CN104667289A - Antitumor drug carrier and application method thereof - Google Patents
Antitumor drug carrier and application method thereof Download PDFInfo
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- CN104667289A CN104667289A CN201410042443.7A CN201410042443A CN104667289A CN 104667289 A CN104667289 A CN 104667289A CN 201410042443 A CN201410042443 A CN 201410042443A CN 104667289 A CN104667289 A CN 104667289A
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Abstract
The invention relates to the field of biological medicines, and particularly provides an antitumor drug carrier and an application method thereof. The antitumor drug carrier is a phycobiliprotein nano particle. On the basis of the difference between normal cells and tumor cells, the phycobiliprotein nano particle without toxicity and immunogenicity is adopted as the antitumor drug carrier; the tumor-targeting capability is given; meanwhile, antitumor drugs are efficiently loaded; the antitumor drugs specifically reach the tumor lesion part; the treatment effects of high selectivity and low toxicity are realized; and the defects that a traditional cytotoxic drug is poor in selectivity and relatively high in toxic and side effect, and the drug resistance is easily generated are overcome. The preparation method provided by the invention is simple and feasible; and the prepared product can be stably stored in a water solution and is beneficial to storage.
Description
Technical field
The present invention relates to biomedicine field, in particular, relate to a kind of antineoplastic drug carrier and using method thereof.
Background technology
Cancer is one of major disease affecting human health, and according to the investigation report display that World Health Organization (WHO) delivers in February, 2013, within 2008, the global number dying from cancer reaches 7,600,000, about has 13,000,000 newly-increased cancer patients every year.At present, cytotoxic drug has been widely used in the chemotherapy of cancer, but these medicines to the healthy cell of cancerous cell and human body exist non-specific, make it while killing and wounding cancerous cell, also have impact on normal body function.Therefore, improve local application's concentration, reducing general toxic reaction is the problem that oncotherapy urgently solves.Exploitation safety non-toxic, there is tumour-specific and can the tumor delivery system of high-efficient carrier antitumor drug imperative.In recent years, it is found that nano-medicament carrier can utilize the dimensional effect of its uniqueness, interfacial effect and macro quanta tunnel effect to strengthen the antitumous effect of medicine, obviously reduce drug-induced toxic and side effects.Such as, Li Yaping (ACS Nano [J], 7 (7): 5858 ~ 5869) has the pipe/polyhenylethylene nano drug-loading system of pH and time-response by building, simultaneously load amycin and disulfiram, work in coordination with and inhibit tumor cell proliferation, finally reverse tumor multi-medicine drug-resistant; Zeyu Xiao etc. devises the self assembly gold nanorods that can respond near infrared light, it can transport chemotherapeutics to specific tumors position, and optionally discharge medicine by the change of response external light, produce heat simultaneously and realize thermochemotherapy collaborative raising antitumor curative effect (Angewandte Chemie [J], 2012,51:11853 – 11857).Shi Jinjin etc. devise Fullerene-nanogold composite material, and to be applied in the neoplasm in situ degree of depth photo-thermal therapy radio frequency or microfiltration irradiates in medicine (China Patent No.: CN201310260339).But, in clinical conversion aspect, if nano medicament carrying system cannot be excreted by usual channel, harmful accumulation will be caused in vivo, have a strong impact on body health.Therefore, the safety of nanoscale medicine delivery system is the problem that people pay close attention to most all the time.Protein nano particle, as the outstanding representative of biocompatibility, is decomposed by homergy in vivo, and nanoparticle surface have many can adorned group, being conducive to the functionalization of nanoparticle, is desirable pharmaceutical carrier.Sungho Bae connects TRAIL and transferrins by the serum albumin nanoparticle surface load amycin people, obviously can suppress the propagation of HCT116, adriamycin-resistant MCF-7 and CAPAN-1 tumor cell, (the Biomaterials [J] on the tumor locus of HCT116 xenograft mouse and medicine can concentrate, 2012,33:1536-1546).Fa-Ming Chen constructs the glucosan-gelatin Nano capsule controlled release system of thermo-responsive, this Nano capsule utilizes and is connected on surperficial N-isopropylacrylamide to the sensitivity of temperature to discharge medicine (Biomaterials [J], 2013,34:6515-6527).The design of these biodegradability nanoprotein pharmaceutical carriers, can carry out the modification of physicochemical property, overcome antitumor drug hydrophilic poor, be difficult to enter the problems such as cell membrane to conventional tumour medicine; Meanwhile, by the finishing of antagonism nanoprotein pharmaceutical carrier, the functions such as antitumor drug targeting, spike, Synergistic treatment can be given, greatly improve the medical value of antitumor drug.
Phycobniliprotein is the water-soluble compound combined by multiple proteins, and be combined with covalent bond as chromophoric photopigment of catching, be mainly present in blue bacterium and some algae, comprise in red algae, hidden algae, Lycoperdon polymorphum Vitt algae and minority dinoflagellate.Its major function is caught as photosynthetic photopigment complex of catching and transmit light energy, then carries out photosynthesis with chlorophyll.Phycobniliprotein is mainly divided into four classes, that is: phycocyanin, allophycocyanin, phycoerythrin, phycoerythrocyanin (pec).Phycobniliprotein because of the fluorescent characteristic of its uniqueness be desirable fluorescent marker.In addition, phycobniliprotein has the several functions such as antioxidation, anti-inflammatory, defying age, antiviral, antiallergic, enhancing immunity, Tumor suppression, becomes the focus of people's Recent study.Caroline Costa Moraes etc. obtain the phycocyanin of analytical pure rank by a step chromatographic analysis, ion-exchange chromatography and intermediate processing, the method is simple, not only simplifies extraction step, and improves the purity of phycocyanin.（Bioresource technology[J]，2009,100:5312–5317）。Juen-Haur Hwang etc. find that phycocyanin can treat tinnitus disease by suppressing the expression of NR2B and short inflammation gene expression, show its significant anti-inflammatory effect (Plos one [J], 8 (3): e58215).The allophycocyanin of colibacillary maltose-binding protein with cyanophyceae merges by Lin Liping etc. mutually, find that this recombinant allophycocyanin obviously can suppress Hepatocarcinoma Proliferation, have simultaneously and improve T cell immunologic function, promote T cell propagation, increase the immunostimulant feature (China Patent No.: CN1771925A) of cytokine IL-2 and γ-IFN generation.Cao Minjie etc. have invented the anti-soybean trypsin inhibitor antibody of a kind of phycoerythrin labelling, and this detection method has higher susceptiveness, simplicity, safety (China Patent No.: CN103087197A) than chemoluminescence method.The enzymatic hydrolysate that our seminar has set forth phycocyanin in the enzymatic hydrolysate and application patent thereof of Spirulina phycocyanin has effect of obvious antioxidation, scavenging free radicals, individual freedom base level can be reduced, prevention free radical disease (China Patent No.: CN101928743A).Be a kind of safe, nontoxic and have the biomacromolecule of good physiologically active based on phycobniliprotein, therefore its application in antineoplastic drug carrier has bright prospect.
Therefore, improve local application's concentration, reducing general toxic reaction is the problem that oncotherapy urgently solves.Exploitation safety non-toxic, there is tumour-specific and can the tumor delivery system of high-efficient carrier antitumor drug imperative.
Summary of the invention
Technical problem to be solved by this invention is, provides a kind of antineoplastic drug carrier and using method thereof, solves the problems such as tumor local application's concentration in chemotherapy course is low, general toxic reaction is violent.
The technical scheme solved this technical problem is: provide a kind of antineoplastic drug carrier, and described antineoplastic drug carrier is phycobniliprotein nanoparticle.
In antineoplastic drug carrier provided by the invention, the raw material of described phycobniliprotein is plant-derived phycobniliprotein, be of a size of 100 ~ 300 nanometers, and surface has active group.
In antineoplastic drug carrier provided by the invention, described phycobniliprotein is the one in phycocyanin, allophycocyanin, phycoerythrin, phycoerythrocyanin (pec).
In antineoplastic drug carrier provided by the invention, described active group comprises at least one in amino, carboxyl, hydroxyl, sulfydryl.
