradiated in five minutes, DITSL and ITSL temperature highest is raised to 54.2 DEG C and 54.1 DEG C respectively.And DTSL and PBS groups only increase 9.7 DEG C and 9.3 DEG C, finally rise to 34 DEG C or so.When lacking photo-thermal preparation, laser can not Heat energy is effectively converted into, so DTSL and PBS groups only have a small amount of temperature rise.And DITSL and ITSL are due to IR-780's There is temperature rise substantially and rapid.ITSL and DITSL are after temperature raises a period of time, and temperature can slowly decline, this be by The self-quenching between molecule is there occurs afterwards for a period of time in IR-780 illumination.

2.3 DITSL drug release characteristics

We are incubated DITSL with the metal bath of different temperatures, test the DITSL sensitive release characteristics of temperature.In DITSL DOX release has obvious difference between body temperature and hyperpyrexia.DOX accumulative release rate has two nodes, one be In 1min, discharge the rapidest, 1min is heated at 42 DEG C, DITSL releases about 40% medicine.Second node be 5min, constant temperature is at 42 DEG C, and DOX preparation increase trend near-linear between heating 1min and 5min, release is rapid, Increase to 70% from 40%.Heating 5min after, DOX release slightly slows down, heat from 5min to 20min, preparation from 66.9% ± 4.0% increases to 73.9% ± 3.9%.DOX sustained releases with extending heating time, in heating 30min Afterwards, DITSL releases 78% DOX.Discharged faster in medicine 1min at 50 DEG C, release about 61% DOX, 1min is arrived 30min releasing trend and situation at 42 DEG C are similar, after heating 30min, DOX release rate about 90%.And other conditions are identical In the case of, DITSL drug release rate and accumulative release rate are significantly lower than 42 DEG C and 50 DEG C at 37 DEG C, are finally released in 30min About 22.8% DOX is put.

Then we test the insoluble drug release situation of near-infrared laser induction.If without laser irradiation, DITSL medicine Thing release rate is relatively low, release rate about 8.9% after 5 minutes.Drug release rates of the DTSL under laser irradiation is with DITSL without laser feelings Condition is similar, the drug release rate after 5 minutes about 11.2%.DITSL drug release rate increases with the time that laser is irradiated, Increased rapid in one minute, reached 56.0%, then increased and slightly slow down, after 5 minutes, drug release rate about 80.1%, Much it is higher by other groups.

2nd, the experiment in vitro of DITSL Hyperthermic Chemotherapies breast cancer

1. method

1.1 cell culture

With DMEM high glucose mediums (adding 10% hyclone and 1% penicillin and 1% streptomysin) in 37 DEG C, 5% CO₂4T1 cells are cultivated in incubator, culture medium, the passage of 0.25% Trypsin Induced is changed within every 3 days.

1.2 cell in vitro are absorbed

Breast cancer 4T1 cell seedings are in 6 orifice plates (1.2 × 10⁶Individual/hole), orifice plate is placed in cell culture incubator and is incubated 24h, after cell is completely adherent, it is 5 μ g/ml, 10 μ g/ml or 20 μ g/ml DITSL fresh culture to be replaced by content of dispersion It is incubated the PBS solution that isodose is added in 1h, 2h or 3h, control group.PBS is washed three times after incubation, washes away the lipid not swallowed Body.Pancreatin digests and collects cell, with flow cytometry analysis intracellular IR-780 and DOX fluorescent value.Each sample selects one Ten thousand cells, data analysis uses Flow Jo softwares.

It is aobvious we used laser co-focusing to further look at the Subcellular Localization of DTSL, ITSL and DITSL in 4T1 Micro mirror.Take the logarithm growth period 4T1 breast cancer cells kind in the 8 special orifice plates of hole laser co-focusing, each plate hole cell number is 2×10⁴, orifice plate is placed in cell culture incubator and is incubated 24h, after cell is completely adherent, (the 10 μ g/ml containing DTSL respectively are changed DOX), ITSL (10 μ g/ml IR-780), and DITSL (10 μ g/ml DOX, 10 μ g/ml IR-780) fresh culture.It is right According to the PBS solution that isodose is added in group.Cultivated in incubator after 3h, suck culture supernatants, PBS is washed 3 times, 4% poly DAPI nuclear targetings 5min under the conditions of formaldehyde fixation 15min, lucifuge, PBS are washed 3 times.DAPI can be combined with DNA strengths, Blue-fluorescence is sent under fluorescence microscope.DAPI can pass through complete cell membrane, and it can be used for living cells and fixed cell Dyeing.Orifice plate is placed under laser co-focusing and detects intake situations of the DITSL in 4T1 cells.DAPI excitation wavelength is 340nm, launch wavelength is 488nm, and IR-780 excitation wavelength is 708nm, and launch wavelength is 760 nm.

