CN104274834A - Environment-sensitive tumor-targeting polymer micelle and preparation method thereof - Google Patents
Environment-sensitive tumor-targeting polymer micelle and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, relates to environment-sensitive tumor-targeting polymer micelle and a preparation method thereof, and especially relates to a cell microenvironment-sensitive oxidized ascorbic acid-modified tumor-targeting polymer micelle administration system and a preparation method thereof. An administration system realizes delivery in tumor by a glucose transporter mechanism, the polymer micelle of which the inner core has a disulfide bond crosslinked barrier is designed by use of the GSH characteristics of high density in the cell microenvironment, drug release characteristics trigged by the cell microenvironment are obtained, and the environment-sensitive tumor-targeting polymer micelle has a high transport capability, a fast transport rate and difficult saturation. Compared with polypeptide protein molecules, the environment-sensitive tumor-targeting polymer micelle has the characteristics of simple synthesis preparation, high stability and no immunogenicity. Through synergism of targeting and structure stabilization, drug delivery to a target position and a target cell is realized so that drug delivery efficiency is greatly improved and thus the environment-sensitive tumor-targeting polymer micelle has a good clinical application prospect.
Description
Technical field
The invention belongs to biological technical field, relate to tumour target polymer micelle, tumour target polymer micelle relating to a kind of environment sensitive and preparation method thereof, particularly relate to a kind of cell micro-environment sensitivity, the tumour target polymer micelle administration system that oxidized type dehydroascorbic acid is modified and preparation method thereof.
Background technology
At present, the clinical treatment of malignant tumor adopts the treatment means that (chemotherapy, radiotherapy or operative treatment) in many ways combines.Have the features such as infiltration, transfer because of malignant tumor, some micro metastasis are difficult to be detected usually, and therefore, chemotherapy remains one of main path of clinical treatment tumor.But, problems such as most of chemotherapeutics exists non-selectivity in poorly water-soluble, body, toxic and side effects is large, metabolism is fast, drug effect is low and seriously limit clinical practice.For overcoming above-mentioned defect, researcheres are devoted to the research and development of the chemotherapeutics delivery strategies of intelligence being used to efficient Nano medication delivery system always.
For improving the delivery efficiency of chemotherapeutics to tumor region, current targeting drug delivery system adopts ligand modified strategy mostly, namely enters born of the same parents through receptor-mediated endocytic pathway.Research display, part is macromolecular polypeptides mostly, in vivo likely by enzymolysis, and the synthesis relative complex of polypeptide.Surface of cell membrane also expresses a class transporter albumen, the transhipment of primary responsibility nutrient substance, research confirms, tumor cell is different from one of Normocellular key character, be exactly high-caliber glucose uptake rate and metabolic rate, the glucose demand of malignant cell is far above normal cell, therefore, Glucose transporter-1 (facilitative glucose transporter1, GLUT1) become the important path of tumor cell transhipment and metabolizable glucose, it is at most tumor cell surface process LAN.Separately studies have found that, oxidized type dehydroascorbic acid (dehydroascorbic acid, DHA), due to similar to D-Glucose structure, can be transported by GLUT1, its advantage has high-affinity with GLUT1, can constantly accumulate in born of the same parents because avoiding the outer row of GLUT1.
In order to improve the targeted delivery efficiency of drug-supplying system, not only need to find efficient targeted molecular, also in the urgent need to improving nanoparticle stability in vivo, to reduce the leakage of medicine at non-target site; Especially for chemotherapeutics, reduce chemotherapeutics in the accumulation of non-target position, periphery, can toxicity be reduced.Therefore, for target site or the different characteristic of target cell microenvironment, the such as change of pH, redox characteristic, temperature or enzyme etc., drug-supplying system is designed to environment sensitive type by increasing researcher, namely has the drug release characteristics of environmental response.