In antineoplastic drug carrier provided by the invention, the preparation method of described phycobniliprotein nanoparticle comprises the steps:
S1, prepare phycobniliprotein aqueous solution;
S2, use pH adjusting agent, regulate the isoelectric point, IP of pH away from phycobniliprotein of described phycobniliprotein aqueous solution;
S3, use desolventizing, make phycobniliprotein precipitation from described phycobniliprotein aqueous solution be formed as nanoparticle;
S4, use cross-linking agent, the phycobniliprotein nanoparticle of crosslinked precipitation, obtains the aqueous solution of crosslinked phycobniliprotein nanoparticle closely.
In antineoplastic drug carrier provided by the invention, in described step S1, described phycobniliprotein is the one in phycocyanin, allophycocyanin, phycoerythrin, phycoerythrocyanin (pec), and the concentration of described phycobniliprotein aqueous solution is 2 ~ 200mg/mL.
In antineoplastic drug carrier provided by the invention, in described step S2, described pH adjusting agent is at least one in sodium hydrate aqueous solution, aqueous phosphatic, carbonate aqueous solution, bicarbonate aqueous solution, and after regulating, the pH of described phycobniliprotein aqueous solution is 7.0 ~ 10.
In antineoplastic drug carrier provided by the invention, in described step S3, described desolventizing is at least one in ethanol, methanol, propanol, isopropyl alcohol, acetone, oxolane, acetonitrile, and the speed that adds of desolventizing is 0.5 ~ 3mL/min.
In antineoplastic drug carrier provided by the invention, in described step S4, described cross-linking agent is at least one in glutaraldehyde, vanillin, glyceraldehyde, cross-linking reaction 1 ~ 72 hour.
In antineoplastic drug carrier provided by the invention, also comprise described phycobniliprotein nanoparticle after described step S4 and collect step, described collection step comprises centrifugal, resuspended.
The using method of the antineoplastic drug carrier that the present invention also provides, use in above-mentioned antineoplastic drug carrier, stir to targeted molecular aqueous solution and add 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate, lucifuge, after having reacted, reactant liquor is added in the aqueous solution of described antineoplastic drug carrier phycobniliprotein nanoparticle, stir 1 ~ 72 hour, add antineoplastic drug solution again, stir 1 ~ 72 hour, centrifugal, to precipitate dry or resuspended, obtain the phycobniliprotein nanoparticle of load antitumor drug.
In the using method of antineoplastic drug carrier provided by the invention, described targeted molecular is folic acid, integrin, transferrins, the one of wearing in film peptide, MUC-1 aptamer, galactosamine, new vessels targeting peptides.
In the using method of antineoplastic drug carrier provided by the invention, the mol ratio of described targeted molecular and described 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate is 1 ﹕ 5.
In the using method of antineoplastic drug carrier provided by the invention, described antitumor drug concentration of aqueous solution is 8 ~ 800mg/mL, and centrifuge RPMs is 5000 ~ 20000rpm.
In the using method of antineoplastic drug carrier provided by the invention, the preserving type of the phycobniliprotein nanoparticle of described load antitumor drug preserves with colloidal sol or powder morphology at 1 ~ 30 DEG C.
In the using method of antineoplastic drug carrier provided by the invention, described antitumor drug is the one in alkylating agent class medicine, antimetabolitas, antibiotics, natural origin antitumor drug, polypeptide drug, platinum complex, organic selenium derivant, ruthenium complex; Described alkylating agent class medicine comprises cyclophosphamide, ifosfamide, phosphinothioylidynetrisaziridine, semustine, mustine hydrochlcride, busulfan, chlorambucil, formylmerphalan, carmustine, lomustine, melphalan, nitrocaphane; Described antimetabolitas comprises cytosine arabinoside, fluorouracil, methotrexate, hydroxyurea, ftorafur, Meisoindigotin, mercaptopurine; Described antibiotics comprises Actinomycin D, mitomycin, doxorubicin hydrochloride, Bleomycin A5 hydrochloride., epirubicin hydrochloride, NSC 654509, daunorubicin hydrochloride; Described natural origin antineoplastic agent comprises homoharringtonine, vincristine sulfate, hydroxycamptothecin, etoposide, vindesine sulfate, vinblastine sulfate, Vinorelbine monotartrate, paclitaxel; Described polypeptide class antineoplastic agent comprises aminoglutethimide, tamoxifen, flutamide, gonadorelin, leuprorelin acetate, letrozole; Described platinum complex comprises cisplatin, carboplatin, oxaliplatin, naphthalene reach platinum; Described ruthenium complex comprises ruthenium complex NAMI-A, ruthenium complex KP1019, Polypyridine class ruthenium complex, benzimidazole ruthenium complex; Described organic selenium derivant comprises selenole derivant, selenium phenol, selenocystine, ebselen.
Present invention also offers the using method of another kind of antineoplastic drug carrier, use in the antineoplastic drug carrier as described in claim 1-9, antitumor drug aqueous solution is added in the aqueous solution of described antineoplastic drug carrier phycobniliprotein nanoparticle, stir 1 ~ 72 hour, centrifugal, to precipitate dry or resuspended, obtain the phycobniliprotein nanoparticle of load antitumor drug.
In the using method of another kind of antineoplastic drug carrier provided by the invention, described antitumor drug concentration of aqueous solution is 8 ~ 800mg/mL, and centrifuge RPMs is 5000 ~ 20000rpm.
In the using method of another kind of antineoplastic drug carrier provided by the invention, the preserving type of the phycobniliprotein nanoparticle of described load antitumor drug preserves with colloidal sol or powder morphology at 1 ~ 30 DEG C.
In the using method of another kind of antineoplastic drug carrier provided by the invention, it is characterized in that, described antitumor drug is the one in alkylating agent class medicine, antimetabolitas, antibiotics, natural origin antitumor drug, polypeptide drug, platinum complex, organic selenium derivant, ruthenium complex; Described alkylating agent class medicine comprises cyclophosphamide, ifosfamide, phosphinothioylidynetrisaziridine, semustine, mustine hydrochlcride, busulfan, chlorambucil, formylmerphalan, carmustine, lomustine, melphalan, nitrocaphane; Described antimetabolitas comprises cytosine arabinoside, fluorouracil, methotrexate, hydroxyurea, ftorafur, Meisoindigotin, mercaptopurine; Described antibiotics comprises Actinomycin D, mitomycin, doxorubicin hydrochloride, Bleomycin A5 hydrochloride., epirubicin hydrochloride, NSC 654509, daunorubicin hydrochloride; Described natural origin antineoplastic agent comprises homoharringtonine, vincristine sulfate, hydroxycamptothecin, etoposide, vindesine sulfate, vinblastine sulfate, Vinorelbine monotartrate, paclitaxel; Described polypeptide class antineoplastic agent comprises aminoglutethimide, tamoxifen, flutamide, gonadorelin, leuprorelin acetate, letrozole; Described platinum complex comprises cisplatin, carboplatin, oxaliplatin, naphthalene reach platinum; Described ruthenium complex comprises ruthenium complex NAMI-A, ruthenium complex KP1019, Polypyridine class ruthenium complex, benzimidazole ruthenium complex; Described organic selenium derivant comprises selenole derivant, selenium phenol, selenocystine, ebselen.
The invention has the advantages that, as phycobniliprotein nanoparticle good stability, convenient storage, the good biocompatibility in aqueous of antineoplastic drug carrier, simultaneously, there is various functional group in particle surface, be conducive to carrying out finishing or surface-functionalized to it, as engaged targeted molecular in phycobniliprotein nanoparticle surface, make it have active targeting ability.
Accompanying drawing explanation
In order to be illustrated more clearly in the technical scheme in the embodiment of the present invention, below the accompanying drawing used required in describing embodiment is briefly described, apparently, accompanying drawing in the following describes is only one embodiment of the present of invention, for those of ordinary skill in the art, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to these accompanying drawings.
