CN103585133B - A kind ofly prepare the method that cancerous cell targeting imitates cell microcapsule - Google Patents
A kind ofly prepare the method that cancerous cell targeting imitates cell microcapsule Download PDFInfo
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- CN103585133B CN103585133B CN201310566240.3A CN201310566240A CN103585133B CN 103585133 B CN103585133 B CN 103585133B CN 201310566240 A CN201310566240 A CN 201310566240A CN 103585133 B CN103585133 B CN 103585133B
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Abstract
The invention discloses and a kind ofly prepare the method that cancerous cell targeting imitates cell microcapsule.Select heparin-agarose affinity chromatography to wear amino imitative cell microcapsule as raw material preparation table, be dispersed in the aptamer solution containing aldehyde radical; After hatching a period of time, aptamer labelling, to imitative cell microcapsule surface, then washs with phosphate buffer; Imitative cell microcapsule after process is immersed in the drug solution of doxorubicin hydrochloride, by electrostatic interaction drug loading, finally obtains the imitative cell microcapsule of different drug loading.Preparation method of the present invention is easy to be controlled, and material source is extensive, and production efficiency is high, can regulate the drug loading of imitative cell microcapsule easily, have the feature of target cancer cell simultaneously, have good application prospect.
Description
Technical field
The present invention relates to and a kind ofly prepare the method that cancerous cell targeting imitates cell microcapsule.Especially aptamer modified imitative cell microcapsule is utilized to realize the method for cancerous cell targeting.
Background technology
The targeting of pharmaceutical carrier is a very important concept.Pharmaceutical carrier can improve the concentration of medicine at tumor tissues to the specific recognition of cancerous cell, therefore significantly can reduce dosage, thus reduces the toxic and side effects of normal tissue or cell.Therefore, the targeting realizing pharmaceutical carrier has very important significance in drug delivery field.
The common pharmaceutical carrier with target function mainly uses the polymer of synthetic or the protein of natural origin and polysaccharide as raw material, and it is relatively simple for structure.But often poor biocompatibility, is more easily removed by the immune system recognition of human body, have targeting cancerous tissue and the not strong shortcoming of cell ability, these shortcomings limit its propagation and employment.
Imitative cell microcapsule space in capsule and the capsule external space is kept apart by cell membrane the spherical material formed.Utilize cytochalasin b process mammalian cell, destroy the assembling of the actin maintaining cellular morphology, then the imitative cell microcapsule of preparation under the effect of mechanical shearing.
Imitative cell microcapsule has plurality of advantages as the pharmaceutical carrier of cancerous cell targeting: be that small-molecule drug can be embedded on microcapsule inside or cyst wall by the effect such as hydrogen bond, hydrophobic interaction because microcapsule surface essence is one deck class membrane structure on the one hand; Therefore effectively can avoid the generation of medicine violent release phenomenon, also can reduce the murder by poisoning of drug molecule to cell simultaneously.On the other hand, be different from the micro-nano carrier that traditional chemical is modified, imitative cell microcapsule derives from cell, therefore has splendid biocompatibility, and is expected to the scavenging action reducing autoimmune system, has better clinical value.
Aptamer AS1411 is the oligonucleotide being rich in G, and it can be combined with the paranuclein of cancer cell surfaces specifically, thus realizes target function.Experiment before proves that it can be enriched to cancerous tissue and cell, and the propagation of anticancer.
Summary of the invention
The object of this invention is to provide and a kind ofly easyly prepare the method that cancerous cell targeting imitates cell microcapsule fast.
The method preparing the imitative cell microcapsule of cancerous cell targeting of the present invention, comprises the following steps:
1) have in the culture plate of heparin-agarose affinity chromatography and add in cultivation the cytochalasin b solution that 20 μ L concentration are 1mg/mL, at 37 DEG C, hatch 30min; Phosphate buffer is washed for several times, collected by trypsinisation cell, vortex process gained cell, and centrifuging and taking supernatant, obtains imitative cell microcapsule suspension;
2) the imitative cell microcapsule suspension getting 1mg step 1) gained joins end modifiedly to be had in the aptamer solution of aldehyde radical, makes the concentration of aptamer be 1 μ g/mL; 4h is hatched at 37 DEG C; Collected by centrifugation gained microgranule, washing, obtains aptamer modified imitative cell microcapsule;
3) 200 μ g steps 2 are got) the imitative cell microcapsule of gained joins in doxorubicin hydrochloride solution, makes the concentration of doxorubicin hydrochloride be 40 ~ 200 μ g/mL, hatch 3 ~ 24h for 37 DEG C; Collected by centrifugation gained microgranule, washing, the cancerous cell targeting obtaining loading doxorubicin hydrochloride imitates cell microcapsule.
