CN110755625B - Targeting type drug carrier based on nano-hydroxyapatite and construction method of nano-drug delivery system - Google Patents
Targeting type drug carrier based on nano-hydroxyapatite and construction method of nano-drug delivery system Download PDFInfo
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Abstract
The invention relates to the field of targeted drug carriers, and provides a targeted drug carrier based on nano hydroxyapatite and a construction method of a nano drug delivery system aiming at the problem of small application range of a silane hydrolysis medium, wherein the technical scheme is as follows: the molecular formula of the targeting drug carrier is Ca 10 (PO 4 ) 6 (OH) 4 (Si 1 C 6 H 12 O 3 ) Man; preparing mesoporous spherical hydroxyapatite HA by a coprecipitation method by taking ethylene diamine modified carbon quantum dots N-CQDs as a template, preparing modified nano hydroxyapatite HA-GTS by taking GTS as a coupling agent, and modifying the modified nano hydroxyapatite HA-GTS by mannose to prepare a tumor targeting vector HA-GTS-Man; the targeted drug carrier has better quality, so that the targeted drug carrier has better effect of treating the tumor.
Description
Technical Field
The invention relates to the field of targeted drug carriers, in particular to a targeted drug carrier based on nano hydroxyapatite and a construction method of a nano drug delivery system.
Background
In recent years, the incidence rate and the death rate of malignant tumors are obviously increased, the targeting research of the antitumor drugs is more and more emphasized by people, and the nano drug delivery system has become the research focus in the field of antitumor drug delivery.
The construction of a nano-targeting drug delivery system mainly comprises tumor active targeting and tumor passive targeting, wherein the active targeting is to modify the drug delivery system by utilizing a specific ligand to combine with a receptor overexpressed on the surface of a tumor cell and transfer a substance specifically combined to the drug delivery system to achieve the effect of targeted delivery, and the passive targeting is mainly based on the EPR effect of tumor tissues on macromolecules.
The mannose receptor is C-type, is one of animal lectins, and can effectively and rapidly recognize mannose and glycoprotein at the end of fucose to form an organic defense system. Research shows that mannose receptor is over-expressed in colorectal cancer, therefore mannose is an ideal candidate object as a tumor cell ligand, mannose molecules only contain hydroxyl, and are not easy to form stable chemical bonds with other chemical groups, and in many modification methods, mannose with modified structure is used for realizing the next reaction, and the steps are complicated.
Hydroxyapatite is the main inorganic component of human and animal bones, can realize chemical bond combination with organism tissues on an interface, has certain solubility in vivo, can release ions harmless to the organism, can participate in vivo metabolism, has stimulation or induction effect on hyperosteogeny, can promote the repair of defective tissues, and shows bioactivity.
At present, a plurality of silane coupling agents (such as aminosilane and the like) containing different chemical groups are applied to the field of composite materials, the silane coupling agents are usually used as 'modification fulcrums', corresponding modification can be realized through proper hydrolysis, many researches mainly focus on modifying silane containing amino groups to hydroxyapatite, and then modifying hydroxyl hydroxyapatite to form a drug carrier through a receptor, and a large number of researches show that the hydrolysis of the silane is mostly carried out in 'specific environment', for example, in systems such as absolute ethyl alcohol or an aqueous ethyl alcohol solution (ethyl alcohol: water = 9:1) and the like, the silane is carried out in a small range of surface modification of the hydroxyapatite, so that the prepared drug carrier is poor in quality, and an improvement space exists.
Disclosure of Invention
Aiming at the defects in the prior art, the first purpose of the invention is to provide a targeting type drug carrier based on nano hydroxyapatite, which has a good quality.
In order to achieve the purpose, the invention provides the following technical scheme:
a targeting drug carrier based on nano-hydroxyapatite has a molecular formula of Ca 10 (PO 4 ) 6 (OH) 4 (Si 1 C 6 H 12 O 3 ) Man, structural formula:
by adopting the technical scheme, ca 10 (PO 4 ) 6 (OH) 4 (Si 1 C 6 H 12 O 3 ) Man (HA-GTS-Man for short) is adopted, the targeted drug carrier contains mannose molecules, and the mannose molecules only contain hydroxyl and are stably bonded with epoxy groups of silane under an alkaline condition, so that the mannose forms stable chemical bonds to be well combined on hydroxyapatite, the range of the silane on the surface modification of the hydroxyapatite is enlarged, the targeted drug carrier is guaranteed to have good quality, and the effect of the targeted drug carrier on treating tumors is good.