Fig. 1 is the particle size distribution figure of the present invention first preferred embodiment gained phycobniliprotein nanoparticle;
Fig. 2 is the electrokinetic potential figure of the present invention first preferred embodiment gained phycobniliprotein nanoparticle;
Fig. 3 is the transmission electron microscope picture of the present invention first preferred embodiment gained phycobniliprotein nanoparticle;
Fig. 4 is the Fourier infrared spectrum figure of the present invention first preferred embodiment gained phycobniliprotein nanoparticle of carrying medicament;
Fig. 5 is the particle size distribution figure of the present invention second preferred embodiment gained phycobniliprotein nanoparticle of carrying medicament;
Fig. 6 is the particle size distribution figure of the present invention the 3rd preferred embodiment gained phycobniliprotein nanoparticle of carrying medicament;
Fig. 7 is the particle size distribution figure of the present invention the 4th preferred embodiment gained phycobniliprotein nanoparticle of carrying medicament;
Fig. 8 is the particle size distribution figure of the present invention the 5th preferred embodiment gained phycobniliprotein nanoparticle of carrying medicament;
Fig. 9 is the electrokinetic potential figure of the present invention second preferred embodiment gained phycobniliprotein nanoparticle of carrying medicament;
Figure 10 is the electrokinetic potential figure of the present invention the 3rd preferred embodiment gained phycobniliprotein nanoparticle of carrying medicament;
Figure 11 is the electrokinetic potential figure of the present invention the 4th preferred embodiment gained phycobniliprotein nanoparticle of carrying medicament;
Figure 12 is the electrokinetic potential figure of the present invention the 5th preferred embodiment gained phycobniliprotein nanoparticle of carrying medicament;
Figure 13 is the transmission electron microscope picture of the present invention second preferred embodiment gained phycobniliprotein nanoparticle of carrying medicament;
Figure 14 is the transmission electron microscope picture of the present invention the 3rd preferred embodiment gained phycobniliprotein nanoparticle of carrying medicament;
Figure 15 is the transmission electron microscope picture of the present invention the 4th preferred embodiment gained phycobniliprotein nanoparticle of carrying medicament;
Figure 16 is the transmission electron microscope picture of the present invention the 5th preferred embodiment gained phycobniliprotein nanoparticle of carrying medicament;
Figure 17 is the Fourier infrared spectrum figure of the present invention second preferred embodiment gained phycobniliprotein nanoparticle of carrying medicament;
Figure 18 is the Fourier infrared spectrum figure of the present invention the 3rd preferred embodiment gained phycobniliprotein nanoparticle of carrying medicament;
Figure 19 is the Fourier infrared spectrum figure of the present invention the 4th preferred embodiment gained phycobniliprotein nanoparticle of carrying medicament;
Figure 20 is the Fourier infrared spectrum figure of the present invention the 5th preferred embodiment gained phycobniliprotein nanoparticle of carrying medicament;
Figure 21 is phycobniliprotein nanoparticle autofluorescence image in tumor cell of the present invention second preferred embodiment gained carrying medicament;
Figure 22 is phycobniliprotein nanoparticle autofluorescence image in tumor cell of the present invention the 3rd preferred embodiment gained carrying medicament;
Figure 23 is phycobniliprotein nanoparticle autofluorescence image in tumor cell of the present invention the 4th preferred embodiment gained carrying medicament;
Figure 24 is phycobniliprotein nanoparticle autofluorescence image in tumor cell of the present invention the 5th preferred embodiment gained carrying medicament;
Figure 25 is the drug absorption rate figure of phycobniliprotein nanoparticle in adriamycin-resistant human liver cancer cell, human liver cancer cell, Human normal hepatocyte of the present invention the 3rd preferred embodiment gained carrying medicament;
Figure 26 is the cell survival rate figure of the present invention the 3rd preferred embodiment gained the suppression adriamycin-resistant human liver cancer cell of the phycobniliprotein nanoparticle of carrying medicament, human liver cancer cell, Human normal hepatocyte;
The mitochondrion fracture that Figure 27 occurs when being the induction adriamycin-resistant apoptosis of human hepatoma cell of the present invention the 3rd preferred embodiment gained phycobniliprotein nanoparticle of carrying medicament and karyopyknosis figure;
Figure 28 is the cell cycle distribution figure of the present invention the 3rd preferred embodiment gained phycobniliprotein nanoparticle process adriamycin-resistant human liver cancer cell of carrying medicament;
Figure 29 is the correlative protein expression figure of the present invention the 3rd preferred embodiment gained induction adriamycin-resistant apoptosis of human hepatoma cell of the phycobniliprotein nanoparticle of carrying medicament.
Detailed description of the invention
Below in conjunction with accompanying drawing, be clearly and completely described the technical scheme in the embodiment of the present invention, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
The present invention is based on the difference between normal cell and tumor cell, use nontoxic, non-immunogenicity phycobniliprotein nanoparticle as antineoplastic drug carrier, and give the ability of its target tumor, high-efficient carrier antitumor drug simultaneously, make antitumor drug single-minded arrival tumor focus position, achieve high selectivity, hypotoxic therapeutic effect, overcome the shortcomings such as comparatively strong, the easy generation drug resistance of the poor selectivity of conventional cell cytotoxic drug, toxic and side effects.Preparation method of the present invention is simple, and the product of preparation can stablize preservation in aqueous, is beneficial to storage.
The invention provides a kind of antineoplastic drug carrier, described antineoplastic drug carrier is phycobniliprotein nanoparticle.Preferably, the phycobniliprotein used is phycocyanin, one in allophycocyanin, phycoerythrin, phycoerythrocyanin (pec).The phycobniliprotein used is separated by a kind of mode of simple and fast cheapness and obtains, be specially, use the algae containing above-mentioned all kinds of phycobniliprotein, antibacterial and commercially available dry powder thereof, ultrasonication after multigelation, dialyse with after 30%, 65% fraction precipitation of ammonium sulphate respectively, employing hydroxyapatite is filler, the phosphate buffer solution of variable concentrations is the column chromatography of eluant, disposable acquisition purity is the phycocyanin of more than 4.5 and the phycobniliprotein of more than 3.0 and contains selenium, tellurium analog, and lyophilization obtains egg albumen powder.
The above-mentioned raw materials of phycobniliprotein used in the present invention is all plant-derived phycobniliprotein, has nontoxic, non-immunogenicity, and has the advantage of good biocompatibility, is the ideal source of antineoplastic drug carrier, be made into the nanoparticle being of a size of 100 ~ 300 nanometers, not only each series antineoplastic medicament of the interaction such as electrostatic or hydrogen bond high-efficient carrier can be passed through, there is general applicability, and there is hydrophilic radical due to nanoparticle surface, the intake of cancerous cell to medicine carrying phycobniliprotein nanoparticle can be significantly improved, and be beneficial to the passive target effect playing medicine, improve the drug level of local organization, hydrophilic radical also helps and carries out finishing or surface-functionalized to it, as engaged targeted molecular in phycobniliprotein nanoparticle surface, make it have active targeting ability.
The active group of above-mentioned phycobniliprotein nanoparticle surface comprises at least one in amino, carboxyl, hydroxyl, sulfydryl, wherein the existence of the hydrophilic radical such as amino, hydroxyl can significantly improve above-mentioned passive target effect, the functional groups such as amino, carboxyl, hydroxyl, sulfydryl can be modification group or functionalization group, and the phycobniliprotein nanoparticle-based of finishing or surface-functionalized back loading antitumor drug can possess active targeting ability.
Above-mentioned phycobniliprotein is made the phycobniliprotein nanoparticle of antineoplastic drug carrier of the present invention, preparation method comprises the steps:
S1, prepare phycobniliprotein aqueous solution;
S2, use pH adjusting agent, regulate the isoelectric point, IP of pH away from phycobniliprotein of described phycobniliprotein aqueous solution;
S3, use desolventizing, make phycobniliprotein precipitation from described phycobniliprotein aqueous solution be formed as nanoparticle;
S4, use cross-linking agent, the phycobniliprotein nanoparticle of crosslinked precipitation, obtains the aqueous solution of crosslinked phycobniliprotein nanoparticle closely.
Wherein step S1 is specially, and uses the desiccation protein powder of above-mentioned phycobniliprotein, soluble in water, and the concentration of phycobniliprotein aqueous solution is 2 ~ 200mg/mL.