In the present invention, said aptamer is AS1411, its sequence: 5 '-GGTGGTGGTGGTTGTGGTGGT-GGTGG.
Principle of the present invention: add aptamer AS1411 solution in imitative cell microcapsule, by aptamers with aldehyde radical and the amino of imitative cell microcapsule surface protein react AS1411 be grafted to imitative cell microcapsule surface.Again it is mixed with doxorubicin hydrochloride solution, carry out drug loading.Aptamer and doxorubicin hydrochloride give imitative cell microcapsule respectively with targeting and the function of killing and wounding cancerous cell.
Beneficial effect of the present invention is:
Present invention process process is simple, and preparation speed is fast, and controllability is good, and material source, in n cell, has superior biocompatibility and transmits performance; Aptamer molecule and imitative cell microcapsule with active reactive group is utilized to carry out reaction in-situ, conservation and density by controlling the surperficial aptamers of rate of charge regulation and control microcapsule; The imitative cell microcapsule obtained, owing to having aptamer and doxorubicin hydrochloride simultaneously, is provided with better targeting and the character of killing and wounding cancerous cell.
Accompanying drawing explanation
Fig. 1 is the fluorescence photo of the imitative cell microcapsule being marked with cell membrane red fluorescence probe DiI, and microcapsule is dispersed in phosphate buffer.
Fig. 2 is the fluorescence photo of the aptamer modified imitative cell microcapsule being grafted with orchil Cy3 labelling, and microcapsule is dispersed in phosphate buffer.
Fig. 3 is the fluorescence photo after cancerous cell targeting microcapsule load doxorubicin hydrochloride, and microcapsule is scattered in phosphate buffer.
Fig. 4 is the drug loading of cancerous cell targeting microcapsule and the relation of drug level of the load doxorubicin hydrochloride of embodiment 1-3 gained.
Fig. 5 is the drug loading of cancerous cell targeting microcapsule and the relation of incubation time of the load doxorubicin hydrochloride of embodiment 4-6 gained.
Detailed description of the invention
Further illustrate the present invention below in conjunction with example, but these examples are not used for limiting the present invention.
Embodiment 1
1) have in the culture plate of heparin-agarose affinity chromatography add 20 μ L cytochalasin b solution (concentration is 1mg/mL) in cultivation, after hatching 30min at 37 DEG C, wash three times, trypsinization, centrifugal collecting cell with phosphate buffer; Vortex process gained cell 1min; Collected by centrifugation gained supernatant is imitative cell microcapsule suspension.
The imitative cell microcapsule of the above-mentioned gained of 200 μ g is dispersed in 1mL phosphate buffer; Add the cell membrane orchil DiI of 10 μ L1mg/mL wherein, make its mix homogeneously, hatch 15min; Gained is imitated cell microcapsule centrifuge washing for several times with remove unmarked on dyestuff; The fluorescence photo being dispersed in the imitative cell microcapsule in phosphate buffer is shown in Fig. 1.
2) the imitative cell microcapsule suspension getting 1mg step 1) gained joins end modifiedly to be had in the aptamer solution of aldehyde radical, and make the concentration of aptamer be 1 μ g/mL, hatch 4h at 37 DEG C, centrifugal, phosphate buffer is washed for several times; The fluorescence photo obtaining aptamer modified imitative cell microcapsule is shown in Fig. 2.
3) 200 μ g steps 2 are got) the imitative cell microcapsule of gained joins in doxorubicin hydrochloride solution, makes the concentration of doxorubicin hydrochloride be 40 μ g/mL, hatch 12h for 37 DEG C; Centrifugal phosphate buffer washes three times.Obtain loading the fluorescence photo that the cancerous cell targeting of doxorubicin hydrochloride imitates cell microcapsule and see Fig. 3.The drug loading obtaining this microcapsule according to the absorbance at 543nm place in ultraviolet spectrogram is 1.5 μ g, sees Fig. 4.
Embodiment 2
With embodiment 1, difference is step 3), gets 200 μ g steps 2) the imitative cell microcapsule of gained joins in doxorubicin hydrochloride solution, makes the concentration of doxorubicin hydrochloride be 80 μ g/mL, hatch 12h for 37 DEG C; Centrifugal phosphate buffer washes three times.The cancerous cell targeting obtaining loading doxorubicin hydrochloride imitates cell microcapsule.The drug loading obtaining this microcapsule according to the absorbance at 543nm place in ultraviolet spectrogram is 2.1 μ g, sees Fig. 4.
