CN108395537A - A kind of tetravalence platinum macromolecular prodrug PDA, tetravalence platinum macromolecular calcium phosphate nanoparticles and its application - Google Patents
A kind of tetravalence platinum macromolecular prodrug PDA, tetravalence platinum macromolecular calcium phosphate nanoparticles and its application Download PDFInfo
- Publication number
- CN108395537A CN108395537A CN201810203247.1A CN201810203247A CN108395537A CN 108395537 A CN108395537 A CN 108395537A CN 201810203247 A CN201810203247 A CN 201810203247A CN 108395537 A CN108395537 A CN 108395537A
- Authority
- CN
- China
- Prior art keywords
- macromolecular
- platinum
- pda
- tetravalence platinum
- calcium phosphate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G79/00—Macromolecular compounds obtained by reactions forming a linkage containing atoms other than silicon, sulfur, nitrogen, oxygen, and carbon with or without the latter elements in the main chain of the macromolecule
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/52—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an inorganic compound, e.g. an inorganic ion that is complexed with the active ingredient
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Inorganic Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Polymers & Plastics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention discloses a kind of tetravalence platinum macromolecular prodrug PDA, tetravalence platinum macromolecular calcium phosphate nanoparticles and its applications, synthesis tetravalence platinum macromolecular prodrug PDA first, then tetravalence platinum macromolecular prodrug PDA is embedded with calcium phosphate again, surface carries out the nano particle of modification acquisition with hydrophilic block polymer again.Tetravalence platinum macromolecular nano-calcium phosphate granule stability prepared by the present invention is good, biocompatibility is high, have good pH responses releasing effect and preferable tumor cytotoxicity effect, for solve stability present in current tetravalence platinum prodrug is low, easily removed by kidney the shortcomings of open up a new way.
Description
Technical field
The present invention relates to a kind of tetravalence platinum macromolecular prodrug PDA, tetravalence platinum macromolecular calcium phosphate nanoparticles and its application,
Belong to organic chemistry and field of nano material preparation.
Background technology
Cis-platinum as a kind of anticancer drug acting on DNA, be now widely used in all kinds of cancers for the treatment of (such as oophoroma,
Cervical carcinoma, cancer of the brain etc.).However it includes renal toxicity and neurotoxicity with very strong toxic side effect and is also easy to produce drug resistance limit
Its application in antitumor drug is made.In order to reduce the toxicity to normal cell and overcome the drug resistance of cancer cell, scientific research
Workers have synthesized many chemical inertnesses, avirulent tetravalence platinum prodrug, and research shows that tetravalence platinum molecule into cancer
Can platinous form be reduced into achieve the effect that anticancer by intracellular such as glutathione, ascorbic acid etc. after cell.
In recent years, polymerization macromolecular drug is increasingly subject to the concern of scientists.Yang Jun et al. synthesized first two kinds by
Tetravalence platinum small molecule DHP and DSP derived from cis-platinum, three kinds of tetravalence platinum macromolecule polyalcohol P (DHP-DA) of one-step synthesis of going forward side by side-
PEG, P (DSP-PA) and P (DSP-EDA).These three tetravalence platinum macromolecule polyalcohols are demonstrated in document can be by tumour cell
It absorbs and their cytotoxicity is generally higher than its corresponding parent small molecule.Jing Xiabin seminars have synthesized one end band first
The tetravalence platinum compounds of carboxyl, is then bonded with the mPEG-b-PCL-b-PLL with amino cation, to self assembly
Formed polymer micelle, and research shows that the cytotoxicity of the polymer micelle than cis-platinum is with its corresponding matrix polymer
By force.However since most of macromolecule polyalcohols are all not biodegradable, biology peace when recycling in vivo in vivo
Full property need to be investigated.
Calcium phosphate is naturally occurring inorganic mineralizing material simultaneously.It is well known that calcium phosphate is hard group of tooth and bone etc.
The inorganic component part knitted, therefore have the characteristics that non-toxic, degradability and fabulous biocompatibility, so nanometer phosphoric acid
The concern for more and more people that calcium is caused with its outstanding physicochemical characteristics.Since nano-calcium phosphate has outstanding physics
And chemical characteristic, this keeps the integrality of itself when it is transported in vivo, so in nano-medicament carrier field, nanometer
The application of calcium phosphate is also more and more.
Based on this, the present invention has synthesized a kind of new tetravalence platinum macromolecular prodrug PDA first, utilization numerous carboxyl thereon
Condition is provided for the mineralising of calcium phosphate, surface is again with polyethyleneglycol modified to reach macrocyclic effect, to prepare one kind
Novel tetravalence platinum macromolecular calcium phosphate nanoparticles simultaneously study its drug release and antitumous effect.
Invention content
The present invention is to avoid existing deficiencies in the technology, it is desirable to provide a kind of tetravalence platinum macromolecular prodrug
PDA, tetravalence platinum macromolecular calcium phosphate nanoparticles and its application.Tetravalence platinum macromolecular nano-calcium phosphate prepared by the present invention
Grain stability is good, and biocompatibility is high, has good pH responses releasing effect and preferable tumor cytotoxicity effect, is
Solve that the stability present in current tetravalence platinum prodrug is low, the shortcomings of easily being removed by kidney opens up a new way.
Tetravalence platinum macromolecular prodrug PDA of the present invention, structural formula are：
The molecular weight ranges of the tetravalence platinum macromolecular prodrug PDA are 8000~12000.