1.3 intracellular insoluble drug releases

Take the logarithm growth period 4T1 breast cancer cells kind in the 8 special orifice plates of hole laser co-focusing, each plate hole cell number For 2 × 10⁴, orifice plate is placed in cell culture incubator and is incubated 24h, after cell is completely adherent, then with (the 10 μ g/ml containing DITSL DOX, 10 μ g/ml IR-780) medium culture 1h, PBS, which is washed, uses DAPI nuclear targetings after 3 times, be placed in laser co-focusing and show Micro- Microscopic observation.The cell after dyeing is divided into three groups again, respectively laser irradiation (808nm, 0.8W/cm², 5min), 37 DEG C or 42 DEG C of incubation 5min, again with the situation of intracellular DOX releases in the case of confocal laser scanning microscope different disposal.

1.4 extrinsic heat therapies, Hyperthermic Chemotherapy effect

Take the logarithm growth period 4T1 cells kind in 96 orifice plates (4 × 10³Individual/hole), it is incubated overnight in cell culture incubator.More Change respectively containing blank liposome, DTSL, ITSL and DITSL PBS or only PBS (per μ l of hole 100), it is ensured that last each hole DOX Concentration with IR-780 is in 0 or 10 μ g/ml.After 37 DEG C of culture 3h, PBS is washed 3 times.Each group is not added with laser treatment respectively or laser shines Penetrate 5min (808nm, 0.8W/cm²), 10 groups altogether.The group irradiated using only adding PBS without laser is blank control group, to add It is laser group to enter the group that laser is irradiated after PBS, to add DTSL groups as chemotherapy group, using ITSL+ laser groups as thermotherapy group, with DITSL+ laser groups are thermochemotherapy group.Cultivate after 2h, add and contain in 10 μ l CCK-8 reagent reactings 1h, CCK-8 reagents per hole Have 2- (2- methoxyl group -4- nitrobenzophenones) -3- (4- nitrobenzophenones) -5- (2,4- disulfonic acid benzene) -2H- tetrazolium monosodium salts, it Can be by the dehydrogenation in living cells mitochondria in the presence of 1- methoxyl group -5- toluphenazines dimethyl suflfates (1-Methoxy PMS) Enzyme is reduced to yellow formazans product (Formazan), and the quantity of quantity and living cells of the dead cell without this function , formazan things It is directly proportional.The absorbance value of reaction product is determined at 450nm wavelength, living cells quantity can be reflected indirectly.Each group is inhaled according to it Receipts value calculates the cytoactive relative to blank group.

In order to intuitively observe thermotherapy, the therapeutic effect of thermochemotherapy, We conducted direct cell dyeing.4T1 is thin first Born of the same parents are planted in 24 orifice plates (7 × 10⁴Individual/hole) overnight incubation, change PBS or only PBS respectively containing DTSL, ITSL and DITSL (per μ l of hole 400), final DOX and IR-780 concentration are in 0 or 10 μ g/ml.After 3 hours, part treatment group carries out laser spoke According to being grouped as follows：PBS groups (blank control group), PBS+ laser groups (laser group), DTSL groups (chemotherapy group), ITSL groups, ITSL+ Laser group (thermotherapy group), DITSL groups, DITSL+ laser groups (thermochemotherapy group).It is further cultured for after 2h, cell carries out Calcein-AM/ PI is dyed, micro- Microscopic observation.Calcein-AM acetoxymethyl ester lipophilicity is very high, it is can pass through cell membrane, passes through work Intracellular esterase effect, Calcein-AM can slough AM bases, and the calcein of generation can send strong green fluorescence, therefore Calcein-AM is only dyed to living cells.PI is a kind of nucleic acid dye, and it can not pass through complete cell membrane, but in apoptosis The cell and dead cell in late period, PI can make the red dye of nucleus through cell membrane.Because calcein and PI can quilts 490nm is excited, therefore available fluorescence microscope observes living cells and dead cell simultaneously.