In view of the defects in the prior art, the second objective of the present invention is to provide a preparation method of a targeted drug carrier, which comprises the following steps:
s1, dissolving citric acid in water to obtain CQDs, and uniformly mixing the CQDs with anhydrous ethylenediamine to obtain an ethylenediamine modified carbon quantum point N-CQDs;
s2, adding N-CQDs and Ca (NO) into the ethylene diamine modified carbon quantum dots 3 ) 2 ·4H 2 Mixing O to form solution A, adding Na 2 HPO 4 Dissolving in pure water to form solution B; dripping the B solution into the A solution, stirring, precipitating, centrifuging, drying and calcining to form a mesoporousSpherical-like hydroxyapatite HA;
s3, ultrasonically dispersing the mesoporous quasi-spherical hydroxyapatite HA into toluene, and adding 3- (2,3-glycidoxy) propyltrimethoxysilane GTS to form HA-GTS;
s4, ultrasonically dispersing the HA-GTS in an organic solvent, adding mannose to modify the HA-GTS, and uniformly stirring to form the tumor targeting vector HA-GTS-Man.
By adopting the technical scheme, mesoporous sphere-like Hydroxyapatite (HA) is prepared by a coprecipitation method by taking carbon quantum dots (N-CQDs) modified by ethylenediamine as a template, modified nano hydroxyapatite (HA-GTS) is prepared by taking GTS as a coupling agent, and then the modified nano hydroxyapatite (HA-GTS) is modified by mannose to prepare the tumor targeting vector (HA-GTS-Man) (see figure 1).
The method is characterized in that 3- (2,3-glycidoxy) propyl trimethoxy silane (GTS) is modified on Hydroxyapatite (HA) in a toluene solvent, the surface modification range of the 3- (2,3-glycidoxy) propyl trimethoxy silane (GTS) on the Hydroxyapatite (HA) is increased, and the technical blank in the application of the 3- (2,3-glycidoxy) propyl trimethoxy silane (GTS) at present is filled.
The ring opening of the epoxy group on the 3- (2,3-glycidoxy) propyltrimethoxysilane (GTS) is carried out under alkaline conditions, and meanwhile, the mannose molecule only contains hydroxyl, so that the epoxy group of the 3- (2,3-glycidoxy) propyltrimethoxysilane and the hydroxyl group of the mannose form a stable ether bond under alkaline conditions, the mannose can be combined on hydroxyapatite by a stable chemical bond, the mannose has stable targeting performance, and the effect of treating diseases is better.
The invention is further configured to: and (3) after the CQDs and the anhydrous ethylenediamine are uniformly mixed in the step S1, carrying out hydrothermal reaction for 24-72h at 70 ℃.
By adopting the technical scheme, the CQDs and the anhydrous ethylenediamine are uniformly mixed and then subjected to hydrothermal reaction at 70 ℃, so that the reaction speed is high and the mixing is uniform.
The invention is further configured to: adding Ca (NO) in the step S2 3 ) 2 ·4H 2 Before O, diluting N-CQDs with pure waterAnd (5) releasing.
By adopting the technical scheme, the carbon quantum dots (N-CQDs) modified by the ethylenediamine are diluted by pure water, so that the diluted carbon quantum dots (N-CQDs) modified by the ethylenediamine and Ca (NO) are mixed 3 ) 2 ·4H 2 The O is mixed more uniformly and the reaction speed is higher.
The invention is further configured to: adding Ca (NO) in the step S2 3 ) 2 ·4H 2 O, adjusting the pH of the solution to pH =8-13.