Tetravalence platinum macromolecular calcium phosphate nanoparticles of the present invention are to embed the tetravalence platinum macromolecular prodrug with calcium phosphate
PDA, surface carry out the nano particle of modification acquisition with hydrophilic block polymer again.
The preparation method of tetravalence platinum macromolecular calcium phosphate nanoparticles of the present invention, includes the following steps：
Step 1：Cis-platinum is added to the water, is warming up to 60 DEG C, hydrogen peroxide (30wt%) is then added and aoxidizes, is stirred to react
4h；Cooling is stood after reaction, faint yellow solid DHP is lyophilized to obtain at -50 DEG C, for axial small point of tetravalence platinum for carrying hydroxyl
Son；
The structural formula of DHP is as follows：
Step 2：It weighs DHP and pyromellitic acid anhydride (PMDA) is added in dimethyl sulfoxide (DMSO), under the conditions of 60 DEG C
It is stirred to react 2h；After reaction, (MWCO, 3500) dialysis is placed reaction liquid into bag filter for 24 hours, to be lyophilized, obtain at -50 DEG C
Tetravalence platinum macromolecular prodrug PDA；
The structural formula of PDA is：
Step 3：It weighs hydrophilic block polymer and PDA is added in distilled water and dissolves, pH is adjusted with 0.1M NaOH solutions
To 9,0.1M Ca (NO are then added dropwise into reaction solution3)2Solution is stirred to react 5h, 0.1M is then added dropwise into reaction solution again
Na2HPO4Solution is stirred to react 10h, dialyses 2 days, and freeze-drying obtains target product.
In step 1, H in hydrogen peroxide2O2It is 159 with the ratio between the amount of substance of cis-platinum：1.
In step 2, the ratio between amount of substance of DHP and PMDA is 1：1.
In step 2, the molecular weight of PDA is 8000~12000.
In step 3, the hydrophilic block polymer is PEG5k- PAA or PEG5k-PAA-5-AF.When hydrophilic block is poly-
Conjunction object is PEG5kWhen-PAA-5-AF, prepares the tetravalence platinum calcium phosphate nano with fluorescence and carry medicine particle.
In step 3, the ratio between amount of substance of carboxyl is 5~6 on hydrophilic block polymer and PDA：1；Ca(NO3)2With
Na2HPO4The ratio between the amount of substance be 1：2；The upper carboxyls of PDA and Ca (NO3)2The ratio between the amount of substance be 1：2~3.
The grain size of tetravalence platinum macromolecular calcium phosphate nanoparticles prepared by the present invention is 50~100nm.
The synthetic route of PDA of the present invention is as follows：
Wherein PEG5k- PAA is the hydrophilic high molecular material of inventor's synthesis, PEG5k- PAA-5-AF be by 5-AF with
PEG5kThe high molecular material with fluorescein that-PAA is bonded.Preparation process is as follows：
Hydrophilic block copolymer PEG5kThe synthesis of-PAA：
1, ethyl mercaptan (24.85g, 400mmol) and 60mL distilled water are mixed and stirred for, then under condition of ice bath slowly
32g mass concentrations are added dropwise and are 50% NaOH solution (wherein NaOH 16g, 400mmol), then 20mL is added under condition of ice bath
Acetone stirs 30min, then adds carbon disulfide (34.2g, 450mmol) solution and become clear crocus, and stir
30min；
2,2 bromopropionic acid (62.73g, 410mmol) is slowly added dropwise under condition of ice bath, it is dense then to add dropwise 32g mass
The NaOH solution (wherein NaOH 16g, 400mmol) that degree is 50%；Heat release removes ice bath after stopping, and 60mL distillations are then added
Water reacts for 24 hours at ambient temperature；60mL distilled water is added under condition of ice bath after reaction, its pH is adjusted with concentrated hydrochloric acid
It is 2, gained grease is extracted 3 times with dichloromethane, and revolving removes dichloromethane, is then recrystallized, is repeated 3 times with n-hexane,
Obtain light yellow crystal ETP；
3,5000 monomethyl ether of taking polyethylene glycol (10g, 2mmol) is dissolved in 60mL toluene, and dicyclohexyl carbon is then added
Diimine (DCC, 4.5386g, 22mmol) and 4-dimethylaminopyridine (DMAP, 25.9mg, 0.212mmol), use constant pressure addition
The toluene solution (20mL) of ETP (4.2068g, 20mmol) is added dropwise in funnel, reacts 12h；After reaction, it is filtered to remove insoluble matter
DCU, revolving remove part toluene, are then precipitated in ice ether, 3 times repeatedly, centrifuge, drain to obtain ETP-PEG5K；
4, by ETP-PEG5K(2.1616g, 0.4163mmol), two isobutyl of acrylic acid (AA, 3g, 41.63mmol) and azo
Nitrile (AIBN, 13.67mg, 0.08326mmol) is dissolved in 30mL Isosorbide-5-Nitraes-dioxane, and N is led under condition of ice bath230min is moved
To being reacted for 24 hours in 60 DEG C of oil bath pans；Revolving removes part Isosorbide-5-Nitrae-dioxane after reaction, is precipitated 3 times with anhydrous ether, from
The heart drains to obtain product PEG5k-PAA。
It is connected to the hydrophilic block copolymer PEG of fluorescein5kThe synthesis of-PAA-5-AF
1,5- Aminofluoresceins (0.05g, 0.144mmol) are dissolved in 20mL DMF, dicyclohexyl carbon is then added
Diimine (DCC, 0.0594g, 0.288mmol) and 4-dimethylaminopyridine (DMAP, 35mg, 0.0288mmol)；
2, the DMF solution (10mL) of PEG5k-PAA (0.1927g, 1.44mmol), reaction are added dropwise with constant pressure funnel
12h；It is filtered to remove insoluble matter DCU after reaction, is then precipitated in ice ether, 3 times repeatedly, production is drained to obtain in centrifugation
Object PEG5k-PAA-5-AF。
The purposes of tetravalence platinum macromolecular calcium phosphate nanoparticles of the present invention, is the application in preparing anticancer drug.