Glutathion (glutathione, GSH), is often referred to reduced glutathion, is to be combined by glutamic acid, cysteine and glycine, the tripeptides containing sulfydryl, is the reducing agent with antioxidation and integration Detoxication important in cell.Sulfydryl on cysteine is the active group of GSH, can be oxidized to disulfide bond and other be contained disulfide bond material to be reduced into sulfydryl by self, thus plays the effect of antioxidation and removing toxic substances.Research finds, in cell cytosol, GSH concentration (~ 10mM) is 5000 times of intercellular substance (~ 2 μMs).Therefore, utilize this cell micro-environment characteristic, drug-supplying system can be designed to the drug release characteristics with GSH triggering.
Summary of the invention
The object of this invention is to provide a kind of tumour target polymer micelle, tumour target polymer micelle being specifically related to a kind of environment sensitive and preparation method thereof, particularly relate to a kind of cell micro-environment sensitivity, the tumour target polymer micelle administration system that oxidized type dehydroascorbic acid is modified and preparation method thereof.
The mechanism that the present invention utilizes glucose transporter to mediate, constructs a kind of new and effective tumour target polymer micelle administration system for cell micro-environment characteristics design.The present invention utilizes the DHA molecule with glucose transporter high-affinity as cancer target head base, by covalently bound modification to polymer micelle surface, builds tumour target polymer micelle.
Add cross-linking agent in the tumour target polymer micelle that cell micro-environment of the present invention triggers, make micelle inner core form disulfide bond crosslinking barrier, realize the drug release of GSH sensitivity in endochylema.
Tumour target polymer micelle administration system of the present invention, the DHA molecule that its targeting head base used is glucose transporter high-affinity.
Polymeric material of the present invention is polyethylene glycol-lysine-polyphenylalanine (MPEG-pLys-pPhe) composition, wherein PEG molecular weight is 5000, polylysine (pLys) fragment n=10, polyphenylalanine (pPhe) fragment is 20, and whole piece polymeric material molecular weight is about 9700.
Concrete, polymer micelle of the present invention adopts polyamino acid base polymer as carrier material, in one embodiment of the present of invention, with the polyethylene glycol-lysine-polyphenylalanine (MPEG-pLys-pPhe) of amphipathic three blocks for carrier material, wherein polyphenylalanine fragment (pPhe) has hydrophobicity, as medicine carrying kernel; Polylysine fragment (pLys), free amine group and the cross-linking agent of its side chain react, and obtain the disulfide bond crosslinking barrier that antiradiation drug is revealed, PEG block is water-wet side, realize long circulating and surface-functionalized.In embodiments of the invention, for realizing the modification of targeted molecular, azide functionalization is carried out on PEG surface, namely synthesis obtains N
3-PEG-pLys-pPhe, then passes through Click reaction forming with the DHA molecule of 2-propargylation, obtains cross linked polymer micelle DHA-PEG-pLys-pPhe.
In the present invention, the DHA molecule adopting 2-propargyl to replace and polymeric material make with the Click reaction forming of Cu (I) catalysis the targeting drug delivery system that oxidized type dehydroascorbic acid modifies;
The present invention, the targeting vector that DHA modifies, wherein DHA degree of modification is respectively 10,20 and 40mol%.
In the present invention, in its body of the carrier material of polymer micelle, safety is high, good biocompatibility, and can need according to design the degree of polymerization adjusting each block.
The feature of the smart polymer micelle of the environment sensitive that the present invention builds is: form polymer micelle by hydrophobic interaction between polymer chain and between polymer chain and insoluble drug molecule, the cross-linking agent adding GSH sensitivity again makes the further covalent cross-linking of polymer lateral chain, this crosslinked covalent bond plays barrier action, reduces medicine and reveals.Under hydrophobic, covalently bound combined effect, compact siro spinning technology between polymer chain and chain, the cross linked polymer micelle obtained: first, can keep high structural stability in blood circulation, before reaching target site, reduces unnecessary leakage medicine; Secondly, under the special environment of target site target cell, breaking of covalent bonds, discharges medicine gradually.