2. result

2.1 cell in vitro are absorbed

After DITSL of the 4T1 through various concentrations handles the different times, the quantitative result that flow cytometry is surveyed is shown： 4T1 cellular uptakes DTSL and ITSL have dosage and time dependence.In same time point the fluorescence intensity of (3h) cell with The increase of drug concentration and strengthen.Meanwhile, these cells are at same drug concentration (10 μ g/ml), with medicine incubation time Extension, cell has stronger fluorescence intensity.Intake of the tumour cell to liposome is in time dependence.4T1 takes the photograph to DITSL Take and be slightly different, when drug concentration is 5 μ g/ml, 10 μ g/ml and 15 μ g/ml, ingestion of medicines amount increases with concentration, and 20 μ It is declined slightly during g/ml.Simultaneously in same drug concentration, when 1h, 2h and 3h, intakes of the 4T1 to medicine gradually increases, and during 4h It is declined slightly.

Each liposome is shown in 4T1 Subcellular Localization result.The intracellular visible IR- of 4T1 after ITSL and DITSL incubations 780 fluorescence, IR-780 is evenly distributed in the endochylema of cell, and after DTSL and DITSL is treated, it is seen that DOX is main It is gathered in tumor cell core.The DOX of DITSL parcels exists simultaneously in the cytoplasm and nucleus of 4T1 cells.

2.2 intracellular insoluble drug releases

We further study release and cellular uptake of the medicine in cellular level with laser confocal microscope.4T1 Cell adds the PBS of preheating and keeps constant temperature or directly carry out laser irradiation, to compare not equality of temperature after DITSL is incubated The influence that degree and mode discharge to cellular uptake and Intracellular drug.After laser irradiation 5min, intracellular visible DOX fluorescence letter Number it is remarkably reinforced more at room temperature.Similar DOX fluorescence signals enhancing situation is also shown after 5 minutes in 42 DEG C of constant temperature.And 37 DEG C of perseverances After warm 5min, intracellular DOX fluorescence signal and difference very little at room temperature.The DOX fluorescence intensities of each group before and after the processing have quantified Processing, after laser irradiation, the fluorescence intensity increase of adriamycin is obvious, and after 37 DEG C of constant temperature 5min, the fluorescence of adriamycin is strong Degree is not obvious compared with before processing increase.

2.3 In Vitro Chemotherapies, thermotherapy, Hyperthermic Chemotherapy

For chemotherapy, thermotherapy, the cell killing situation of thermochemotherapy in the case of evaluating in vitro, we employ the survey of CCK-8 methods Cytoactive.Shown in Fig. 3, liposome vectors and PBS control group ratio and no cytotoxicity (P>0.05).DTSL(10μg/ml DOX) after chemotherapy, cytoactive substantially lowers, active about 59.4%, the DTSL chemotherapy laser irradiation simultaneously compared with PBS control group Afterwards, cytoactive indifference (P after cytoactive about 53.3%, with chemotherapy>0.05).This is due to without effective in DTSL Photo-thermal preparation, laser irradiation after heat up it is unobvious, it is impossible to reach the effect of fire damage.ITSL (10 μ g/ml IR-780) is incubated Afterwards, 4T1 cytoactives and control group indifference, and after ITSL incubations in the case of the irradiation of row laser, cytoactive significantly lowers, Drop to about 43.6%.Illustrate that independent thermotherapy has certain lethality to cell.It is worth noting that, being incubated adding DITSL And after laser irradiation, cytoactive is reduced to 17.9%, (P low compared with other group of cytoactive<0.01).Increase laser intensity (0.8 W/cm afterwards²), thermotherapy group and thermochemotherapy group cytoactive further lower.The cytoactive of thermochemotherapy group drops from 17.8% It is low to 9.5%.