Beneficial effects of the present invention are embodied in：
1, solution used is aqueous systems during the present invention prepares tetravalence platinum macromolecular nano-calcium phosphate particle, safe nothing
Poison ring is protected；
2, the tetravalence platinum macromolecular prodrug PDA synthesized by the present invention carries a large amount of carboxyls, it is easier to Ca2+Bondage, can be with
The drugloading rate of raising system；
3, the tetravalence platinum macromolecular nano-calcium phosphate granule stability prepared by the present invention is good, and biocompatibility is high, has
Good pH responses releasing effect and preferable tumor cytotoxicity effect.
Description of the drawings
Fig. 1 is nucleus magnetic hydrogen spectrum figures of the tetravalence platinum macromolecular prodrug PDA in deuterated DMSO；
Fig. 2 is measured GPC figures of the PDA-Na in gel aqueous phase permeation chromatography；
Fig. 3 is hydrophilic block copolymer PEG5k- PAA is in D2Nucleus magnetic hydrogen spectrum figure in O；
A is the hydrophilic block copolymer PEG for being connected to fluorescein in Fig. 45k- PAA-5-AF is in D2Nucleus magnetic hydrogen spectrum figure in O,
B is nucleus magnetic hydrogen spectrum figures of the 5-AF in deuterated DMSO；
A is the PEG measured in Fig. 55kThe fluorescence curve of-PAA-5-AF, b are the fluorescence curve of 5-AF, and c is band fluorescence tetravalence
The fluorescence curve of platinum macromolecular nano-calcium phosphate particle；
A in Fig. 6, b are the DLS number distributions of three kinds of prepared tetravalence platinum macromolecular nano-calcium phosphate grain diameters respectively
Figure and DLS intensity distributions；
Fig. 7 is the transmission electron microscope picture of tetravalence platinum macromolecular nano-calcium phosphate particle；
Curve a in Fig. 8, b be tetravalence platinum macromolecular nano-calcium phosphate particle respectively by DLS measure in PBS (pH=
7.4) and in DMEM solution grain size changes over time figure；
Curve a in Fig. 9, b, c, d are PBS solution of the tetravalence platinum macromolecular nano-calcium phosphate particle in pH=7.4, pH respectively
It is surveyed using ICP-MS in the PBS+5mM Vc solution of=7.4 PBS+5mM Vc solution, the PBS solution of pH=5.0 and pH=5.0
The drug release patterns obtained；
A in Figure 10, b are that the tetravalence platinum macromolecular calculated with XPSpeak softwares on the basis of surveyed XPS data is received respectively
Rice calcium phosphate granules divalent platinum, platinic relative amount of different time points and in pH=in the PBS solution of pH=5.0
Divalent platinum, the platinic relative amount of different time points in 5.0 PBS+5mM Vc solution；
Figure 11 is various concentration tetravalence platinum macromolecular nano-calcium phosphate particle through the ultraviolet counted haemolysis Value Data of test；
Figure 12 is DHP, PDA containing different Pt concentration and tetravalence platinum macromolecular nano-calcium phosphate particle to MDA-MB-
231 cytotoxicity values；
Figure 13 is cell of the naked nano-calcium phosphate particle to MDA-MB-231 that various concentration is not surrounded by tetravalence platinum macromolecular
Toxicity value；
Figure 14 is that the tetravalence platinum macromolecular nano-calcium phosphate particle of fluorescent marker is thin through streaming in MDA-MB-231 cells
The curve graph that born of the same parents' instrument measures；
Figure 15 be in DHP, PDA and tetravalence platinum macromolecular nano-calcium phosphate particle platinum element by MDA-MB-231 cells
Quantitatively absorb result.
Specific implementation mode
Technical solution of the present invention is further analyzed and described below by specific embodiment.Following embodiments are with this
Implemented under premised on inventive technique scheme, gives detailed embodiment and specific operating process, but the present invention
Protection domain is not limited to following embodiments.
Embodiment 1：The synthesis of tetravalence platinum macromolecular prodrug PDA
1, it weighs cis-platinum 400mg (1.334mmol) to be added in 12mL water, 24mL 30% is added dropwise after being warming up to 60 DEG C
The hydrogen peroxide of (212mmol), system is yellow solution after reacting 5min, a small amount of pale yellow precipitate occurs after 1h, later without phenomenon
Variation, is stirred to react 4h；Reaction solution is cooled to room temperature after reaction, ice bath stands overnight to obtain pale yellow precipitate, filtering, ice
Faint yellow solid DHP is lyophilized to obtain in water washing；
2, it weighs DHP 150mg (0.45mmol) and PMDA 97.92mg (0.45mmol) to be added in 15mL DMSO, 60
It is stirred to react 2h under the conditions of DEG C, excessive ethyl alcohol sealing end is then added；After reaction, it places reaction liquid into bag filter
(MWCO, 3500) dialyses for 24 hours, and freeze-drying obtains tetravalence platinum macromolecular prodrug PDA.