The tumour target polymer micelle that cell micro-environment of the present invention triggers, polymer micelle finishing targeted molecular DHA, polymer micelle core design becomes disulfide bond crosslinking barrier, under the two combined effect, obviously can increase drug delivery efficiency, make this smart polymer micelle realize the drug delivery of tumor region even in tumor cell, have that turn-over capacity is high, transport speed fast and be difficult to advantages such as being saturated.
The tumour target polymer micelle that cell micro-environment of the present invention triggers, can be used for Bao Zaiyi class hydrophobicity antineoplastic small-molecule drug, such as paclitaxel (paclitaxel, PTX) etc.
The present invention prepares the tumour target polymer micelle administration system of cell micro-environment response as follows:
Dialysis is adopted to prepare polymer micelle.By DHA-PEG-pLys-pPhe, MPEG-pLys-pPhe and hydrophobic drug with certain proportion, be dissolved in DMF, after mix homogeneously, be transferred to bag filter (MW
cutoff1000), be placed in 1L deionized water and dialyse 12h, collect the solution of clear in bag filter, obtain the non-crosslinked drug-carrying polymer micelle that DHA modifies, i.e. DHA-PLysP.DHA-PLysP polymer micelle is transferred in PBS pH8.0 solution, the cross-linking agent of amount of calculation is added further in prescription, two (sulfonic acid the Succinimidyl Propionate) (Dithiobis (sulfosuccinimidylpropionate) of dithio, DTSSP), wherein the mol ratio of DTSSP:Lys is 1: 1, after reaction 3h, be placed in 1L deionized water and dialyse 3h to remove unreacted DTSSP completely, collect the cross linked polymer micelle obtaining DHA and modify, i.e. DHA-PLys
(s-s)p.
The advantage that the present invention gives prominence to is to utilize glucose transporter mediated mechanism to realize the targeted delivery of antitumor drug, and it is high that transporter has turn-over capacity, the feature that transport speed is fast.Targeted molecular is oxidized type dehydroascorbic acid, is micromolecular compound, has to synthesize to prepare simply compared with polypeptide protein quasi-molecule, stable high, the advantages such as non-immunogenicity.For the GSH characteristic of cell micro-environment middle and high concentration, the disulfide bond crosslinking " medicine barrier " of design, has the feature of " born of the same parents stablize, release in born of the same parents " outward, therefore can realize the drug targeting of tumor region even in tumor cell and send, improve drug delivery efficiency.
Accompanying drawing explanation
Fig. 1 hydrogen spectrum authenticating compound structure,
Wherein, A:N
6-Lys (Z)-NCA
B：Phe-NCA。
Fig. 2 hydrogen spectrum qualification polymer architecture,
Wherein, a:MPEG-pLys (Z)
b：MPEG-pLys(Z)-pPhe
c：MPEG-pLys-pPhe
d：DHA-PEG-pLys-pPhe。
Fig. 3 infrared spectrum IR identifies each compound structure,
Wherein, A:N
6-Lys (Z)-NCA
B：Phe-NCA
C：MPEG-NH
2
D：MPEG-pLys-pPhe
E：N
3-PEG-pLys-pPhe。
Fig. 4 atomic force microscope figure,
Wherein, A:DHA-PLysP/PTX polymer micelle
B:DHA-PLys
(s-s)p/PTX polymer micelle
Table 1DHA-PLysP/PTX and DHA-PLys
(s-s)p/PTX polymer micelle particle diameter and z-potential measurement result.
Fig. 5 carries 6-coumarin DHA-PLys
(s-s)p polymer micelle external glutathion sensitive medicaments release characteristics is investigated,
Wherein, A: along with GSH concentration in medium raises, DHA-PLys
(s-s)p polymer micelle drug release feature
B: after adding 10mM GSH, DHA-PLys
(s-s)p polymer micelle drug release feature generation significance changes.