Wherein step S2 is specially, regulate the pH of above-mentioned phycobniliprotein aqueous solution, make pH away from the isoelectric point, IP of phycobniliprotein, phycobniliprotein is well dispersed in aqueous solution, the pH adjusting agent used is sodium hydrate aqueous solution, aqueous phosphatic, carbonate aqueous solution or bicarbonate aqueous solution, and after regulating, the pH of described phycobniliprotein aqueous solution is 7.0 ~ 10.
Wherein step S3 is specially, desolventizing is used to make phycobniliprotein precipitation from described phycobniliprotein aqueous solution, the desolventizing used is ethanol, one in methanol, propanol, isopropyl alcohol, acetone, oxolane and acetonitrile, the speed that adds of desolventizing is 0.5 ~ 3mL/min, described desolventizing all belongs to organic solvent, the hydration shell of protein aqueous solution molecule can be destroyed, make its coagulation be nanoparticle.
Wherein step S4 is specially, and the one in use glutaraldehyde, vanillin, glyceraldehyde, as cross-linking agent, is cross-linked the phycobniliprotein of precipitation, cross-linking reaction 1 ~ 72 hour.Products therefrom of the present invention can be adjusted by change desolventizing rate of addition and crosslinker ratio according to the needs in application as the particle diameter of the phycobniliprotein nanoparticle of antineoplastic drug carrier, and operation is simple.Vanillin is similar to glutaraldehyde effect group, vanillin forms covalent bond by a terminal aldehyde group and nanoprotein amino, one terminal hydroxy group and another nanoprotein amino form hydrogen bond and carry out cross-linked proteins, glutaraldehyde forms by two terminal aldehyde groups and two nanoproteins the object that covalent bond reaches cross-linked proteins, glyceraldehyde, oxidized dextran form covalent bond (SchiffShi key) by a terminal aldehyde group and nanoprotein amino, formed with other molecules solid netted crosslinked again, reach crosslinked object.
After above-mentioned steps terminates, namely phycobniliprotein nanoparticle has been prepared, the collection of phycobniliprotein nanoparticle can adopt centrifugal, resuspended mode to obtain, be preferably 5 take turns centrifugal, centrifuge RPMs is 5000 ~ 20000rpm, often wheel continue 10 minutes, centrifugal complete after the resuspended colloidal suspension obtaining phycobniliprotein nanoparticle of use redistilled water, be designated as PCNPs.
Present invention also offers a kind of using method of antineoplastic drug carrier, use above-mentioned phycobniliprotein nanoparticle as antineoplastic drug carrier, antitumor drug aqueous solution is added in the aqueous solution of described phycobniliprotein nanoparticle, stir 1 ~ 72 hour, centrifugal, to precipitate dry or resuspended, obtain the phycobniliprotein nanoparticle of load antitumor drug.Through stirring, antitumor drug is fully contacted with phycobniliprotein nanoparticle, both are by the interaction such as electrostatic or hydrogen bond high-efficient carrier.
Preferably, described antitumor drug concentration of aqueous solution is 8 ~ 800mg/mL, and centrifuge RPMs is 5000 ~ 20000rpm.
Preferably, the preserving type of the phycobniliprotein nanoparticle of described load antitumor drug preserves with colloidal sol or powder morphology at 1 ~ 30 DEG C.
Above-mentioned antitumor drug can be any one in alkylating agent class medicine, antimetabolitas, antibiotics, natural origin antitumor drug, polypeptide drug, platinum complex, organic selenium derivant, ruthenium complex, above-mentioned antitumor drug all belongs to micromolecular compound, according to himself band point nature difference, be combined with phycobniliprotein nanoparticle by electrostatic interaction, realize the load of nanoprotein carrier to antitumor drug.Wherein, alkylating agent class medicine comprises cyclophosphamide, ifosfamide, phosphinothioylidynetrisaziridine, semustine, mustine hydrochlcride, busulfan, chlorambucil, formylmerphalan, carmustine, lomustine, melphalan, nitrocaphane; Antimetabolitas comprises cytosine arabinoside, fluorouracil, methotrexate, hydroxyurea, ftorafur, Meisoindigotin, mercaptopurine; Antibiotics comprises Actinomycin D, mitomycin, doxorubicin hydrochloride, Bleomycin A5 hydrochloride., epirubicin hydrochloride, NSC 654509, daunorubicin hydrochloride; Natural origin antineoplastic agent comprises homoharringtonine, vincristine sulfate, hydroxycamptothecin, etoposide, vindesine sulfate, vinblastine sulfate, Vinorelbine monotartrate, paclitaxel; Polypeptide class antineoplastic agent comprises aminoglutethimide, tamoxifen, flutamide, gonadorelin, leuprorelin acetate, letrozole; Platinum complex comprises cisplatin, carboplatin, oxaliplatin, naphthalene reach platinum; Ruthenium complex comprises ruthenium complex NAMI-A, ruthenium complex KP1019, Polypyridine class ruthenium complex, benzimidazole ruthenium complex; Organic selenium derivant comprises selenole derivant, selenium phenol, selenocystine, ebselen.
Above-mentioned alkylating agent class medicine is structurally similar, and all containing one or two alkyl, be combined with phycobniliprotein nanoparticle by electrostatic interaction, weak interaction can realize the load of medicine whereby.Functionally, the alkyl that such alkylating agent class medicine exists all can be transformed into the active intermediate product of electron deficiency, electron group (as amino, sulfydryl, hydroxyl, carboxylic acid group, the phosphate etc.) covalent bond contained in the biomacromolecule (DNA, RNA and protein) of these products and cell, there is alkylation reaction, make these cell component ineffective in cellular metabolism, thus the composition of cell is morphed, affect cell division, cause cell death.Therefore, this type of alkylating agent medicine all can efficiently grow by Tumor suppression, and phycobniliprotein nanoparticle is combined with this type of alkylating agent medicine all can play its good anti-tumor activity.
Above-mentioned antimetabolitas structure is similar, existing bibliographical information bovine serum albumin nanoparticle is by electrostatic interaction high-efficient carrier 5-Fluorouracil, and cytosine arabinoside, fluorouracil, methotrexate, hydroxyurea, ftorafur, Meisoindigotin, mercaptopurine, functionally, this type of antimetabolite can affect the activity of the enzyme system of tumor cell, make the precursor biosynthesis block of DNA or RNA, finally reach DNA or RNA and form obstacle, affect the biosynthesis of nucleic acid, cause growth of tumour cell breeding to be suppressed, impel apoptosis.Therefore, this type of antimetabolite of phycobniliprotein nanometer particle load can reach the object of efficient Tumor suppression activity.
Above-mentioned antibiotics antitumor drug can by suppressing DNA, RNA, the synthesis of protein carry out Tumor suppression activity, but take in a large number and can cause body multidrug resistance, and with serious toxic and side effects.Phycobniliprotein nanoparticle and antibiotics antitumor drug are combined by electrostatic interaction can overcome the multidrug resistance of body to medicine, obviously reduces physical toxicity.
Above-mentioned natural origin antineoplastic agent all derives from natural plants, with phycobniliprotein nanoparticle, all there is stronger native affinity, can be combined by electrostatic interaction or hydrogen bond etc. and phycocyanin nanoparticles, existing about the report of bovine serum albumin nanoparticle by electrostatic interaction high-efficient carrier hydroxy camptothecin at present.
Aforementioned polypeptides class drug oral is easily degraded, usually not entering tumor focus is just gone out external by metabolism, polypeptide drug can be combined by hydrogen bond or electrostatic interaction by functional group and the phycocyanin nanoparticles of self is sub, in the transport of phycobniliprotein nanoparticle down to lesions position.
The anti-tumor activity of above-mentioned platinum complex, organic selenium derivant, ruthenium complex is obvious, apoptosis is caused by being combined or activating tumor cell p53 path with the DNA of tumor cell, its common feature is poorly water-soluble, and selectivity is low, therefore by electrostatic interaction or hydrogen bond, drug loading can be realized on phycobniliprotein nanoparticle the object of antitumor drug high-efficiency low-toxicity.