In order to intuitively observe the therapeutic efficiency of each processing method, Calcein-AM/PI dyeing is carried out after cell processing, it is living Cell can be dyed by Calcein-AM, and in green under fluorescence microscope, and the cell of death/late apoptic can be dyed by PI, Take on a red color under the microscope.After the irradiation of independent laser, almost without cell death, illustrate used laser exposure intensity phase To safety.After DTSL chemotherapy, death/non-viable apoptotic cell quantity increase is similar to after DITSL processing, illustrates in no laser In the case of irradiation, DITSL therapeutic effects are similar to independent chemotherapy.And ITSL laser irradiation after, death/non-viable apoptotic cell Quantity increase is obvious.DITSL adds after laser irradiation, due to heat killing and DOX rapid release, and most cells are killed, can See that whole visual field takes on a red color, only a few cell is still survived.Each treatment group result is consistent with CCK-8 quantitative analysis results.Thermalization Treatment group dead cell number substantially increases compared with chemotherapy or thermotherapy group, illustrates that DITSL can be by laser spoke to the toxicity of 4T1 cells The aobvious increase of illumination, thermochemotherapy has the therapeutic effect that monotherapy hardly matches.

3rd, depression effects of the DITSL to breast cancer transplantable tumor

1. method

The strain of 1.1 knurls and experimental animal

Female SPF BALB/c mouse, the week old of mouse age 8~10,18~22g of body weight, by Guangdong Province, animal center is provided (SCXY [Guangdong] 2013-0002), is raised in SPF grades of laboratories of Shenzhen Xianjin Technology Academe of the Chinese Academy of Sciences.Feed, drinking-water, bedding and padding Through 121 DEG C of high pressure steam sterilization, 20min sterilization treatments, water and the free diet of feed.

1.2 cell culture

With DMEM high glucose mediums (adding 10% hyclone and 1% penicillin and 1% streptomysin) in 37 DEG C, 5% CO₂4T1 cells are cultivated in incubator, culture medium, the passage of 0.25% Trypsin Induced is changed within every 3 days.Growth period of taking the logarithm is thin Born of the same parents are tested.

1.3 subcutaneous transplantation knurl models are set up

Zooscopy is agreed to by Chinese Academy of Sciences's Shenzhen advanced technology research animal love and using the committee.Right side of mice Belly preserved skin, phase cell of taking the logarithm centrifuges 3 times after pancreatin digestion and washes away serum, and it is 1 × 10 to be prepared into cell number⁷Individual/ml nothing Serum cell suspension, every mouse web portion is subcutaneously injected into 0.1ml cell suspension.Every three days measurement mouse body weight and Gross tumor volume, mouse inoculation tumour cell 6 days or so, treats that gross tumor volume grows to about 100mm³Shi Jinhang is treated, gross tumor volume meter Formula (3) is shown in calculation.

Gross tumor volume=1/2 × major diameter × minor axis² (3)

1.4 experiment packet

The preparation method of each drug-loaded liposome and the characteristic of liposome are as previously described.Treat that gross tumor volume grows to about 100mm³ Start experiment.Tumor-bearing mice is randomly divided into seven groups at random：(1) blank control group：Every mouse intratumor injection 0.1ml 0.9% Physiological saline；(2) pure laser group：After mouse intratumor injection 0.1ml 0.9% physiological saline, tumor region is placed in sharp 5min (808nm 0.8W/cm are irradiated in optical range²)；(3) chemotherapy group：Injection 0.1ml 400 μ g/ in every mouse tumor Ml DTSL solution；(4) ITSL groups：Every μ g/ml of mouse intratumor injection 0.1ml 400 ITSL solution.(5) thermotherapy group：Often After μ g/ml of mouse intratumor injection 0.1ml 400 ITSL solution, then laser irradiation 5min is carried out to tumor region (808nm, 0.8W/cm²).(6) DITSL groups：Every μ g/ml of mouse intratumor injection 0.1ml 400 DITSL solution.(7) thermalization Treatment group：After every μ g/ml of mouse intratumor injection 0.1ml 400 ITSL solution, then laser irradiation 5min is carried out to tumor region (808nm, 0.8W/cm²)

1.5 in body heat chemotherapy temperature monitoring

Laser parameter：Wavelength：808nm；Power：0.8W/cm²；The ultrasonic irradiation time： 5min.Mouse tumor volume is grown to About 100mm³Left and right rows oncotherapy, therapeutic modality is as previously described.Pure laser group, thermotherapy group and thermochemotherapy group are to tumor area While domain carries out laser irradiation, every one minute, the temperature of tumor region is measured and recorded with thermal infrared imager.