Fig. 6 carries 6-coumarin DHA-PLys
(s-s)p polymer micelle is investigated in cellular level drug release characteristics,
Wherein, A, B: carry 6-coumarin DHA-PLys
(s-s)p polymer micelle just enters cell cytosol
C, D: carry 6-coumarin DHA-PLys
(s-s)the release in endochylema of P polymer micelle.
Fig. 7 red BODIPY labelling DHA-PLys
(s-s)p polymer micelle cellular uptake and mechanism are investigated.
Wherein, A-D:DHA-PLys
(s-s)the DHA degree of modification of P polymer micelle is respectively 0,10, and 20 and 40mol%
E:40mol%DHA-PLys
(s-s)p adds 0.5 μ g/mL Fei Liping
F:40mo1%DHA-PLys
(s-s)p adds 2.5 μMs of phenylarsenic oxides
G:40mol%DHA-PLys
(s-s)p adds 10mM D-Glucose
H:40mol%DHA-PLys
(s-s)p adds 2.5 μMs of colchicine
I:40mol%DHA-PLys
(s-s)p is hatched under 4 DEG C of conditions.
Fig. 8 red BODIPY labelling DHA-PLys
(s-s)p polymer micelle distribution situation in time in mice with tumor body.
Red BODIPY labelling DHA-PLys is observed under Fig. 9 Laser Scanning Confocal Microscope
(s-s)the distribution of P polymer micelle in brain tumor tissue.
After the treatment of Figure 10 mice with tumor, in body, TUNEL apoptosis detects,
Wherein, A: normal saline group
B: taxol group
C:DHA-PLysP/PTX polymer micelle group
D:PLysP/PTX polymer micelle group
E:PLys
(s-s)p/PTX polymer micelle group
F:DHA-PLys
(s-s)p/PTX polymer micelle group.
Figure 11 mice with tumor body weight change and time-to-live,
Wherein, A: body weight change
B: time-to-live.
Detailed description of the invention
Embodiment 1.
N
6-benzyloxycarbonyl group-1B (N
6-Lys (Z)) and the NCAization of L-Phe (L-Phe) carry out according to Fuchs-Farthing method, by synthetic route 1: take N
6-Lys (Z) 30g (107mmol); be suspended in dry THF (300ml); nitrogen protection; in 50 DEG C of oil baths, balance 10min, add triphosgene solid 10.58g (35.68mmol), 50 DEG C are stirred 3h; until solution clarification; be poured into by reactant liquor in anhydrous n-hexane, separate out white precipitate, sucking filtration drying obtains N
6-Lys (Z)-NCA (compound a), with anhydrous THF/ normal hexane system recrystallization, obtains white crystal, productive rate 98%.Equally, take L-Phe5g (30.3mmol), be suspended in the anhydrous THF of 50ml, operate the same, add 3g triphosgene solid (12.1mmol), reaction 3h, other operations are the same, adopt anhydrous THF/ normal hexane system recrystallization, obtain white solid, Phe-NCA (compound b), productive rate 96%.Adopt
1h NMR (shown in Fig. 1), KBr pressed disc method infrared spectrum carry out Structural Identification (Fig. 3 A and 3B).
Synthetic route 1:
Embodiment 2.
Synthesis N
3-PEG-pLys-pPhe or MPEG-pLys-pPhe polymer, adopt N respectively
3-PEG-NH
2and MPEG-NH (MW5000)
2(MW5000) as initiator, by synthetic route 2: take CH
3o-PEG-NH
2or N
3-PEG-NH
2(1g, 0.2mmol) is dissolved in dry DMF (10ml), constantly stirs, takes N
6-Lys (Z)-NCA (compound a) 736mg (2.4mmol), is added in PEG solution under nitrogen protection, 50 DEG C of reaction 48h.Take Phe-NCA (compound b) 917mg (4.8mmol), be added in reactant liquor, 50 DEG C of reaction 48h.After reaction terminates, be poured into by reactant liquor in absolute ether, separate out white precipitate, sucking filtration drying obtains white solid, productive rate 92%.Be added to by white solid in TFA (10ml)-HBr/HOAc (0.5ml), reaction 3h, to remove N
6-benzyloxycarbonyl protecting group (Z).Be transferred to by product (MW1000) in bag filter, be placed in 1L pure water and dialyse 24h, wherein every 12h changes water once, lyophilization, obtains faint yellow solid.Employing hydrogen spectrum (Fig. 2 a-c), KBr pressed disc method infrared spectrum (as shown in figures 3 d and 3e) characterize.