Present invention also offers the using method of another kind of antineoplastic drug carrier, use above-mentioned phycobniliprotein nanoparticle as antineoplastic drug carrier, stir to targeted molecular aqueous solution and add 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate, lucifuge, after having reacted, reactant liquor is added in the aqueous solution of described antineoplastic drug carrier phycobniliprotein nanoparticle, stir 1 ~ 72 hour, add antineoplastic drug solution again, stir 1 ~ 72 hour, centrifugal, to precipitate dry or resuspended, obtain the phycobniliprotein nanoparticle of load antitumor drug.Targeted molecular and EDC react and generate targeted molecular Acibenzolar, first time stirs rear targeted molecular Acibenzolar and is combined with phycobniliprotein nanoparticle, namely targeting phycobniliprotein nanoparticle is defined, second time makes antitumor drug fully contact with targeting phycobniliprotein nanoparticle after stirring, and both are by the interaction such as electrostatic or hydrogen bond high-efficient carrier.
Preferably, the targeted molecular used is folic acid, integrin, transferrins, the one of wearing in film peptide, MUC-1 aptamer, galactosamine, new vessels targeting peptides.
Preferably, the mol ratio of the targeted molecular used and described 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate is 1 ﹕ 5.
The preparation of embodiment 1 phycobniliprotein nanoparticle
Normal temperature and pressure (15 ~ 35 DEG C, 1 normal atmosphere) under, configuration quality concentration is the water solublity phycoerythrocyanin (pec) solution of 2mg/mL, phycoerythrocyanin (pec) pH value of solution to 7.0 is regulated with the sodium hydrate aqueous solution of 0.1N, slowly instill 8mL dehydrated alcohol under continuous stirring, rate of addition is 0.5mL/min, makes phycoerythrocyanin (pec) desolventizing form nanoprotein colloidal sol.After dropwising, add 8%(volume fraction) glutaraldehyde 23.5 μ L, mix homogeneously under magnetic stirring apparatus, sustained response 1 hour.After reaction terminates, phycoerythrocyanin (pec) nanoparticle is taken turns centrifugal (centrifugal speed is 5000rpm, and often wheel continues 10 minutes) through 2 and, afterwards with the resuspended colloidal suspension obtaining phycoerythrocyanin (pec) nanoparticle of 8mL redistilled water, is designated as PCNPs.
Normal temperature and pressure (15 ~ 35 DEG C, 1 normal atmosphere) under, configuration quality concentration is the water solublity phycoerythrin solution of 200mg/mL, phycoerythrin pH value of solution to 10 is regulated with the aqueous phosphatic of 0.1N, slowly instill 8mL anhydrous propanone under continuous stirring, rate of addition is 3mL/min, makes phycoerythrin desolventizing form nanoprotein colloidal sol.After dropwising, add 8%(volume fraction) glyceraldehyde 23.5 μ L, mix homogeneously under magnetic stirring apparatus, sustained response 72 hours.After reaction terminates, phycoerythrin nanoparticle is taken turns centrifugal (centrifugal speed is 20000rpm, and often wheel continues 10 minutes) through 3 and, afterwards with the resuspended colloidal suspension obtaining phycoerythrin nanoparticle of 8mL redistilled water, is designated as PCNPs.
Normal temperature and pressure (15 ~ 35 DEG C, 1 normal atmosphere) under, configuration quality concentration is the water solublity allophycocyanin solution of 20mg/mL, allophycocyanin pH value of solution to 8.4 is regulated with the carbonate aqueous solution of 0.1N, slowly instill 8mL dehydrated alcohol under continuous stirring, rate of addition is 0.8mL/min, makes allophycocyanin desolventizing form nanoprotein colloidal sol.After dropwising, add 8%(volume fraction) vanillin 23.5 μ L, mix homogeneously under magnetic stirring apparatus, sustained response 24 hours.After reaction terminates, allophycocyanin nanoparticle is taken turns centrifugal (centrifugal speed is 12000rpm, and often wheel continues 10 minutes) through 4 and, afterwards with the resuspended colloidal suspension obtaining allophycocyanin nanoparticle of 8mL redistilled water, is designated as PCNPs.
Normal temperature and pressure (15 ~ 35 DEG C, 1 normal atmosphere) under, configuration quality concentration is the water solublity phycocyanin (phycocyanin of 20mg/mL, PC) solution, phycocyanin pH value of solution to 8.4 is regulated with the bicarbonate aqueous solution of 0.1N, slowly instill 8mL anhydrous propanone under continuous stirring, rate of addition is 0.8mL/min, makes phycocyanin desolventizing form nanoprotein colloidal sol.After dropwising, add 8%(volume fraction) glutaraldehyde 23.5 μ L, mix homogeneously under magnetic stirring apparatus, sustained response 24 hours.After reaction terminates, phycocyanin nanoparticles is taken turns centrifugal (centrifugal speed is 12000rpm, and often wheel continues 10 minutes) through 5 and, afterwards with the resuspended colloidal suspension obtaining phycocyanin nanoparticles of 8mL redistilled water, is designated as PCNPs.
Above-mentioned gained four kinds of PCNPs characteristics close to and all there is better performance, be placed in 4 DEG C of Refrigerator stores.Now only characterize its characteristic for the PCNPs that the 4th kind of mode is obtained, be specially, characterize change of granularity (Fig. 1) and electrokinetic potential (zeta) current potential (Fig. 2) thereof of PCNPs aqueous solution with Nano-ZS (MalvernInsruments Limited); The shape appearance figure (see figure 3) of PCNPs is characterized with Hitachi H-7650 type transmission electron microscope; The infrared spectrum (see figure 4) of PCNPs is characterized with Fourier transformation infrared spectrometer.
The desolventizing used in the present embodiment also can play identical effect for ethanol and acetone, identical methanol, propanol, isopropyl alcohol, oxolane and acetonitrile, and model of action is also same as described above.
Phycobniliprotein nanoparticle in the present embodiment can also be targeting phycobniliprotein nanoparticle, be specially and engage targeted molecular on the phycobniliprotein nanoparticle of above-mentioned PCNPs, now illustrate for the PCNPs that above-mentioned 4th kind of mode is obtained: the folic acid (folate getting 16mg, FA) 0.8mL0.1N sodium hydroxide solution is dissolved in, 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDC) 0.2mL(is added by folic acid: EDC mol ratio is 1:5) under stirring, lucifuge reacts 2 hours, obtains folic acid Acibenzolar.Slowly joined by folic acid Acibenzolar in PCNPs solution, lucifuge stirs 24 hours, obtains folacin coupled phycocyanin nanoparticles (FA-PCNPs).Similar, integrin, transferrins, wear the aqueous solution of other targeted moleculars such as film peptide, MUC-1 aptamer, galactosamine, new vessels targeting peptides, when can be also 1 ﹕ 5 to Fen ﹕ EDC mol ratio by target, targeted molecular and EDC react and generate targeted molecular Acibenzolar, under stirring, the phycobniliprotein nanoparticle that targeted molecular Acibenzolar and EDC activate is combined, and covalency forms targeted molecular-nanoprotein conjugate, namely defines targeting phycobniliprotein nanoparticle.
Embodiment 2 phycobniliprotein nanometer particle load medicine
Normal temperature and pressure (15 ~ 35 DEG C, 1 normal atmosphere) under, configuration quality concentration is the water solublity phycocyanin (phycocyanin of 20mg/mL, PC) solution, phycocyanin pH value of solution to 8.4 is regulated with the bicarbonate aqueous solution of 0.1N, slowly instill 8mL anhydrous propanone under continuous stirring, rate of addition is 0.8mL/min, makes phycocyanin desolventizing form nanoprotein colloidal sol.After dropwising, add 8%(volume fraction) vanillin 23.5 μ L, mix homogeneously under magnetic stirring apparatus, sustained response 24 hours.After reaction terminates, phycocyanin nanoparticles is taken turns centrifugal (centrifugal speed is 12000rpm, and often wheel continues 10 minutes) through 5 and, afterwards with the resuspended colloidal suspension obtaining phycocyanin nanoparticles of 8mL redistilled water, is designated as PCNPs.