1.6 thermochemotherapy shorts

The short term efficacy of each therapeutic modality is evaluated using the method for hematoxylin eosin staining.Every group of three tumor-bearing mices, Each group puts to death mouse and takes out tumour, fixed with formalin, wax stone is embedded into through dehydration, transparent, waxdip after treatment 48h.In Cut into slices on paraffin slicing machine, slice thickness is 6 μm.

H＆E dyeing observation tumor tissues morphological changes

H＆E dyeing flows：

A sections dewax 15min in dimethylbenzene.

B moves into dimethylbenzene and absolute alcohol (1:1) 15min in mixed liquor.

C is put into 100%, 95%, 85%, 70% alcohol, and the time is respectively 5min.Most it is transferred to dye liquor through distilled water afterwards.

D haematines dye liquor dyes 5min.

E washings slide 5min washes away unnecessary dye liquor, 0.5~1% hydrochloride alcohol (70% alcohol) color separation a moment.

Microscopy is controlled, untill chromatin in nucleus and core is clear, approximate number 10s.

G distilled water is short to be washed.

The eosin stains of H 0.1~0.5% dye 1min.

I is successively through 70%, 85%, 95%, 100% dehydration of alcohol, and at different levels is 2~3min, the wine of concentration below 95% Yihong is easily decolourized in essence, should suitably shorten the time.

J dimethylbenzene is transparent (secondary), altogether about 30min.

K mountings：Unnecessary dimethylbenzene around section is wiped, appropriate neutral gum is added dropwise, slide sealing is capped.Microscopic observation Cellular morphology changes.

In order to detect the apoptosis situation of tissue, the apoptosis situation of cell in each group tumor tissues is detected with TUNEL reagents. TUNEL experimental principles：Cell is when occurring apoptosis, 3 '-OH the urging in terminal deoxynucleotidyl transferase of the DNA exposures of fracture Under change, and fluorescein-labeled dUTP reactions.Probe blue light under fluorescence microscope is excited into green glow.Its operating procedure：Take out Tumor tissues fixed through formalin after be prepared into paraffin section, slice thickness is 8mm.The paraffin section de-waxing for preparing, After aquation, take out sample and be put into the staining jar containing penetrating liquid (0.1%Triton-X-100 is dissolved in 0.1% liquor sodii citratis) In, room temperature, reaction time 8min.PBS 3 times.Put samples into containing antigen retrieval buffers (0.1M citric acid+citric acid Sodium pH of buffer 6.0) staining jar in, 92-98 DEG C, reaction time 3-4min.PBS 3 times.Each sample adds 50 μ l TUNEL reaction solutions, negative control, which is added under 50 μ l marking fluid, normal temperature lucifuge wet environment, reacts 60min.PBS 3 times. DBA nitrite ions are added dropwise, in monitoring colour developing degree under microscope, in good time terminating reaction, running water is rinsed.Haematoxylin redyes cell Core, micro- Microscopic observation karyon returns running water after basket and rinsed.Fast Field ethanol, ethanol, absolute ethyl alcohol dehydration, dimethylbenzene are saturating It is bright.

Long-term treatment in 1.7 bodies

Mouse tumor volume grows to about 100mm³Left and right rows oncotherapy, every group of 7 tumor-bearing mices, for example preceding institute of therapeutic modality State.The mouse treatment same day is designated as the 0th day, was then measured every 3 days and records a mouse tumor volume.Submethod is to use electronics The major diameter and minor axis of vernier caliper measurement tumour, calculate the volume of tumour.To being weighed to mouse while gross tumor volume is measured, remember Record the change of mouse weight.The measurement of gross tumor volume and mouse weight continues to treatment the 30th day, according to animal care with making With plan, mouse tumor volume is more than 1000mm³Euthanasia is sentenced to mouse.Using the mouse tumor Volume Changes in 21 days as The evaluation of long-term efficacy after oncotherapy, the existence Appreciation gist after treatment is used as using mouse survival situation in 30 days.

1.8 tumor vessel immunohistochemical analysis

In order to detect the capilary situation of tissue, immunohistochemical staining detection each group tumour is carried out with rabbit-anti mouse CD31 antibody The apoptosis situation of cell in tissue.CD31 is thin by vascular endothelial cell, blood platelet, monocyte, neutrophil leucocyte and T lymphs The adhesion molecule of cellular expression, its structure contactin member.