By adopting the technical scheme, the pH of the solution is adjusted to be 8-13, so that the solution is in an alkaline state, the epoxy group of the 3- (2,3-glycidoxy) propyl trimethoxy silane and the hydroxyl group of the mannose easily form a stable ether bond, and the mannose can be combined on hydroxyapatite by a stable chemical bond, so that the mannose has stable targeting property, and the effect of treating diseases is better.
The invention is further configured to: and in the step S2, when the solution B is dropwise added to the solution A, stirring is required while dropwise adding.
Through adopting above-mentioned technical scheme, through dropwise drip B liquid when adding A liquid, the limit is dripped the limit and is stirred, increases the area of contact of B liquid and A liquid for B liquid and A liquid reaction are comparatively even, and then make B liquid and A liquid reaction comparatively abundant, have accelerated the speed of reaction.
The invention is further configured to: the calcining temperature in the step S2 is 350-650 ℃, and the calcining time is 3-8h.
By adopting the technical scheme, the calcination temperature is 350-650 ℃ and the calcination time is 3-8h, so that the mesoporous spherical Hydroxyapatite (HA) HAs better quality, the structure of the mesoporous spherical Hydroxyapatite (HA) is more stable, and better functions are achieved.
The invention is further configured to: and (3) adding 3- (2,3-glycidoxy) propyl trimethoxy silane (GTS) in the step S3, and reacting for 2-6h at 80 ℃.
By adopting the technical scheme, after 3- (2,3-glycidoxy) propyltrimethoxysilane (GTS) is added, the reaction is carried out for 2-6h at 80 ℃, the mixing speed is promoted, so that the 3- (2,3-glycidoxy) propyltrimethoxysilane (GTS) is better dissolved in toluene, and the 3- (2,3-glycidoxy) propyltrimethoxysilane (GTS) is more fully hydrolyzed.
The invention is further configured to: and step S4, adding mannose, and reacting at 90 ℃ under the protection of nitrogen.
By adopting the technical scheme, after the mannose is added, the reaction is carried out at 90 ℃ under the protection of nitrogen, so that the reaction of the raw materials in the solution is stable, the raw materials in the solution are protected, and the reaction rate is accelerated.
In view of the defects of the prior art, the third object of the present invention is to provide a method for constructing a nano drug delivery system, which comprises the following steps:
(1) Adding curcumin solution into a tumor targeting carrier HA-GTS-Man, and carrying out ultrasonic treatment for 5-20min to form a premix;
(2) Stirring the premix in the dark, centrifugally washing and collecting supernatant;
(3) And detecting the absorbance of the supernatant.
Through adopting above-mentioned technical scheme, through carrying out the light-resistant stirring with the premix, reduce the condition that light influences the premix, avoid the condition that the premix dissolves under the light irradiation condition, guaranteed the quality and the effect of premix for the premix has the stability of preferred.
In conclusion, the invention has the following beneficial effects:
1. according to the targeting type drug carrier based on the nano-hydroxyapatite, the targeting type drug carrier has better quality, the steps of preparing the targeting type drug carrier are reduced, the cost of an experiment is lower, and meanwhile, the effect of treating tumors by the targeting type drug carrier is better;
2. by modifying 3- (2,3-epoxypropoxy) propyltrimethoxysilane (GTS) on Hydroxyapatite (HA) in a toluene solvent, the surface modification range of 3- (2,3-epoxypropoxy) propyltrimethoxysilane (GTS) on Hydroxyapatite (HA) is increased, so that the application range of 3- (2,3-epoxypropoxy) propyltrimethoxysilane (GTS) is wider;
3. when dropwise adding the B liquid to the A liquid, the stirring is carried out while the B liquid is dropwise added, the contact area of the B liquid and the A liquid is increased, the B liquid and the A liquid are enabled to react uniformly, the B liquid and the A liquid are enabled to react sufficiently, and the reaction speed is accelerated.
Drawings
FIG. 1 is a synthesis diagram of HA-GTS-Man;
FIG. 2 is an XRD spectrum of HA-GTS-Man in examples 1 to 4;
FIG. 3 is a FT-IR spectrum of HA-GTS-Man in examples 1 to 4;
FIG. 4 is the drug release profile of Curr @ HA-GTS-Man;
FIG. 5 shows a TEM image of HA-GTS-Man.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples.