Get the folic acid (folate of 16mg, FA) 0.8mL0.1N sodium hydroxide solution is dissolved in, 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDC) 0.2mL(is added by folic acid: EDC mol ratio is 1:5) under stirring, lucifuge reacts 2 hours, obtains folic acid Acibenzolar.Slowly joined by folic acid Acibenzolar in PCNPs solution, lucifuge stirs 24 hours, obtains folacin coupled phycocyanin nanoparticles (FA-PCNPs).
Preparation cyclophosphamide (CTX) aqueous solution, the amount of phycobniliprotein nanometer particle load CTX and the concentration relationship of CTX aqueous solution not remarkable, so the concentration of CTX aqueous solution is the arbitrary value in 8 ~ 800mg/mL, as 8mg/mL, 54mg/mL, 100mg/mL, 400mg/mL, 800mg/mL, if the concentration of the CTX aqueous solution used is larger, then suitably reduce liquor capacity.Added by above-mentioned CTX aqueous solution in above-mentioned FA-PCNPs solution, under magnetic agitation, lucifuge reaction 1 ~ 72 hour, specifically can adjust according to carrying medicament amount, as 1 hour, 12 hours, 24 hours, 36 hours, 72 hours.After reaction terminates, centrifugal segregation unreacted FA, EDC and antitumor drug, centrifuge RPMs is 5000 ~ 20000rpm, and 3 take turns centrifugal (often wheel 10 minutes, rotating speed 12000rpm; Often take turns 20 minutes, rotating speed 5000rpm; Often take turns 5 minutes, rotating speed 20000rpm).Precipitation 8mL distilled water is resuspended, obtains targeting phycocyanin nanoparticles of load CTX, i.e. the sub-load ring phosphamide (FA-PCNPs-CTX) of folate-targeted phycocyanin nanoparticles.Change of granularity (Fig. 5) and electrokinetic potential (zeta) current potential (Fig. 9) thereof of FA-PCNPs-CTX aqueous solution is characterized with Nano-ZS (Malvern Insruments Limited); The shape appearance figure (see Figure 13) of FA-PCNPs-CTX is characterized with Hitachi H-7650 type transmission electron microscope; The infrared spectrum (see Figure 17) of FA-PCNPs-CTX is characterized with Fourier transformation infrared spectrometer; Using Hela human cervical carcinoma tumor cell as cell model, enter the situation (see Figure 21) of cell with fluorescence microscope Real-Time Monitoring medicine.
Identical, other alkylating agent class medicine as ifosfamide, phosphinothioylidynetrisaziridine, semustine, mustine hydrochlcride, busulfan (Busulfan), chlorambucil, formylmerphalan, carmustine, lomustine, melphalan, nitrocaphane etc. also can make in the same way with phycobniliprotein nanometer particle load, and play respective drug effect, due to the method for carrying medicament and above-mentioned just the same, do not repeat them here.
Embodiment 3 phycobniliprotein nanometer particle load medicine
Normal temperature and pressure (15 ~ 35 DEG C, 1 normal atmosphere) under, preparation mass concentration is the water solublity phycocyanin (phycocyanin of 20mg/mL, PC) solution, phycocyanin pH value of solution to 8.4 is regulated with the bicarbonate aqueous solution of 0.1N, slowly instill 8mL anhydrous propanone under continuous stirring, rate of addition is 0.8mL/min, makes phycocyanin desolventizing form nanoprotein colloidal sol.After dropwising, add 8%(volume fraction) vanillin 23.5 μ L, mix homogeneously under magnetic stirring apparatus, sustained response 24 hours.After reaction terminates, phycocyanin nanoparticles is taken turns centrifugal (centrifugal speed is 12000rpm, and often wheel continues 10 minutes) through 5 and, afterwards with the resuspended colloidal suspension obtaining phycocyanin nanoparticles of 8mL redistilled water, is designated as PCNPs.
Get the folic acid (folate of 16mg, FA) 0.8mL0.1N sodium hydroxide solution is dissolved in, 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDC) 0.2mL(is added by folic acid: EDC mol ratio is 1:5) under stirring, lucifuge reacts 2 hours, obtains folic acid Acibenzolar.Slowly joined by folic acid Acibenzolar in PCNPs solution, lucifuge stirs 24 hours, obtains folacin coupled phycocyanin nanoparticles (FA-PCNPs).
Dimethyl sulfoxide (DMSO) solution of preparation amycin (DOX), the amount of phycobniliprotein nanometer particle load DOX and the concentration relationship of DOX solution not remarkable, so the concentration of DOX solution is the arbitrary value in 8 ~ 800mg/mL, as 8mg/mL, 54mg/mL, 100mg/mL, 400mg/mL, 800mg/mL, if the concentration of the DOX solution used is larger, then suitably reduce liquor capacity.Added by above-mentioned DOX solution in above-mentioned FA-PCNPs solution, under magnetic agitation, lucifuge reaction 1 ~ 72 hour, specifically can adjust according to carrying medicament amount, as 1 hour, 12 hours, 24 hours, 36 hours, 72 hours.After reaction terminates, centrifugal segregation unreacted FA, EDC and antitumor drug, centrifuge RPMs is 5000 ~ 20000rpm, and 3 take turns centrifugal (often wheel 10 minutes, rotating speed 12000rpm; Often take turns 20 minutes, rotating speed 5000rpm; Often take turns 5 minutes, rotating speed 20000rpm).Precipitation 8mL distilled water is resuspended, obtains targeting phycocyanin nanoparticles of load DOX, i.e. the sub-load ring phosphamide (FA-PCNPs-DOX) of folate-targeted phycocyanin nanoparticles.Change of granularity (Fig. 6) and electrokinetic potential (zeta) current potential (Figure 10) thereof of FA-PCNPs-DOX solution is characterized with Nano-ZS (Malvern Insruments Limited); The shape appearance figure (see Figure 14) of FA-PCNPs-DOX is characterized with Hitachi H-7650 type transmission electron microscope; The infrared spectrum (see Figure 18) of FA-PCNPs-DOX is characterized with Fourier transformation infrared spectrometer; Using Hela human cervical carcinoma tumor cell as cell model, enter the situation (see Figure 22) of cell with fluorescence microscope Real-Time Monitoring DOX; Detect absorbance (Figure 25) in RHepG2 adriamycin-resistant human liver cancer cell, HepG2 human liver cancer cell, L02 Human normal hepatocyte of targeting phycocyanin nanoparticles load amycin and cell survival rate (Figure 26); The mitochondrion fracture occurred when detecting targeting phycocyanin nanoparticles load amycin induction RHepG2 adriamycin-resistant apoptosis of human hepatoma cell with Mitotracker staining and DAPI staining and karyopyknosis figure (Figure 27); By the cell cycle distribution (Figure 28) of Epics XL-MCL flow cytometry analysis RHepG2 adriamycin-resistant human liver cancer cell after the process of targeting phycocyanin nanoparticles load amycin; The correlative protein expression (Figure 29) of targeting phycocyanin nanoparticles load amycin induction RHepG2 adriamycin-resistant apoptosis of human hepatoma cell is analyzed with Western blot.
Identical, other antibiotics as Actinomycin D, mitomycin, doxorubicin hydrochloride, Bleomycin A5 hydrochloride., epirubicin hydrochloride, NSC 654509, daunorubicin hydrochloride etc. also can make in the same way with phycobniliprotein nanometer particle load, and play respective drug effect, due to the method for carrying medicament and above-mentioned just the same, do not repeat them here.
Embodiment 4 phycobniliprotein nanometer particle load medicine
Normal temperature and pressure (15 ~ 35 DEG C, 1 normal atmosphere) under, preparation mass concentration is the water solublity phycocyanin (phycocyanin of 20mg/mL, PC) solution, phycocyanin pH value of solution to 8.4 is regulated with the bicarbonate aqueous solution of 0.1N, slowly instill 8mL anhydrous propanone under continuous stirring, rate of addition is 0.8mL/min, makes phycocyanin desolventizing form nanoprotein colloidal sol.After dropwising, add 8%(volume fraction) vanillin 23.5 μ L, mix homogeneously under magnetic stirring apparatus, sustained response 24 hours.After reaction terminates, phycocyanin nanoparticles is taken turns centrifugal (centrifugal speed is 12000rpm, and often wheel continues 10 minutes) through 5 and, afterwards with the resuspended colloidal suspension obtaining phycocyanin nanoparticles of 8mL redistilled water, is designated as PCNPs.