Fig. 1 is nucleus magnetic hydrogen spectrum figures of the tetravalence platinum macromolecular prodrug PDA in deuterated DMSO, is PDA through analyzing product.
Fig. 2 is measured GPC figures of the tetravalence platinum macromolecular prodrug PDA-Na in gel aqueous phase permeation chromatography.Specifically
It is 9 or so that tetravalence platinum macromolecular prodrug PDA is adjusted to pH with 0.1MNaOH solution, then is matched to it with gel aqueous phase permeation chromatography
At aqueous solution tested, GPC aqueous phase flow rates be 1mL/min.
Table 1 is tetravalence platinum macromolecular prodrug PDA-Na through the given corresponding data of gel aqueous phase permeation chromatography test.It can
To find out the molecular weight of PDA-Na for 9000 or so, PDI 2.993.
Table 1
| Compound | Retention time/min | Mp | Mw | Mn | PDI |
| PDA-Na | 25.436 | 8943 | 12752 | 4261 | 2.993 |
Embodiment 2：Hydrophilic block copolymer PEG5kThe synthesis of-PAA
1, ethyl mercaptan (24.85g, 400mmol) and 60mL distilled water are mixed and stirred for, then under condition of ice bath slowly
32g mass concentrations are added dropwise and are 50% NaOH solution (wherein NaOH 16g, 400mmol), then 20mL is added under condition of ice bath
Acetone stirs 30min, then adds carbon disulfide (34.2g, 450mmol) solution and become clear crocus, and stir
30min；
2,2 bromopropionic acid (62.73g, 410mmol) is slowly added dropwise under condition of ice bath, it is dense then to add dropwise 32g mass
The NaOH solution (wherein NaOH 16g, 400mmol) that degree is 50%；Heat release removes ice bath after stopping, and 60mL distillations are then added
Water reacts for 24 hours at ambient temperature；60mL distilled water is added under condition of ice bath after reaction, its pH is adjusted with concentrated hydrochloric acid
It is 2, gained grease is extracted 3 times with dichloromethane, and revolving removes dichloromethane, is then recrystallized, is repeated 3 times with n-hexane,
Obtain light yellow crystal ETP；
3,5000 monomethyl ether of taking polyethylene glycol (10g, 2mmol) is dissolved in 60mL toluene, and dicyclohexyl carbon is then added
Diimine (DCC, 4.5386g, 22mmol) and 4-dimethylaminopyridine (DMAP, 25.9mg, 0.212mmol), use constant pressure addition
The toluene solution (20mL) of ETP (4.2068g, 20mmol) is added dropwise in funnel, reacts 12h；After reaction, it is filtered to remove insoluble matter
DCU, revolving remove part toluene, are then precipitated in ice ether, 3 times repeatedly, centrifuge, drain to obtain ETP-PEG5K；
4, by ETP-PEG5K(2.1616g, 0.4163mmol), two isobutyl of acrylic acid (AA, 3g, 41.63mmol) and azo
Nitrile (AIBN, 13.67mg, 0.08326mmol) is dissolved in 30mL Isosorbide-5-Nitraes-dioxane, and N is led under condition of ice bath230min is moved
To being reacted for 24 hours in 60 DEG C of oil bath pans；Revolving removes part Isosorbide-5-Nitrae-dioxane after reaction, is precipitated 3 times with anhydrous ether, from
The heart drains to obtain product PEG5k-PAA。
Fig. 3 is hydrophilic block copolymer PEG5k- PAA is in D2Nucleus magnetic hydrogen spectrum figure in O is PEG through analyzing product5k-
PAA, and the AA unit numbers connected are 87.
Embodiment 3：It is connected to the hydrophilic block copolymer PEG of fluorescein5kThe synthesis of-PAA-5-AF
1,5- Aminofluoresceins (0.05g, 0.144mmol) are dissolved in 20mL DMF, dicyclohexyl carbon is then added
Diimine (DCC, 0.0594g, 0.288mmol) and 4-dimethylaminopyridine (DMAP, 35mg, 0.0288mmol)；
2, the DMF solution (10mL) of PEG5k-PAA (0.1927g, 1.44mmol), reaction are added dropwise with constant pressure funnel
12h；It is filtered to remove insoluble matter DCU after reaction, is then precipitated in ice ether, 3 times repeatedly, production is drained to obtain in centrifugation
Object PEG5k-PAA-5-AF。
A is the hydrophilic block copolymer PEG for being connected to fluorescein in Fig. 45k- PAA-5-AF is in D2Nucleus magnetic hydrogen spectrum figure in O,
B is nucleus magnetic hydrogen spectrum figures of the 5-AF in deuterated DMSO.
A is the PEG measured in Fig. 55kThe fluorescence curve of-PAA-5-AF, b are the fluorescence curve of 5-AF, and c is band fluorescence tetravalence
The fluorescence curve of platinum macromolecular nano-calcium phosphate particle.Complex chart 4 and Fig. 5's as a result, understanding that 5-AF is successfully bonded to
PEG5k- PAA is upper and it is made to carry fluorescent effect.