In the invention, citric acid is C805019 product number citric acid sold by Shanghai Michelin Biotechnology Limited;
in the invention, 3- (2,3-glycidoxy) propyl trimethoxy silane (GTS) is gamma-glycidoxypropyl trimethoxy silane sold by Shanghai Michelin Biotechnology, inc.;
in the present invention, mannose (Man) is available from Shanghai Maxin Biochemical technology Ltd;
in the invention, the disodium hydrogen phosphate is sold by Shanghai Maxin Biochemical technology Limited;
in the invention, the sodium hydroxide is sodium hydroxide sold by Shanghai Maxlin Biochemical technology Co., ltd;
in the present invention, N, N-Dimethylformamide (DMF) available from Tianjin Dalochi chemical Co., ltd is used as N, N-Dimethylformamide (DMF);
in the invention, the anhydrous ethylenediamine is sold by Shanghai Lengfeng chemical company Limited;
in the present invention, toluene sold by Guangzhou chemical reagent works is used as toluene.
Example 1
A targeting type drug carrier based on nano hydroxyapatite comprises the following steps:
s1, adding 30mmol of citric acid into a stirring kettle, dissolving the citric acid in 280mL of pure water, carrying out hydrothermal reaction for 3h at 180 ℃, dialyzing the mixture for 24h in pure water by using a dialysis bag (Mw: 500 Da) to obtain Carbon Quantum Dots (CQDs), then adding 4g of anhydrous ethylenediamine, uniformly mixing, stirring at the rotation speed of 80r/min for 10min in the stirring kettle, carrying out hydrothermal reaction for 3h at 70 ℃, dialyzing in pure water by using a dialysis bag (Mw: 500 Da) for 24h, and freeze-drying to obtain the ethylenediamine modified carbon quantum dots (N-CQDs);
s2. 20mg of ethylenediamine-modified carbon quantum dots (N-CQDs) were diluted to 90mL with pure water to form a diluted solution, and 4mmol of Ca (NO) was added to the diluted solution 3 ) 2 ·4H 2 Mixing O, adjusting the pH value of the diluent to 8, stirring uniformly to form solution A, stirring at the rotating speed of 85r/min for 15min in a stirring kettle, adding 2.4mmol of disodium hydrogen phosphate into another stirring kettle, dissolving in 20mL of pure water, stirring uniformly to form solution B, and stirring at the rotating speed of 75r/min for 12min in the stirring kettle; dropwise adding the solution B into the solution A while stirring, aging at room temperature of 37 ℃ for 6h after stirring, centrifuging the precipitate, washing with water for several times, drying at 60 ℃, and calcining at 350 ℃ for 3h to form mesoporous quasi-spherical Hydroxyapatite (HA);
s3, ultrasonically dispersing 0.2g of mesoporous sphere-like Hydroxyapatite (HA) into 100mL of toluene, adding 2mL of 3- (2,3-glycidoxy) propyl trimethoxy silane (GTS), reacting for 2h at 80 ℃, washing for several times by using toluene, and drying at 50 ℃ to form HA-GTS;
s4, ultrasonically dispersing 0.5g of HA-GTS in 100mL of N, N-Dimethylformamide (DMF), adding 0.8g of mannose, uniformly stirring, adding 0.4g of NaOH, reacting at 90 ℃ for 6 hours under the protection of nitrogen, sequentially washing with DMF, water and ethanol, and drying at 50 ℃ to form the tumor targeting carrier HA-GTS-Man.