CD31 staining procedures：

A paraffin sections are in 60 DEG C of drying in oven 1h, abundant wax melting.

B conventional xylenes dewax (dimethylbenzene I, each 15min of dimethylbenzene II).

C graded ethanols are dehydrated, and are put into 100%, 95%, 85%, 70% alcohol, and the time is respectively 5min, last distilled water and PBS.

D Proteinase Ks working solution (10-20in 10mM Tris/HCL, pH7.4-8) is in 37 DEG C of antigen retrieval 15-30min. Subsequent PBS 2 times

Rabbit-anti mouse CD31 monoclonal antibodies (1 are added dropwise in E：200), PBS is cleaned 3 times after 4 DEG C of overnight incubations.

The universal secondary antibody of horseradish enzyme mark, 37 DEG C of incubation 20min, PBS 3 times is added dropwise in F.Seen under fluorescence microscope Examine CD31 green fluorescence staining conditions.

CD31 Fluorescence image analysis uses Scn Image image analysis softwares.

2. result

The internal thermogenic action of 2.1 thermotherapies and thermochemotherapy

High temperature plays an important role in thermotherapy and thermochemotherapy, therefore we have first checked for tumour when thermotherapy and thermochemotherapy Interior temperature conditions.Mouse is after intratumor injection ITSL, and during two points of halfs of laser pre-irradiation, temperature linearly rises, and is increased to 61 DEG C or so.Two points of half temperature are constant at 60 DEG C or so afterwards.It is similar when thermochemotherapy situation is to thermotherapy, in two points of laser pre-irradiation In clock, temperature rapid increase in knurl reaches that temperature is declined slightly in 61.3 DEG C or so, latter three minutes, about about 60 DEG C.In knurl Inject after PBS, temperature is slowly raised when laser irradiates, and is finally reached 37.3 DEG C.

2.2 Anticancer effect in vivo

In order to study the antitumor action of thermochemotherapy, each tumor-bearing mice is divided into seven groups, receive respectively PBS, laser, chemotherapy, The treatment of thermotherapy, ITSL, DITSL and thermochemotherapy, evaluates the short term efficacy and long-term efficacy of antineoplaston respectively.

2.2.1 the short effect of oncotherapy

Evaluated with histological stain and experimental mouse taking-up tumor tissues making paraffin section is put to death after short term efficacy, treatment 48h. From H＆E coloration results, blank control group, the nucleus of pure laser group and ITSL the groups tumour cell under brazilwood extract dyeing Complete display, cytoplasm pinkiness, tissue line is disorderly, and nucleus increase, dyeing is deepened, special in obvious cancer pathology Levy.In DITSL groups and DITSL tumor tissue section, due to tumor cell necrosis, the nucleus amount of tumour cell in section Significantly reduce, thermotherapy group tumor biopsy pathological staining situation is similar with chemotherapy group.And the section of thermochemotherapy group is visible, tumour cell Cell karyorrhexis, dissolving, the nucleus of only a small amount of cell is still visible, but most cells have not seen nucleus, only The cytoplasm of pink is remained, tumour cell is downright bad on a large scale.Thermalization therapy, which has, significantly kills tumor effect.

Tumor tissues paraffin section carries out the apoptosis situation of tumor tissues after 7 different experiments processing of TUNEL experiments detection. Comprising DAB colour developings in this TUNEL dyeing, redye again by haematoxylin, DAB can dye apoptotic cell brown, haematoxylin can be by Nuclei dyeing au bleu, the apoptosis situation of tumor tissues is detected with this.Blank control group, pure laser group and ITSL groups exist Brazilwood extract dyeing undertissue arrangement disorder, nucleus increase, form is irregular, in obvious cancer pathology feature, apoptotic cell It is less.In DITSL groups and DITSL tumor tissue section, the tumor apoptotic cell of visible brown color is dispersed in distribution in section, swells Knurl increasing apoptosis is more.Thermotherapy group tumour immunity histochemical staining result visible part tumour cell is still survived, apoptosis of tumor cells quantity Increase.And the section of thermochemotherapy group is visible, blue nucleus is significantly reduced, and the apoptotic cell showed increased of brown, is entirely regarded Brown is presented in wild scope, and the nucleus of only a small amount of cell is still visible, and most cells have not seen nucleus, only surplus palm fibre The apoptotic cell of color, a wide range of apoptosis of tumour cell.The dyeing of Apoptosis situation and cell pathology is consistent, it was demonstrated that thermalization Treat short-term to kill tumor effect obvious.