Embodiment 4：The preparation of tetravalence platinum macromolecular nano-calcium phosphate particle
Weigh 16.92mg PEG5k-PAA(-COOH：0.1220mmol), 6.25mg PDA (- COOH：0.02272mmol)
In 25mL single port bottles, 10mL is added and distills water dissolution, pH to 9 is adjusted with 0.1M NaOH solutions；Then dropwise into reaction solution
0.1M Ca (NO are added dropwise at a slow speed3)21.250mL 0.1M Na are added dropwise at a slow speed after stirring 5h in solution 0.625mL dropwise again2HPO4It is molten
Liquid stirs 10h, dialyses 2 days, product is lyophilized to obtain.
When 1.692mg PEG are added in we before the reaction5k- PAA-5-AF, it is big that we have prepared the tetravalence platinum with fluorescence
Molecule nano calcium phosphate granules.In addition, we change the inventory of PDA into 5mg and 3.13mg, above-mentioned same method system is utilized
Obtained other two kinds of tetravalences platinum macromolecular nano-calcium phosphate particle.
A in Fig. 6, b are the DLS number distributions of three kinds of prepared tetravalence platinum macromolecular nano-calcium phosphate grain diameters respectively
Figure and DLS intensity distributions.
Fig. 7 is the transmission electron microscope picture of prepared tetravalence platinum macromolecular nano-calcium phosphate grain diameter, it can be seen that particle
Size is uniform, and grain size is in 40nm or so.
Curve a in Fig. 8, b are to choose PDA inventorys as tetravalence platinum macromolecular nano-calcium phosphate made from 6.25mg respectively
Grain changes over time figure by the grain size in PBS (pH=7.4) and DMEM solution that DLS is measured, it can be seen that granule stability
It is good.
Table 2 is three kinds of tetravalence platinum macromolecular nano-calcium phosphate particles by the DLS particle size datas measured and passes through ICP-MS
The drugloading rate and carrier medicine carrying efficiency data measured.
Table 2
Embodiment 5：The drug release experiment of tetravalence platinum macromolecular nano-calcium phosphate particle
It is that tetravalence platinum macromolecular nano-calcium phosphate particle made from 6.25mg is released accordingly that we, which choose PDA inventorys,
Experiment is put, specific embodiment is as follows：
1,4 groups of 2mL 1.0mg/mL tetravalence platinum macromolecular nano-calcium phosphate particle solutions are measured to be added in bag filter (thoroughly
Analysing bag uses preceding boiling water boiling 5min, bag filter one end to be tightened with rope, one end clip), the PBS for preparing pH=7.4 respectively is molten
The PBS+5mM Vc solution of liquid, the PBS solution of pH=5.0, the PBS+5mM Vc solution of pH=7.4, pH=5.0, each time
It selects and the above-mentioned buffer solutions of 15mL is respectively taken to be added in centrifuge tube, centrifuge tube is placed in constant temperature oscillation case, and temperature is set as 37 DEG C, in difference
Time point by bag filter taking-up put into fresh buffer solution, time point 0h, 2h, 4h, 12h, for 24 hours, 48h, 72h, collect
Centrifuge tube be placed on -20 DEG C of preservations, be lyophilized, the sample being lyophilized measures the wherein content of Pt with ICP-MS.
2, in addition we measure 2 groups of 2mL3.0mg/mL tetravalence platinum macromolecular nano-calcium phosphate particle solutions and are distinguished
It is added to the PBS solution of the pH=5.0 of 10mL and the PBS+5mM Vc solution of the pH=5.0 of 10mL, point is each in different times
2mL sample liquids are taken out, freeze-drying is collected and measure wherein divalent platinum and platinic relative amount with XPS.
Curve a in Fig. 9, b, c, d are PBS solution of the tetravalence platinum macromolecular nano-calcium phosphate particle in pH=7.4, pH respectively
It is surveyed using ICP-MS in the PBS+5mM Vc solution of=7.4 PBS+5mM Vc solution, the PBS solution of pH=5.0 and pH=5.0
The drug release patterns obtained, it can be seen that the tetravalence platinum in the PBS solution of pH=7.4 and the PBS+5mM Vc solution of pH=7.4
Drug in macromolecular nano-calcium phosphate particle does not discharge substantially, is discharged comparatively fast, in pH=5.0 in the PBS solution of pH=5.0
PBS+5mM Vc drug in solution release faster.
A in Figure 10, b are the tetravalence platinum macromolecular calculated with XPSpeak softwares on the basis of the XPS data surveyed respectively
Nano-calcium phosphate particle divalent platinum, platinic relative amount of different time points and in pH=in the PBS solution of pH=5.0
Divalent platinum, the platinic relative amount of different time points in 5.0 PBS+5mM Vc solution, it can be seen that pH=5.0's
Tetravalence platinum macromolecular nano-calcium phosphate particle discharges substantially in the form of platinic in PBS solution, in the PBS+5mM of pH=5.0
Divalent platinum content gradually increases in Vc solution, and substantially all after 12h is converted into divalent platinum.