Synthetic route 2:
Embodiment 3.
Reacted, by DHA and the N of propargylation by the nitrine-alkynyl Click of Cu (I) catalysis
3-PEG-pLys-pPhe connects, by synthetic route 3: take polymer N
3-PEG-pLys-pPhe500mg (0.1mmol) is dissolved in DMF, nitrogen protection, and the DHA43mg (0.2mmol, 2eq.) taking propargylation is added in polymer solution.Fresh CuI (0.5eq.) and DIPEA (1eq.), is added in reactant liquor, 30 DEG C of reaction 12h.Be transferred to by reactant liquor (MW1000) in bag filter, be placed in 1L10mM EDTA-2Na pH7.0 and dialyse 24h, be transferred in 1L pure water the 24h that dialyses, lyophilization obtains the DHA-PEG-pLys-pPhe of purification; Hydrogen spectrum is adopted to carry out characterizing (as shown in Figure 2 d).
Synthetic route 3:
Embodiment 4.
Adopt dialysis to prepare polymer micelle, take amphipathic three block polyethylene glycol-lysine-polyphenylalanine polymeric material MPEG-pLys-pPhe20mg and 1mg PTX and be dissolved in 2ml DMF, after mix homogeneously, be transferred to bag filter (MW
cutoff1000), be placed in 1L deionized water to dialyse 12h, collect the solution of clear in bag filter, obtain the non-targeted non-crosslinked of PLysP/PTX and carry PTX polymer micelle solution, polymer micelle is made to occur further crosslinked, PLysP/PTX polymer micelle is transferred in PBS pH8.0 solution, adds cross-linking agent DTSSP5.45mg, wherein DTSSP:Lys, mol ratio is 1: 1, after reaction 3h, be placed in 1L deionized water and dialyse 3h to remove unreacted DTSSP completely, collect and obtain PLys
(s-s)the non-targeted crosslinked drug-carrying polymer micelle solution of P/PTX; For preparing target polymer micelle, in micelle prescription, appropriate MPEG-pLys-pPhe is replaced to DHA-PEG-pLys-pPhe, the mol ratio of total carrier material is accounted for by control DHA-PEG-pLys-pPhe, controlling the degree of modification (%) of targeting micelle, in the present embodiment, adopting degree of modification to be that the target polymer micelle of 40% is for investigating; According to the method described above, DHA-PLysP/PTX targeting non-cross-linked polymer micelle and DHA-PLys is prepared respectively
(s-s)p/PTX targeting cross linked polymer micelle, measuring and obtaining envelop rate is 92%, and drug loading is about 4.6%
Embodiment 5.
Preparation DHA-PLysP/PTX and DHA-PLys
(s-s)p/PTX polymer micelle solution, suitably drops on clean mica sheet after dilution, dries the particle diameter that rear employing atomic force microscope measures micelle, observes the form (as shown in Figure 4) of micelle, DHA-PLysP/PTX (Fig. 4 A) and DHA-PLys
(s-s)p/PTX (Fig. 4 figure) polymer micelle all in rule spherical, good dispersion degree, illustrates that drug-carrying polymer micelle is after cross-linking reaction, and the interparticle association body being cross-linked to form more Large stone does not occur.
Embodiment 6.
Preparation DHA-PLysP/PTX and DHA-PLys
(s-s)p/PTX polymer micelle solution, adopts granularity/zeta potential measurement instrument, and measure light scattering particle diameter and the Zeta potential value of micelle, result is as shown in table 1.