Get the folic acid (folate of 16mg, FA) 0.8mL0.1N sodium hydroxide solution is dissolved in, 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDC) 0.2mL(is added by folic acid: EDC mol ratio is 1:5) under stirring, lucifuge reacts 2 hours, obtains folic acid Acibenzolar.Slowly joined by folic acid Acibenzolar in PCNPs solution, lucifuge stirs 24 hours, obtains folacin coupled phycocyanin nanoparticles (FA-PCNPs).
Dimethyl sulfoxide (DMSO) solution of preparation benzimidazole pyridine ruthenium complex (RuPOP), the amount of phycobniliprotein nanometer particle load RUPOP and the concentration relationship of RUPOP solution not remarkable, so the concentration of RUPOP solution is the arbitrary value in 8 ~ 800mg/mL, as 8mg/mL, 54mg/mL, 100mg/mL, 400mg/mL, 800mg/mL, if the concentration of the RUPOP solution used is larger, then suitably reduce liquor capacity.Added by above-mentioned RUPOP solution in above-mentioned FA-PCNPs solution, under magnetic agitation, lucifuge reaction 1 ~ 72 hour, specifically can adjust according to carrying medicament amount, as 1 hour, 12 hours, 24 hours, 36 hours, 72 hours.After reaction terminates, centrifugal segregation unreacted FA, EDC and antitumor drug, centrifuge RPMs is 5000 ~ 20000rpm, and 3 take turns centrifugal (often wheel 10 minutes, rotating speed 12000rpm; Often take turns 20 minutes, rotating speed 5000rpm; Often take turns 5 minutes, rotating speed 20000rpm).Precipitation 8mL distilled water is resuspended, obtains targeting phycocyanin nanoparticles of load RUPOP, i.e. the sub-load ring phosphamide (FA-PCNPs-RUPOP) of folate-targeted phycocyanin nanoparticles.Change of granularity (Fig. 7) and electrokinetic potential (zeta) current potential (Figure 11) thereof of FA-PCNPs-RUPOP solution is characterized with Nano-ZS (Malvern Insruments Limited); The shape appearance figure (see Figure 15) of FA-PCNPs-RUPOP is characterized with Hitachi H-7650 type transmission electron microscope; The infrared spectrum (see Figure 19) of FA-PCNPs-RUPOP is characterized with Fourier transformation infrared spectrometer; Using Hela human cervical carcinoma tumor cell as cell model, enter the situation (see Figure 23) of cell with fluorescence microscope Real-Time Monitoring medicine.
Identical, other platinum complex such as cisplatin, carboplatin, oxaliplatin, naphthalene reach platinum etc., other organic selenium derivant comprises selenole derivant, selenium phenol, selenocystine, ebselen etc., other ruthenium complex as NAMI-A, KP1019, Polypyridine class ruthenium complex, benzimidazole ruthenium complex etc. also can make in the same way with phycobniliprotein nanometer particle load, and play respective drug effect, due to the method for carrying medicament and above-mentioned just the same, do not repeat them here.
Embodiment 5 phycobniliprotein nanometer particle load medicine
Normal temperature and pressure (15 ~ 35 DEG C, 1 normal atmosphere) under, preparation mass concentration is the water solublity phycocyanin (phycocyanin of 20mg/mL, PC) solution, phycocyanin pH value of solution to 8.4 is regulated with the bicarbonate aqueous solution of 0.1N, slowly instill 8mL anhydrous propanone under continuous stirring, rate of addition is 0.8mL/min, makes phycocyanin desolventizing form nanoprotein colloidal sol.After dropwising, add 8%(volume fraction) vanillin 23.5 μ L, mix homogeneously under magnetic stirring apparatus, sustained response 24 hours.After reaction terminates, phycocyanin nanoparticles is taken turns centrifugal (centrifugal speed is 12000rpm, and often wheel continues 10 minutes) through 5 and, afterwards with the resuspended colloidal suspension obtaining phycocyanin nanoparticles of 8mL redistilled water, is designated as PCNPs.
Get the folic acid (folate of 16mg, FA) 0.8mL0.1N sodium hydroxide solution is dissolved in, 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDC) 0.2mL(is added by folic acid: EDC mol ratio is 1:5) under stirring, lucifuge reacts 2 hours, obtains folic acid Acibenzolar.Slowly joined by folic acid Acibenzolar in PCNPs solution, lucifuge stirs 24 hours, obtains folacin coupled phycocyanin nanoparticles (FA-PCNPs).
Prepare biphenyl and dimethyl sulfoxide (DMSO) solution of selenium oxadiazole derivative (PSeD), the amount of phycobniliprotein nanometer particle load PSED and the concentration relationship of PSED solution not remarkable, so the concentration of PSED solution is the arbitrary value in 8 ~ 800mg/mL, as 8mg/mL, 12.5mg/mL, 100mg/mL, 400mg/mL, 800mg/mL, if the concentration of the PSED solution used is larger, then suitably reduce liquor capacity.Added by above-mentioned PSED solution in above-mentioned FA-PCNPs solution, under magnetic agitation, lucifuge reaction 1 ~ 72 hour, specifically can adjust according to carrying medicament amount, as 1 hour, 12 hours, 24 hours, 36 hours, 72 hours.After reaction terminates, centrifugal segregation unreacted FA, EDC and antitumor drug, centrifuge RPMs is 5000 ~ 20000rpm, and 3 take turns centrifugal (often wheel 10 minutes, rotating speed 12000rpm; Often take turns 20 minutes, rotating speed 5000rpm; Often take turns 5 minutes, rotating speed 20000rpm).Precipitation 8mL distilled water is resuspended, obtains targeting phycocyanin nanoparticles of load PSED, i.e. the sub-load ring phosphamide (FA-PCNPs-PSED) of folate-targeted phycocyanin nanoparticles.Change of granularity (Fig. 8) and electrokinetic potential (zeta) current potential (Figure 12) thereof of FA-PCNPs-PSED solution is characterized with Nano-ZS (Malvern Insruments Limited); The shape appearance figure (see Figure 16) of FA-PCNPs-PSED is characterized with Hitachi H-7650 type transmission electron microscope; The infrared spectrum (see Figure 20) of FA-PCNPs-PSED is characterized with Fourier transformation infrared spectrometer; Using Hela human cervical carcinoma tumor cell as cell model, enter the situation (see Figure 24) of cell with fluorescence microscope Real-Time Monitoring medicine.
Identical, other antimetabolitas is as cytosine arabinoside, fluorouracil, methotrexate, hydroxyurea, ftorafur, Meisoindigotin, mercaptopurine etc., natural origin antineoplastic agent is as homoharringtonine, vincristine sulfate, hydroxycamptothecin, etoposide, vindesine sulfate, vinblastine sulfate, Vinorelbine monotartrate, paclitaxel etc., polypeptide class antineoplastic agent is as aminoglutethimide, tamoxifen, flutamide, gonadorelin, leuprorelin acetate, letrozole etc. also can make in the same way with phycobniliprotein nanometer particle load, and play respective drug effect, due to the method for carrying medicament and above-mentioned just the same, do not repeat them here.
Embodiment 6 phycobniliprotein nanometer particle load medicine
In embodiment 2-5, the integrating step of targeted molecular and phycobniliprotein nanoparticle removed, namely the direct load antitumor drug of non-target tropism phycobniliprotein nanoparticle, specifically the same, do not repeat them here.
Claims (20)
1. an antineoplastic drug carrier, is characterized in that, described antineoplastic drug carrier is phycobniliprotein nanoparticle.
2. antineoplastic drug carrier according to claim 1, is characterized in that, the raw material of described phycobniliprotein is plant-derived phycobniliprotein, be of a size of 100 ~ 300 nanometers, and surface has active group.
3. antineoplastic drug carrier according to claim 2, is characterized in that, described phycobniliprotein is the one in phycocyanin, allophycocyanin, phycoerythrin, phycoerythrocyanin (pec).