Embodiment 6：The hemolytic experiment of tetravalence platinum macromolecular nano-calcium phosphate particle
1, the fresh mouse blood of 0.8mL is first centrifuged into (2500rpm, 10min), pours out supernatant, adds 1mL 0.01M
PBS (pH=7.4) solution dilutes to obtain diluted blood；
2,5mL 0.1mg/mL, 0.01mg/mL, 0.001mg/mL tetravalence platinum macromolecular nano-calcium phosphates are prepared respectively again
Each three groups of PBS (pH=7.4) solution of grain, negative control, three groups of 5mL deionizations are done with three groups of 5mL PBS solutions (pH=7.4)
Water does positive control；
3, above-mentioned 15 groups of solution is placed in constant temperature oscillation case, temperature is set as 37 DEG C, time 30min, then adds diluted blood
It shakes up, 37 DEG C of culture 1h survey ultraviolet, peak value at record 540nm.
Figure 11 is 0.1mg/mL, 0.01mg/mL and 0.001mg/mL tetravalence platinum macromolecular nano-calcium phosphate particles are through ultraviolet
Test counted haemolysis Value Data, it can be seen that when no more than 0.1mg/mL, hemolysis value is no more than the concentration of the particle
2%, it is preferable with blood compatibility.
Embodiment 7：The cytotoxicity experiment of tetravalence platinum macromolecular nano-calcium phosphate particle
We have chosen tetravalence platinum small molecule DHP, tetravalence platinum macromolecular prodrug PDA and tetravalence platinum macromolecular calcium phosphate nano
It carries medicine particle (Pt (IV) CPNP) and their cytotoxicities to MDA-MB-231 is investigated by MTT experiment, while we also investigate
Naked calcium phosphate nano carries the cytotoxicity of medicine particle to MDA-MB-231, and specific embodiment is as follows：
(1) by MDA-MB-231 cell inoculations to 96 orifice plates, 1 × 10 is inoculated with per hole5A cell, then be added in every hole
100 μ LDMEM complete mediums are placed in 37 DEG C of incubators and cultivate 24 hours；
(2) by PBS, DHP, PDA and Pt (IV) CPNP with DMEM complete mediums be diluted to different concentration be made into it is molten
Liquid, every group of solution take 100 μ L to be added in orifice plate, and parallel group is set as 3 groups, is further continued for being placed in 37 DEG C of incubators and cultivates 48h；
(3) culture terminates, and sucks culture medium and adds the DMEM complete mediums of same volume.25 μ L will be added in every hole
MTT liquid storages (5mg mL-1In PBS solution), 25 μ L PBS buffer solution are added in blank well, continue culture 2 hours；
(4) 100 μ LDMSO are added in every hole and are incubated half an hour；
(5) with microplate reader measure solution 570nm absorbance (Abs).Cell viability is calculated with following formula：
Wherein, Abs (sample) is the absorption value of sample well；Abs (blank) is the absorption value of blank well；Abs
(control) it is the light absorption value in PBS control hole.
Figure 12 is DHP, PDA containing different Pt concentration and tetravalence platinum macromolecular nano-calcium phosphate particle to MDA-MB-
231 cytotoxicity values, it can be seen that compared to DHP and PDA, tetravalence platinum macromolecular nano-calcium phosphate under same platinum concentration
The cytotoxicity of grain is stronger, and fragmentation effect is more preferable.
Figure 13 is cell of the naked nano-calcium phosphate particle to MDA-MB-231 that various concentration is not surrounded by tetravalence platinum macromolecular
Toxicity value, it can be seen that the cytotoxicity of naked nano-calcium phosphate particle is low, and safety is good.
Embodiment 8：The qualitative intake experiment of cell of tetravalence platinum macromolecular nano-calcium phosphate particle (Pt (IV) CPNP)
(1) by MDA-MB-231 cell inoculations in 24 orifice plates, 1 × 10 is inoculated with per hole5100 μ L DMEM are added in a cell
Then complete medium is cultivated 24 hours in 37 DEG C of incubators；
(2) after cultivating, culture medium is removed, then new Pt (IV) CPNP containing 5- Aminofluoresceins label of change
The DMEM complete mediums of nano particle co-culture 2h, 4h, 6h under the conditions of 37 DEG C with cell；
(3) culture solution is removed, is cleaned twice with PBS buffer solution, is digested simultaneously using 0.25% trypsase (containing EDTA)
It collects in cell to streaming pipe, using the intracellular green florescent signal of flow cytomery, as a result uses FlowJo_V10 soft
Part is analyzed.
Figure 14 is the curve graph that Pt (IV) CPNP of fluorescent marker are measured in MDA-MB-231 cells through flow cytometer,
It can be seen that Pt (IV) CPNP can be by cellular uptake, and it is ingested substantially completely after 4h.
Embodiment 9：The cell quantification intake experiment of tetravalence platinum macromolecular nano-calcium phosphate particle (Pt (IV) CPNP)
(1) by MDA-MB-231 cells respectively with every hole 5 × 105A cell seeding uses 100 μ LDMEM in 96 orifice plates
Complete medium is cultivated 24 hours in 37 DEG C of incubators；
(2) culture medium is removed, new DMEM complete mediums are changed to, DHP, PDA and Pt (IV) CPNP solution is added, makes platinum
The total concentration of element is 2,4,8 μ g/mL, and 3h is co-cultured with cell under the conditions of 37 DEG C；
(3) culture solution is removed, is cleaned three times using cold PBS, using 0.25% trypsin digestion and cell, collects cell extremely
It in PBS solution, is placed in clean beaker, 500 μ L concentrated nitric acids are added and are evaporated, add 500 μ L chloroazotic acid and be settled to
5mL；
(4) ICP-MS is used to detect the content of intracellular platinum element.