Example 2
A targeting type drug carrier based on nano hydroxyapatite comprises the following steps:
s1, adding 65mmol of citric acid into a stirring kettle, dissolving the citric acid in 280mL of pure water, carrying out hydrothermal reaction for 7.5h at 180 ℃, dialyzing the mixture for 48h in pure water by using a dialysis bag (Mw: 500 Da) to obtain Carbon Quantum Dots (CQDs), then adding 4g of anhydrous ethylenediamine, uniformly mixing, stirring the mixture at the rotation speed of 80r/min in the stirring kettle for 10min, carrying out hydrothermal reaction for 5.5h at 70 ℃, dialyzing the mixture for 48h in pure water by using the dialysis bag (Mw: 500 Da), and freeze-drying to obtain the ethylenediamine modified carbon quantum dots (N-CQDs);
s2, diluting 70mg of ethylenediamine modified carbon quantum dots (N-CQDs) to 90mL with pure water to form a diluted solution, and adding 4mmol of Ca (NO) to the diluted solution 3 ) 2 ·4H 2 Mixing O, adjusting the pH value of the diluent to 10.5, uniformly stirring to form solution A, stirring for 15min at the rotation speed of 85r/min in another stirring kettle, adding 2.4mmol of disodium hydrogen phosphate into the other stirring kettle, dissolving in 20mL of pure water, uniformly stirring to form solution B, stirring at the rotation speed of 75r/min in the other stirring kettle for 12min; dropwise adding the solution B into the solution A while stirring, aging at room temperature of 37 ℃ for 15h after stirring, centrifuging the precipitate, washing with water for several times, drying at 60 ℃, and calcining at 500 ℃ for 5.5h to form mesoporous spherical Hydroxyapatite (HA);
s3, ultrasonically dispersing 0.6g of mesoporous sphere-like Hydroxyapatite (HA) in 100mL of toluene, adding 6mL of 3- (2,3-glycidoxy) propyl trimethoxy silane (GTS), reacting for 4h at 80 ℃, washing for several times by using the toluene, and drying at 50 ℃ to form HA-GTS;
s4, ultrasonically dispersing 0.5g of HA-GTS in 100mL of N, N-Dimethylformamide (DMF), adding 2.8g of mannose, uniformly stirring, adding 0.4g of NaOH, reacting at 90 ℃ for 15 hours under the protection of nitrogen, sequentially washing with DMF, water and ethanol, and drying at 50 ℃ to form the tumor targeting vector HA-GTS-Man.
Example 3
A targeting type drug carrier based on nano hydroxyapatite comprises the following steps:
s1, adding 100mmol of citric acid into a stirring kettle, dissolving the citric acid in 280mL of pure water, carrying out hydrothermal reaction for 12h at 180 ℃, dialyzing the mixture for 72h in pure water by using a dialysis bag (Mw: 500 Da) to obtain Carbon Quantum Dots (CQDs), then adding 4g of anhydrous ethylenediamine, uniformly mixing, stirring at the rotation speed of 80r/min in the stirring kettle for 10min, carrying out hydrothermal reaction for 8h at 70 ℃, dialyzing the mixture for 72h in pure water by using a dialysis bag (Mw: 500 Da), and freeze-drying to obtain the ethylenediamine modified carbon quantum dots (N-CQDs);
s2, diluting 120mg of ethylenediamine modified carbon quantum dots (N-CQDs) to 90mL with pure water to form a diluted solution, and adding 4mmol of Ca (NO) to the diluted solution 3 ) 2 ·4H 2 Mixing O, adjusting the pH value of the diluent to 13, stirring uniformly to form solution A, stirring at the rotating speed of 85r/min for 15min in a stirring kettle, adding 2.4mmol of disodium hydrogen phosphate into another stirring kettle, dissolving in 20mL of pure water, stirring uniformly to form solution B, and stirring at the rotating speed of 75r/min for 12min in the stirring kettle; dropwise adding the solution B into the solution A while stirring, aging at room temperature of 37 ℃ for 24h after stirring, centrifuging the precipitate, washing with water for several times, drying at 60 ℃, and calcining at 650 ℃ for 8h to form mesoporous quasi-spherical Hydroxyapatite (HA);
s3, ultrasonically dispersing 1.0g of mesoporous sphere-like Hydroxyapatite (HA) into 100mL of toluene, adding 10mL of 3- (2,3-glycidoxy) propyl trimethoxysilane (GTS), reacting for 6h at 80 ℃, washing for several times by using toluene, and drying at 50 ℃ to form HA-GTS;
s4, ultrasonically dispersing 0.5g of HA-GTS in 100mL of N, N-Dimethylformamide (DMF), adding 4.8g of mannose, uniformly stirring, adding 0.4g of NaOH, reacting at 90 ℃ for 24 hours under the protection of nitrogen, sequentially washing with DMF, water and ethanol, and drying at 50 ℃ to form the tumor targeting vector HA-GTS-Man.