4. antineoplastic drug carrier according to claim 2, is characterized in that, described active group comprises at least one in amino, carboxyl, hydroxyl, sulfydryl.
5. antineoplastic drug carrier according to claim 1, is characterized in that, the preparation method of described phycobniliprotein nanoparticle comprises the steps:
S1, prepare phycobniliprotein aqueous solution;
S2, use pH adjusting agent, regulate the isoelectric point, IP of pH away from phycobniliprotein of described phycobniliprotein aqueous solution;
S3, use desolventizing, make phycobniliprotein precipitation from described phycobniliprotein aqueous solution be formed as nanoparticle;
S4, use cross-linking agent, the phycobniliprotein nanoparticle of crosslinked precipitation, obtains the aqueous solution of crosslinked phycobniliprotein nanoparticle closely.
6. antineoplastic drug carrier according to claim 5, it is characterized in that, in described step S1, described phycobniliprotein is the one in phycocyanin, allophycocyanin, phycoerythrin, phycoerythrocyanin (pec), and the concentration of described phycobniliprotein aqueous solution is 2 ~ 200mg/mL.
7. antineoplastic drug carrier according to claim 5, it is characterized in that, in described step S2, described pH adjusting agent is at least one in sodium hydrate aqueous solution, aqueous phosphatic, carbonate aqueous solution, bicarbonate aqueous solution, and after regulating, the pH of described phycobniliprotein aqueous solution is 7.0 ~ 10.
8. antineoplastic drug carrier according to claim 5, it is characterized in that, in described step S3, described desolventizing is at least one in ethanol, methanol, propanol, isopropyl alcohol, acetone, oxolane, acetonitrile, and the speed that adds of desolventizing is 0.5 ~ 3mL/min.
9. antineoplastic drug carrier according to claim 5, is characterized in that, in described step S4, described cross-linking agent is at least one in glutaraldehyde, vanillin, glyceraldehyde, cross-linking reaction 1 ~ 72 hour.
10. antineoplastic drug carrier according to claim 5, is characterized in that, also comprise described phycobniliprotein nanoparticle after described step S4 and collect step, described collection step comprises centrifugal, resuspended.
The using method of 11. 1 kinds of antineoplastic drug carriers, it is characterized in that, use in the antineoplastic drug carrier as described in claim 1-9, stir to targeted molecular aqueous solution and add 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate, lucifuge, after having reacted, reactant liquor is added in the aqueous solution of described antineoplastic drug carrier phycobniliprotein nanoparticle, stir 1 ~ 72 hour, add antineoplastic drug solution again, stir 1 ~ 72 hour, centrifugal, to precipitate dry or resuspended, obtain the phycobniliprotein nanoparticle of load antitumor drug.
The using method of 12. antineoplastic drug carriers according to claim 11, is characterized in that, described targeted molecular is folic acid, integrin, transferrins, the one of wearing in film peptide, MUC-1 aptamer, galactosamine, new vessels targeting peptides.
The using method of 13. antineoplastic drug carriers according to claim 11, is characterized in that, the mol ratio of described targeted molecular and described 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate is 1 ﹕ 5.
The using method of 14. antineoplastic drug carriers according to claim 11, is characterized in that, described antitumor drug concentration of aqueous solution is 8 ~ 800mg/mL, and centrifuge RPMs is 5000 ~ 20000rpm.
The using method of 15. antineoplastic drug carriers according to claim 11, is characterized in that, the preserving type of the phycobniliprotein nanoparticle of described load antitumor drug preserves with colloidal sol or powder morphology at 1 ~ 30 DEG C.
The using method of 16. antineoplastic drug carriers according to claim 11, it is characterized in that, described antitumor drug is the one in alkylating agent class medicine, antimetabolitas, antibiotics, natural origin antitumor drug, polypeptide drug, platinum complex, organic selenium derivant, ruthenium complex; Described alkylating agent class medicine comprises cyclophosphamide, ifosfamide, phosphinothioylidynetrisaziridine, semustine, mustine hydrochlcride, busulfan, chlorambucil, formylmerphalan, carmustine, lomustine, melphalan, nitrocaphane; Described antimetabolitas comprises cytosine arabinoside, fluorouracil, methotrexate, hydroxyurea, ftorafur, Meisoindigotin, mercaptopurine; Described antibiotics comprises Actinomycin D, mitomycin, doxorubicin hydrochloride, Bleomycin A5 hydrochloride., epirubicin hydrochloride, NSC 654509, daunorubicin hydrochloride; Described natural origin antineoplastic agent comprises homoharringtonine, vincristine sulfate, hydroxycamptothecin, etoposide, vindesine sulfate, vinblastine sulfate, Vinorelbine monotartrate, paclitaxel; Described polypeptide class antineoplastic agent comprises aminoglutethimide, tamoxifen, flutamide, gonadorelin, leuprorelin acetate, letrozole; Described platinum complex comprises cisplatin, carboplatin, oxaliplatin, naphthalene reach platinum; Described ruthenium complex comprises ruthenium complex NAMI-A, ruthenium complex KP1019, Polypyridine class ruthenium complex, benzimidazole ruthenium complex; Described organic selenium derivant comprises selenole derivant, selenium phenol, selenocystine, ebselen.
The using method of 17. 1 kinds of antineoplastic drug carriers, it is characterized in that, use in the antineoplastic drug carrier as described in claim 1-9, antitumor drug aqueous solution is added in the aqueous solution of described antineoplastic drug carrier phycobniliprotein nanoparticle, stir 1 ~ 72 hour, centrifugal, will precipitate dry or resuspended, obtain the phycobniliprotein nanoparticle of load antitumor drug.
The using method of 18. antineoplastic drug carriers according to claim 17, is characterized in that, described antitumor drug concentration of aqueous solution is 8 ~ 800mg/mL, and centrifuge RPMs is 5000 ~ 20000rpm.
The using method of 19. antineoplastic drug carriers according to claim 17, is characterized in that, the preserving type of the phycobniliprotein nanoparticle of described load antitumor drug preserves with colloidal sol or powder morphology at 1 ~ 30 DEG C.
The using method of 20. antineoplastic drug carriers according to claim 17, it is characterized in that, it is characterized in that, described antitumor drug is the one in alkylating agent class medicine, antimetabolitas, antibiotics, natural origin antitumor drug, polypeptide drug, platinum complex, organic selenium derivant, ruthenium complex; Described alkylating agent class medicine comprises cyclophosphamide, ifosfamide, phosphinothioylidynetrisaziridine, semustine, mustine hydrochlcride, busulfan, chlorambucil, formylmerphalan, carmustine, lomustine, melphalan, nitrocaphane; Described antimetabolitas comprises cytosine arabinoside, fluorouracil, methotrexate, hydroxyurea, ftorafur, Meisoindigotin, mercaptopurine; Described antibiotics comprises Actinomycin D, mitomycin, doxorubicin hydrochloride, Bleomycin A5 hydrochloride., epirubicin hydrochloride, NSC 654509, daunorubicin hydrochloride; Described natural origin antineoplastic agent comprises homoharringtonine, vincristine sulfate, hydroxycamptothecin, etoposide, vindesine sulfate, vinblastine sulfate, Vinorelbine monotartrate, paclitaxel; Described polypeptide class antineoplastic agent comprises aminoglutethimide, tamoxifen, flutamide, gonadorelin, leuprorelin acetate, letrozole; Described platinum complex comprises cisplatin, carboplatin, oxaliplatin, naphthalene reach platinum; Described ruthenium complex comprises ruthenium complex NAMI-A, ruthenium complex KP1019, Polypyridine class ruthenium complex, benzimidazole ruthenium complex; Described organic selenium derivant comprises selenole derivant, selenium phenol, selenocystine, ebselen.
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Effective date of registration: 20181116 Address after: 510535 Room 101, Production Complex Building, 206 Kaiyuan Avenue, Science City, Guangzhou High-tech Industrial Development Zone, Guangdong Province Patentee after: Guangdong Jichuang Selenium Source Nanometer Research Institute Co., Ltd. Address before: 510632 Whampoa Avenue, Guangzhou, Guangzhou, Guangdong Province, No. 601 Patentee before: Jinan University |