Table 1
Embodiment 7.
The DHA-PLys of 6-coumarin is carried in preparation
(s-s)p polymer micelle solution, measure 100 μ l solution respectively and be added to 400 μ l PBS pH7.4, in 2 μMs of GSH PBS pH7.4 and 10mM glutathion PBS pH7.4 solution, with 37 DEG C, 100rpm is hatched in air table, after 0.0833,0.25,0.5,1,2,4,6,8,12,24 and 48h, adopt spectrofluorophotometer, in λ
ex=497nm, λ
em=523nm place measures fluorescence intensity, often organizes n=4, and to be dissolved in PBS pH7.4 in contrast to carry the DHA-PLysP polymer micelle of 6-coumarin, the drug release characteristics of the outer GSH sensitivity of cross linked polymer glue bundle body as shown in Figure 5A.
Relevant with GSH for confirming cross linked polymer Micellar drug release behavior further, in vitro in drug release experiment, will DHA-PLysP and the DHA-PLys of 6-coumarin be carried
(s-s)p solution, after measuring 100 μ l respectively, be added in 400 μ l PBS pH7.4, with 37 DEG C, 100rpm is hatched in air table, after hatching 2h, to DHA-PLys
(s-s)add 10mM GSH in P group, continue to hatch, adopt the fluorescence intensity of fluorescent spectrophotometer assay 6-coumarin, result as shown in Figure 5 B.
Embodiment 8
By neuronal cell SH-SY5Y with 2.0 × 10
4cells/cm
2density be inoculated in 10mm
2cultivate 2 days in glass bottom Tissue Culture Dish, test after basis of microscopic observation cellular morphology is full.SH-SY5Y cell removes ECCM and Hank ' s liquid (not containing D-Glu) and balances 15min.Year 6-coumarin DHA-PLys of fresh preparation
(s-s)p polymer micelle, 37 DEG C and SH-SY5Y cell incubation 20min, cold Hank ' s liquid (not containing D-Glu) washes 3 times, qualitative observation cellular uptake situation under Laser Scanning Confocal Microscope, continue to hatch 20min in 37 DEG C after cell removes medicinal liquid, the drug release situation (as shown in Figure 6) in endochylema of the polymer micelle after born of the same parents is observed, when carrying the DHA-PLys of 6-coumarin under being again placed in Laser Scanning Confocal Microscope
(s-s)when P polymer micelle starts to enter cell, faint green fluorescence is centered around around nucleus in spot distribution (as shown in Figure 6 A and 6B); Remove medicinal liquid, continue to hatch 20min under 37 DEG C of conditions after, green fluorescence is that dispersivity is distributed in whole cytoplasm, and fluorescence intensity significantly strengthens (as shown in figures 6 c and 6d).
Embodiment 9
U87 cell is inoculated in 24 orifice plates and cultivates, examine under a microscope cellular morphology and density, testing when cell density reaches 70-80% and form is good, in order to investigate the impact that DHA degree of modification absorbs polymer micelle U87, preparing the PLys of red BODIPY labelling respectively
(s-s)p, DHA-PLys
(s-s)the polymer micelle of P (10%, 20% and 40% degree of modification), after U87 cell removes ECCM, at 37 DEG C, 15min is balanced with Hank ' s liquid (not containing D-Glu), micellar solution is formulated in Hank ' s liquid (not containing D-Glu), dosage 500 μ g/ hole, hatch at 37 DEG C with cell respectively, after 30min, cold Hank ' s liquid (not containing D-Glu) washes 3 times, the cellular uptake situation that fluorescence microscopy Microscopic observation is respectively organized, result is as shown in Fig. 7 A-D: after DHA modifies, DHA-PLys
(s-s)the cellular uptake of P polymer micelle on U87 comparatively PLys
(s-s)p is significantly increased, and along with the increase of micellar surface DHA degree of modification, cellular uptake amount increases;