2.2.2 long-term oncotherapy effect

This research have studied the long-term treatment effects of thermochemotherapy simultaneously, was limited with 30 days, while having monitored mouse tumor life Long situation, Survival situation.Mouse tumor Volume Changes situation.After mouse intratumor injection PBS, regardless of whether row laser shines again Penetrate, tumour growing state in nine days is similar to chemotherapy group and DITSL groups, and after 9 days, tumour, which increases, to be accelerated, with ITSL Group is similar.After mouse intratumor injection ITSL, tumour growth is linear, and growth is rapid.Blank control group, laser group and ITSL groups three Without statistics difference between person's tumour growth feelings, illustrate that independent laser either ITSL has no tumor inhibition effect.Chemotherapy group and DITSL groups are suppressed due to DOX lasting antitumor action, the growth of tumour, and gross tumor volume is in the trend being slowly increased, its Gross tumor volume has difference with control group.Thermotherapy group is obviously reduced in the 3rd day gross tumor volume for the treatment of, but after three days, tumour The speed of growth is substantially accelerated, and this is due to that this research only carries out seance, and most of tumour cell is killed after thermotherapy, and The tumor tissues of remaining survival may proceed to propagation due to not follow-up, lasting antineoplaston, the tumour cell of survival, most Tumour is caused to be recurred due to not exclusively eliminating eventually.As seen from the figure, after thermochemotherapy, tumour was completely eliminated, at follow-up 21 days Inside no longer recur.Even if IR-780 photo-thermal therapy can not kill all tumour cells completely, discharge rapidly and be gathered in swollen The suppression tumour growth that the DOX in knurl region can continue.

Mouse tumor picture after each group is treated 21 days, as seen from Figure 4, blank control group, laser group and ITSL groups mouse are swollen Knurl volume is larger, and chemotherapy group, DITSL group gross tumor volumes are smaller, and thermotherapy group is too high due to tumor by local temperature rise, tumor region surface Skin is formed a scab, and remaining tumour recurs near crust.Thermochemotherapy group tumor region surface skin after laser irradiation can also be tied Scab, spends 9-15 days, crust comes off naturally, and cambium normal healing, tumour no longer recurs.

Each group mouse survival rate, according to animal care and application plan, when mouse tumor volume is more than 1000mm³When, it is right Mouse sentences euthanasia, is observation node with 30 days.Blank control group had a mouse tumor volume to be more than at first at the 15th day 1000mm³, by the end of 30 days, blank control group, laser group and all mouse tumors of ITSL groups were all higher than 1000mm³, chemotherapy group Consistent with DITSL group Survivals, thermochemotherapy group survival rate is 100%.

Embodiment 2

Lipid and IR-780 is miscible in chloroform, and in vortex, nitrogen dries up into uniform film, and vacuum pumps residual chlorine It is imitative, add (NH4)₂SO₄Solution, lipid membrane is dissolved, and IR-780 liposomes are made to clarifying in sonic oscillation in a water bath, will Established liposome is extruded repeatedly with mini-extruder extrusion instrument from polycarbonate membrane, is prepared into the liposome of uniform particle sizes, by IR-780 Liposome is loaded in bag filter, is put into PBS solution and is dialysed, the ammonium sulfate of liposome interior is substituted for into PBS, and adriamycin is molten Liquid is added dropwise in liposome, the vibration of side edged, and adriamycin is loaded into liposome kernel using ammonium sulphate gradient, this hair is made Bright nano liposomes.

Described lipid is by DPPC, MPPC and distearyl acyl group phosphatidyl Monoethanolamine-polyethylene glycol 2000 composition, the mol ratio of three is 86: 10: 4.

The mass ratio of IR-780 and lipid is 1: 100.

The time that vacuum takes out chloroform is 3h.

Bath temperature is 65 DEG C.

Described polycarbonate membrane is 200nm.