Figure 15 be in DHP, PDA and Pt (IV) CPNP platinum element by the quantitative intake of MDA-MB-231 cells as a result, from
In it can be seen that Pt (IV) CPNP in platinum element by MDA-MB-231 cellular uptake amounts be higher than DHP and PDA.
The foregoing is merely exemplary embodiment of the present invention, are not intended to limit the invention, all the present invention's
All any modification, equivalent and improvement etc., should all be included in the protection scope of the present invention made by within spirit and principle.
Claims (10)
1. a kind of tetravalence platinum macromolecular prodrug PDA, it is characterised in that its structural formula is：
2. tetravalence platinum macromolecular prodrug PDA according to claim 1, it is characterised in that：
The molecular weight ranges of the tetravalence platinum macromolecular prodrug PDA are 8000~12000.
3. a kind of tetravalence platinum macromolecular calcium phosphate nanoparticles, it is characterised in that：It is with four described in calcium phosphate embedding claim 1
Valence platinum macromolecular prodrug PDA, surface carry out the nano particle of modification acquisition with hydrophilic block polymer again.
4. tetravalence platinum macromolecular calcium phosphate nanoparticles according to claim 3, it is characterised in that：
The grain size of the tetravalence platinum macromolecular calcium phosphate nanoparticles is 50~100nm.
5. a kind of preparation method of the tetravalence platinum macromolecular calcium phosphate nanoparticles described in claim 3, it is characterised in that including
Following steps：
Step 1：Cis-platinum is added to the water, 60 DEG C is warming up to, hydrogen peroxide oxidation is then added, be stirred to react 4h；After reaction
Cooling is stood, faint yellow solid DHP is lyophilized to obtain at -50 DEG C, for the axial tetravalence platinum small molecule for carrying hydroxyl；
Step 2：It weighs DHP and pyromellitic acid anhydride is added in dimethyl sulfoxide, 2h is stirred to react under the conditions of 60 DEG C；Reaction knot
Shu Hou places reaction liquid into dialysis in bag filter and for 24 hours, is lyophilized at -50 DEG C, obtains tetravalence platinum macromolecular prodrug PDA；
Step 3：It weighs hydrophilic block polymer and PDA is added in distilled water and dissolves, pH to 9 is adjusted with 0.1M NaOH solutions,
Then 0.1M Ca (NO are added dropwise into reaction solution3)2Solution is stirred to react 5h, 0.1M is then added dropwise into reaction solution again
Na2HPO4Solution is stirred to react 10h, dialyses 2 days, and freeze-drying obtains target product.
6. preparation method according to claim 5, it is characterised in that：
In step 1, H in hydrogen peroxide2O2It is 159 with the ratio between the amount of substance of cis-platinum：1.
7. preparation method according to claim 5, it is characterised in that：
In step 2, the ratio between amount of substance of DHP and PMDA is 1：1.
8. preparation method according to claim 5, it is characterised in that：
In step 3, the hydrophilic block polymer is PEG5k- PAA or PEG5k-PAA-5-AF。
9. preparation method according to claim 5, it is characterised in that：
In step 3, the ratio between amount of substance of carboxyl is 5~6 on hydrophilic block polymer and PDA：1；Ca(NO3)2And Na2HPO4
The ratio between the amount of substance be 1：2；The upper carboxyls of PDA and Ca (NO3)2The ratio between the amount of substance be 1：2~3.
10. a kind of purposes of the tetravalence platinum macromolecular calcium phosphate nanoparticles described in claim 3, it is characterised in that：It is to make
Application in standby anticancer drug.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810203247.1A CN108395537A (en) | 2018-03-13 | 2018-03-13 | A kind of tetravalence platinum macromolecular prodrug PDA, tetravalence platinum macromolecular calcium phosphate nanoparticles and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810203247.1A CN108395537A (en) | 2018-03-13 | 2018-03-13 | A kind of tetravalence platinum macromolecular prodrug PDA, tetravalence platinum macromolecular calcium phosphate nanoparticles and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108395537A true CN108395537A (en) | 2018-08-14 |
Family
ID=63092468
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810203247.1A Pending CN108395537A (en) | 2018-03-13 | 2018-03-13 | A kind of tetravalence platinum macromolecular prodrug PDA, tetravalence platinum macromolecular calcium phosphate nanoparticles and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108395537A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116059357A (en) * | 2022-12-26 | 2023-05-05 | 中山大学 | PH responsive charge-reversal nano composite preparation and preparation method and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050025969A1 (en) * | 2003-07-31 | 2005-02-03 | Berning Douglas E. | Gold-coated nanoparticles for use in biotechnology applications |
| WO2008043396A1 (en) * | 2006-10-12 | 2008-04-17 | Nm Tech Nanomaterials Microdevice Technology Ltd. | Material, item and products comprising a composition having anti-microbial properties |
| CN102120036A (en) * | 2011-02-22 | 2011-07-13 | 中国科学院长春应用化学研究所 | Nano micelle of biodegradable macromolecular-bonding Pt(IV) anti-cancer medicament and preparation method thereof |
| CN102942594A (en) * | 2012-11-14 | 2013-02-27 | 中国科学技术大学 | Anti-cancer medicinal aspirin platinum complex and preparation method thereof |