Embodiment 3
With embodiment 1, difference is step 3), gets 200 μ g steps 2) the imitative cell microcapsule of gained joins in doxorubicin hydrochloride solution, makes the concentration of doxorubicin hydrochloride be 200 μ g/mL, hatch 12h for 37 DEG C; Centrifugal phosphate buffer washes three times.The cancerous cell targeting obtaining loading doxorubicin hydrochloride imitates cell microcapsule.The drug loading obtaining this microcapsule according to the absorbance at 543nm place in ultraviolet spectrogram is 6.5 μ g, sees Fig. 4.
Embodiment 4
With embodiment 1, difference is step 3), gets 200 μ g steps 2) the imitative cell microcapsule of gained joins in doxorubicin hydrochloride solution, makes the concentration of doxorubicin hydrochloride be 200 μ g/mL, hatch 3h for 37 DEG C; Centrifugal phosphate buffer washes three times.The cancerous cell targeting obtaining loading doxorubicin hydrochloride imitates cell microcapsule.The drug loading obtaining this microcapsule according to the absorbance at 543nm place in ultraviolet spectrogram is 2.1 μ g, sees Fig. 5.
Embodiment 5
With embodiment 1, difference is step 3), gets 200 μ g steps 2) the imitative cell microcapsule of gained joins in doxorubicin hydrochloride solution, makes the concentration of doxorubicin hydrochloride be 200 μ g/mL, hatch 24h for 37 DEG C; Centrifugal phosphate buffer washes three times.The cancerous cell targeting obtaining loading doxorubicin hydrochloride imitates cell microcapsule.The drug loading obtaining this microcapsule according to the absorbance at 543nm place in ultraviolet spectrogram is 7.0 μ g, sees Fig. 5.
Claims (2)
1. prepare the method that cancerous cell targeting imitates cell microcapsule, comprise the following steps:
1) have in the culture plate of heparin-agarose affinity chromatography and add in cultivation the cytochalasin b solution that 20 μ L concentration are 1mg/mL, at 37 DEG C, hatch 30min; Phosphate buffer is washed for several times, collected by trypsinisation cell, vortex process gained cell, and centrifuging and taking supernatant, obtains imitative cell microcapsule suspension;
2) the imitative cell microcapsule suspension getting 1mg step 1) gained joins end modifiedly to be had in the aptamer AS1411 solution of aldehyde radical, makes the concentration of aptamer be 1 μ g/mL; 4h is hatched at 37 DEG C; Collected by centrifugation gained microgranule, washing, obtains the imitative cell microcapsule that aptamer AS1411 modifies;
3) 200 μ g steps 2 are got) the imitative cell microcapsule of gained joins in doxorubicin hydrochloride solution, makes the concentration of doxorubicin hydrochloride be 40 ~ 200 μ g/mL, hatch 3 ~ 24h for 37 DEG C; Collected by centrifugation gained microgranule, washing, the cancerous cell targeting obtaining loading doxorubicin hydrochloride imitates cell microcapsule.
2. according to claim 1ly prepare the method that cancerous cell targeting imitates cell microcapsule, it is characterized in that said aptamer is AS1411, its sequence: 5 '-GGTGGTGGTGGTTGTGGTGGT-GGTGG.
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| CN103920161A (en) * | 2014-03-27 | 2014-07-16 | 浙江大学 | Method for efficiently loading medicines in cell membrane microcapsules |
| CN105311632A (en) * | 2015-10-27 | 2016-02-10 | 浙江大学 | Preparation method of cell membrane vesicles loading photodynamic therapy drugs |
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Non-Patent Citations (3)
| Title |
|---|
| Aptamer-conjugated and doxorubicin-loaded unimolecular micelles for targeted therapy of prostate cancer;W.Xu,et al;《Biomaterials》;20130411;第34卷;第5244-5253页 * |
| Preparation, characterization, and in vivo evaluation of doxorubicin loaded BSA nanoparticles with folic acid modified dextran surface;H.Hao,et al;《International Journal of Pharmaceutics》;20130128;第444卷;第77-84页 * |
| 核酸适配体在抗肿瘤药物主动靶向传递中的应用;周文虎等;《中国医药工业杂志》;20121231;第43卷(第6期);第490-495页 * |
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