The time dialysed in PBS solution is 4h.

The concentration of described Doxorubicin solution is 1mg/ml, and the mass ratio of adriamycin and lipid is 1:10.

Embodiment 3

The mass ratio of IR-780 and lipid is 10：100.

The time that vacuum takes out chloroform is 3h.

Bath temperature is 65 DEG C.

Described polycarbonate membrane is 200nm.

The time dialysed in PBS solution is 4h.

The concentration of described Doxorubicin solution is 1mg/ml, and the mass ratio of adriamycin and lipid is 1:30.

Liposome prepared by embodiment 2 and 3 also has the antitumous effect same with liposome prepared by embodiment 1.

Although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with A variety of changes, modification can be carried out to these embodiments, replace without departing from the principles and spirit of the present invention by understanding And modification, the scope of the present invention is defined by the appended.

Claims

1. a kind of preparation method for integrating photo-thermal and the nano liposomes of chemotherapy, it is characterised in that：The preparation method Comprise the following steps that：

Lipid and IR-780 is miscible in chloroform, and in vortex, nitrogen dries up into uniform film, and vacuum pumps residual chloroform, plus Enter (NH₄)₂SO₄Solution, lipid membrane is dissolved, and to clarifying IR-780 liposomes are made, by shape in sonic oscillation in a water bath Into liposome extruded repeatedly from polycarbonate membrane with mini-extruder extrusion instrument, the liposome of uniform particle sizes is prepared into, by IR-780 lipids Body be loaded on bag filter in, be put into PBS solution dialyse, the ammonium sulfate of liposome interior is substituted for PBS, by Doxorubicin solution by It is added dropwise in liposome, adriamycin is loaded into liposome kernel using ammonium sulphate gradient, is made the present invention's by the vibration of side edged Nano liposomes.

2. the preparation method as claimed in claim 1 for integrating photo-thermal and the nano liposomes of chemotherapy, it is characterised in that：Institute The lipid stated is MPPC and DSPE-poly- by DPPC Ethylene glycol 2000 is constituted, and the mol ratio of three is 86：10：4.

3. the preparation method as claimed in claim 1 for integrating photo-thermal and the nano liposomes of chemotherapy, it is characterised in that： The mass ratio of IR-780 and lipid is 1~10：100.

4. the preparation method as claimed in claim 1 for integrating photo-thermal and the nano liposomes of chemotherapy, it is characterised in that：Very The time that sky takes out chloroform is 3h.

5. the preparation method as claimed in claim 1 for integrating photo-thermal and the nano liposomes of chemotherapy, it is characterised in that：Water Bath temperature is 65 DEG C.

6. the preparation method as claimed in claim 1 for integrating photo-thermal and the nano liposomes of chemotherapy, it is characterised in that：Institute The polycarbonate membrane stated is 200nm.

7. the preparation method as claimed in claim 1 for integrating photo-thermal and the nano liposomes of chemotherapy, it is characterised in that： The time dialysed in PBS solution is 4h.

8. the preparation method as claimed in claim 1 for integrating photo-thermal and the nano liposomes of chemotherapy, it is characterised in that：Institute The concentration for the Doxorubicin solution stated is 1mg/ml, and the mass ratio of adriamycin and lipid is 1:10~30.

9. the preparation method as claimed in claim 1 for integrating photo-thermal and the nano liposomes of chemotherapy, it is characterised in that：Institute The ammonium sulphate gradient stated is after blank liposome is prepared, to remove in the ammonium ion in the external aqueous phase of lipid, interior aqueous phase one An amino molecule and a proton are formed after ammonium ion ionization, amino molecule is equally removed under cross-film diffusion, and this is with regard to shape PH gradient between inside and outside aqueous phase, medicine is not carried and is got ready, and is added after Doxorubicin solution, Doxorubicin molecules are in pH gradient Under driving, cross-film enters in intraliposomal aqueous phase, and with sulfate ion into salt, into salt after form colloidal precipitate, be gathered in In aqueous phase, Doxorubicin molecules and proton combine to form its ionic state in addition, so that membrane permeability is lost, without reversely diffusing out Go.

10. nano liposomes prepared by the preparation method as described in claim any one of 1-9 are used for the medicine for preparing treatment tumour The purposes of thing.