| CN104353083A (en) * | 2014-11-14 | 2015-02-18 | 复旦大学 | Thermal gel controlled-release injection of platinum-containing antitumor drug and preparation method of thermal gel controlled-release injection |
-
2018
- 2018-03-13 CN CN201810203247.1A patent/CN108395537A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050025969A1 (en) * | 2003-07-31 | 2005-02-03 | Berning Douglas E. | Gold-coated nanoparticles for use in biotechnology applications |
| WO2008043396A1 (en) * | 2006-10-12 | 2008-04-17 | Nm Tech Nanomaterials Microdevice Technology Ltd. | Material, item and products comprising a composition having anti-microbial properties |
| CN102120036A (en) * | 2011-02-22 | 2011-07-13 | 中国科学院长春应用化学研究所 | Nano micelle of biodegradable macromolecular-bonding Pt(IV) anti-cancer medicament and preparation method thereof |
| CN102942594A (en) * | 2012-11-14 | 2013-02-27 | 中国科学技术大学 | Anti-cancer medicinal aspirin platinum complex and preparation method thereof |
| CN104353083A (en) * | 2014-11-14 | 2015-02-18 | 复旦大学 | Thermal gel controlled-release injection of platinum-containing antitumor drug and preparation method of thermal gel controlled-release injection |
Non-Patent Citations (4)
| Title |
|---|
| RODNEY P. FEAZELL,ET AL.: ""Soluble Single-Walled Carbon Nanotubes as Longboat Delivery Systems for Platinum(IV) Anticancer Drug Design"", 《J. AM. CHEM. SOC》 * |
| ZHONGMING ZHU: ""A Method to Encapsulate Small Organic Molecules in Calcium Phosphate Nanoparticles Based on the Supramolecular Chemistry of Cyclodextrin"", 《MICROMACHINES》 * |
| 董云雷: ""纳米磷酸钙铂药载体的制备及其抗肿瘤性质研究"", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》 * |
| 郦豪，等: ""抗肿瘤药物顺铂及其纳米载体的研究进展"", 《安徽化工》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116059357A (en) * | 2022-12-26 | 2023-05-05 | 中山大学 | PH responsive charge-reversal nano composite preparation and preparation method and application thereof |
| CN116059357B (en) * | 2022-12-26 | 2023-11-03 | 中山大学 | PH responsive charge-reversal nano composite preparation and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wu et al. | Facile synthesis of sulfur-doped carbon quantum dots from vitamin B1 for highly selective detection of Fe3+ ion | |
| CN110128665B (en) | Amphiphilic block polymer near-infrared fluorescent probe based on azo reductase response and application | |
| CN106362162B (en) | ZnO@PMAA-b-PHPMA CdS quantum dots and its preparation and the application as pharmaceutical carrier | |
| CN104672199B (en) | One kind is containing double iodine cyclic carbonate compounds and preparation method thereof | |
| CN108504348B (en) | Preparation and application of dual-fluorescent polymer quantum dot material | |
| CN109111918A (en) | An a kind of one step preparation method of nitrogen-doped carbon quantum dot | |
| CN111053911A (en) | Reduction response type cross-linking agent and preparation and application of cross-linked hydroxyl drug molecule thereof | |
| CN107641201A (en) | Block copolymer preparation method and applications of the Quick Oxidation/reduction dual responsiveness containing double selenium keys | |
| CN108395537A (en) | A kind of tetravalence platinum macromolecular prodrug PDA, tetravalence platinum macromolecular calcium phosphate nanoparticles and its application | |
| CN111494650B (en) | Preparation method of amphiphilic polymer nanoparticles based on near-infrared fluorescence imaging and reduction response and product thereof | |
| CN114306282A (en) | Diagnosis and treatment integrated drug-loaded nanoparticle for enhancing iron death effect and preparation method and application thereof | |
| Li et al. | Water-soluble chitosan-g-PMAm (PMAA)-Bodipy probes prepared by RAFT methods for the detection of Fe3+ ion | |
| Zhao et al. | Synthesis of composite microgel capsules by ultrasonic spray combined with in situ crosslinking | |
| CN109231183A (en) | A kind of citric acid is the carbon quantum dot and its preparation method and application of carbon source | |
| Guo et al. | Rapid synthesis of amphiphilic europium complexes via ultrasonic treatment-assisted crosslinking reaction | |
| CN112972696A (en) | Fluorescent polypeptide doped metal organic framework nano material, preparation method and application | |
| Zhang et al. | One-step hydrothermal approach to synthesis carbon dots from D-sorbitol for detection of iron (III) and cell imaging | |
| CN112121182A (en) | Nano probe for detecting hypoxic cells and preparation method and application thereof | |
| CN109821025B (en) | Light and redox dual-stimulus response type amphiphilic polymer drug carrier and preparation method and application thereof | |
| CN104650307B (en) | PH five block linear polymers of sensitivity and micella based on PDEAEMA | |
| Zeng et al. | A feasible strategy of fabricating camptothecin (SN38)-loaded holmium ferrite nanocarrier delivery for glioma treatment | |
| CN108424766B (en) | Preparation and application of TPE-PDEAM with multi-responsive polymer quantum dots | |
| CN109400830A (en) | A kind of pH can dissociate lightly crosslinked polymeric object nano material and its preparation method and application | |
| Chen et al. | A novel AIE-active dye for fluorescent nanoparticles by one-pot combination of Hantzsch reaction and RAFT polymerization: synthesis, molecular structure and application in cell imaging | |
| Hasantabar et al. | Fabrication of chitosan based magnetic nanocomposite by click reaction strategy; evaluation of nanometric and cytotoxic characteristics |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20180814 |