Cellular uptake mechanism is investigated: cell and different inhibitor 37 DEG C of preincubate 10min, and transporter inhibitors comprises D-Glucose (D-Glu, 10mM); Or different endocytosis inhibitor, comprise phenylarsenic oxide (PhO, 2.5 μMs), Fei Liping (Fil, 0.5 μ g/mL), colchicine (Col, 2.5 μMs), after preincubate terminates, Hank ' s liquid (not containing D-Glu) washes 1 time, then adds the red BODIPY labelling DHA-PLys containing the corresponding inhibitor of comparable sodium
(s-s)p polymer micelle solution (dosage 500 μ g/ hole), hatches 30min for 37 DEG C, and cold Hank ' s (not containing D-Glu) liquid washes 3 times; Hatch under 4 DEG C of conditions, investigate the impact of temperature on picked-up, after hatching end, be placed in qualitative observation under inverted fluorescence microscope, by cellular uptake Inhibition test result as shown in Fig. 7 (D-I), 10mM D-Glucose can suppress DHA-PLys
(s-s)p polymer micelle is by U87 picked-up (Fig. 7 G); Various endocytosis inhibitor (phenylarsenic oxide, Fei Liping, colchicine) can suppress DHA-PLys to a certain extent
(s-s)the picked-up (as Suo Shi Fig. 7 E, 7F and 7H) of P, the flat inhibition of its Sino-Philippines power the most obviously (Fig. 7 E), illustrates endocytosis mainly caveolae process, under 4 DEG C of conditions, DHA-PLys
(s-s)the picked-up of P is subject to obvious suppression (Fig. 7 I), and DHA-PLys is described
(s-s)p cellular uptake is the process of Energy Dependence.
Embodiment 10
Adopt red fluorescence probe BODIPY tagged polymers micelle DHA-PLys
(s-s)p and PLys
(s-s)p, tumor-bearing mice distribution in vivo fluorescence imaging result as shown in Figure 8,1h, 2h, 8h and 12h upon administration, DHA-PLys
(s-s)p has the accumulation of respective degrees at tumor locus, DHA-PLys
(s-s)p and PLys
(s-s)p compares overall more, and the gathering of tumor locus is more concentrated; The picked-up observing other organs can find, DHA-PLys
(s-s)p compares PLys in liver picked-up
(s-s)p is few, is then slightly more than 12h gets tumor tissue section in the picked-up of heart and pulmonary, and after dyeing, Laser Scanning Confocal Microscope carries out observed result as shown in Figure 9, for DHA-PLys to it
(s-s)p administration group, has the accumulation of micelle in tumor center region, and enters the polymer micelle of tumor region, and major part is absorbed by tumor cell, enters cell cytosol (as shown in Fig. 9 a-9d).
Embodiment 11
The the 12nd, 15 and 18 day is completed, the following preparation of tail vein injection: taxol (Toxal), PLysP/PTX, DHA-PLysP/PTX, PLys at Brain Glioma Model
(s-s)p/PTX and DHA-PLys
(s-s)p/PTX, dosage is 10mg PTX/kg, and normal saline group is set to contrast, often organize 3 nude mices, the 3rd day (i.e. Brain Glioma Model set up after the 21st day) anesthesia execution after administration terminates, gets brain rapidly and prepares the crown frozen section at glioma position, slice thickness 20 μm.TUNEL immunofluorescence apoptosis detection kit description, carries out subsequent slice dyeing, observes under being placed in inverted fluorescence microscope, and as shown in Figure 10, normal saline group (Figure 10 A) has no apoptotic cell to glioma apoptosis testing result; Taxol and PLysP/PTX (Figure 10 B and 10D) group then have a small amount of apoptosis, and mostly occur at glioma edge; , there is obvious apoptosis in targeting micelle group (Figure 10 C and 10F), apoptosis region Relatively centralized; Crosslinked targeting micelle DHA-PLys
(s-s)p/PTX then can produce a large amount of apoptosis (Figure 10 F) in inside tumor.
Embodiment 12
Mice with tumor carries out administration according to above-mentioned dosage regimen, and as shown in figure 11, the time-to-live of normal saline group is 23.5 days for the time-to-live of each experimental group tumor-bearing mice and body weight change, and the time-to-live of taxol group and normal saline group are slightly long, are 25.5 days; PTX micelle group comparatively above two groups increase all to some extent, wherein targeting micelle group is obviously better than non-targeted micelle group, for DHA-PLys
(s-s)p/PTX treatment group, the significant prolongation time-to-live, when observation proceeds to the 50th day, still have the model mouse of 30% to survive, its mean survival time is 46 days (as shown in Figure 11 B).
Claims (8)
1. the tumour target polymer micelle of an environment sensitive, it is characterized in that, described polymer micelle adopts the DHA molecule with glucose transporter high-affinity as cancer target head base, by covalently bound modification to polymer micelle surface, polymer latex is intrafascicular adds cross-linking agent, makes tumour target polymer micelle.
2. by the tumour target polymer micelle of environment sensitive according to claim 1, it is characterized in that, described polymeric material is polyethylene glycol-lysine-polyphenylalanine (MPEG-pLys-pPhe) composition, wherein PEG molecular weight is 5000, polylysine (pLys) fragment n=10, polyphenylalanine (pPhe) fragment is 20, and whole piece polymeric material molecular weight is 9700.
3. by the tumour target polymer micelle of the environment sensitive described in claim 1 or 2, it is characterized in that, the DHA molecule adopting 2-propargyl to replace and described polymeric material make with the Click reaction forming of Cu (I) catalysis the targeting drug delivery system that oxidized type dehydroascorbic acid modifies.
4. by the tumour target polymer micelle of environment sensitive according to claim 3, it is characterized in that, the targeting vector that described DHA modifies, wherein DHA degree of modification is respectively 10,20 and 40mol%.
5. by the tumour target polymer micelle of environment sensitive according to claim 2, it is characterized in that, described polylysine side-chain amino group and cross-linking agent react, and are formed " crosslinked barrier " that antiradiation drug is revealed.
6. the preparation method of the tumour target polymer micelle of the environment sensitive of claim 1, it is characterized in that, it comprises step:
DHA-PEG-pLys-pPhe, MPEG-pLys-pPhe and hydrophobic drug are dissolved in DMF in proportion, after mix homogeneously, are transferred to bag filter (MW
cutoff1000), be placed in deionized water and dialyse 12h, collect clear transparent solutions in bag filter, obtain the non-crosslinked drug-carrying polymer micelle that DHA modifies, i.e. DHA-PLysP;
DHA-PLysP polymer micelle is transferred in PBS pH8.0 solution, add the cross-linking agent DTSSP of amount of calculation, wherein the mol ratio of DTSSP:Lys is 1: 1, after reaction 3h, be placed in the deionized water 3h that dialyses and remove unreacted DTSSP completely, collect the cross linked polymer micelle obtaining DHA and modify, i.e. DHA-PLys
(s-s)p.
7. the tumour target polymer micelle of the environment sensitive of claim 1 sends the purposes in drug-supplying system at the cancer target preparing hydrophobic anticancer drug.
8., by the purposes of claim 8, it is characterized in that, described hydrophobic anticancer drug is paclitaxel (paclitaxel, PTX).
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| CN106265508A (en) * | 2015-06-24 | 2017-01-04 | 复旦大学附属华山医院 | The load paclitaxel targeting cross linked polymer micelle of a kind of DHA modification and preparation method and application |
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| AHN NA KOO ET AL.: ""Tumor accumulation and antitumor efficacy of docetaxel-loaded core-shell-corona micelles with shell-specific redox-responsive cross-links"", 《BIOMATERIALS》 * |
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| CN110652594B (en) * | 2018-06-28 | 2022-12-30 | 复旦大学 | Multi-target-point therapeutic micelle for regulating and controlling Alzheimer disease microenvironment and preparation method thereof |
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