Example 4
A targeting drug carrier based on nano hydroxyapatite comprises the following steps:
s1, adding 88mmol of citric acid into a stirring kettle, dissolving in 280mL of pure water, carrying out hydrothermal reaction for 5h at 180 ℃, dialyzing in pure water by using a dialysis bag (Mw: 500 Da) for 24h to obtain Carbon Quantum Dots (CQDs), then adding 4g of anhydrous ethylenediamine, uniformly mixing, stirring at the rotation speed of 80r/min for 10min in the stirring kettle, carrying out hydrothermal reaction for 5h at 70 ℃, dialyzing in pure water by using a dialysis bag (Mw: 500 Da) for 24h, and freeze-drying to obtain the ethylenediamine modified carbon quantum dots (N-CQDs);
s2, diluting 50mg of ethylenediamine modified carbon quantum dots (N-CQDs) with pure waterTo 90mL to form a dilution, 4mmol Ca (NO) was added to the dilution 3 ) 2 ·4H 2 Mixing O, adjusting the pH value of the diluent to 12, stirring uniformly to form solution A, stirring at the rotating speed of 85r/min for 15min in a stirring kettle, adding 2.4mmol of disodium hydrogen phosphate into another stirring kettle, dissolving in 20mL of pure water, stirring uniformly to form solution B, and stirring at the rotating speed of 75r/min for 12min in the stirring kettle; dropwise adding the solution B into the solution A while stirring, aging at room temperature of 37 ℃ for 12h after stirring, centrifuging the precipitate, washing with water for several times, drying at 60 ℃, and calcining at 500 ℃ for 6h to form mesoporous sphere-like Hydroxyapatite (HA);
s3, ultrasonically dispersing 0.5g of mesoporous sphere-like Hydroxyapatite (HA) in 100mL of toluene, adding 8mL of 3- (2,3-epoxypropoxy) propyl trimethoxy silane (GTS), reacting at 80 ℃ for 6h, washing with toluene for several times, and drying at 50 ℃ to form HA-GTS;
s4, ultrasonically dispersing 0.5g of HA-GTS in 100mL of N, N-Dimethylformamide (DMF), adding 1.6g of mannose, uniformly stirring, adding 0.4g of NaOH, reacting at 90 ℃ for 12 hours under the protection of nitrogen, sequentially washing with DMF, water and ethanol, and drying at 50 ℃ to form the tumor targeting vector HA-GTS-Man (shown in figure 5).
Examples 1-4, the structure of HA-GTS-Man was analyzed by X-ray diffractometer (FIG. 2) and Fourier transform infrared spectroscopy (FIG. 3).
Example 5
A method for constructing a nano drug delivery system comprises the following steps:
(1) Placing 50mg of tumor targeting vector HA-GTS-Man in a test tube, adding 5mL (1 mg/mL) of curcumin solution, and performing ultrasonic treatment for 5-20min to form a premix;
(2) Stirring the premix for 4-12 h at 37 ℃ in a dark condition, centrifuging, washing with ethanol, and collecting precipitate and supernatant;
(3) The absorbance of the supernatant at 426nm was measured.
Example 6
A targeting evaluation of a nano-drug delivery system comprises the following steps:
a. 20mg of Cur @ HA-GTS-Man was placed in a dialysis bag (MW: 8k Da), and 3mL of PBS buffer was added;
b. then placing the dialysis bag in 30mL PBS buffer solution with pH =5.5 and containing 0.5% Tween 80, and stirring at 37 ℃ in a dark place;
c. at the predetermined time point, 5mL of solution was aspirated, and 5mL of fresh PBS was replenished, and the test results showed significant environmental responsiveness (see figure 4 for details).
Example 7
A targeting evaluation of a nano drug delivery system comprises the following steps:
a. placing 20mg Cur @ HA-GTS-Man in a dialysis bag (MW: 8k Da), and adding 3mL of PBS buffer solution;
b. then the dialysis bag is placed in 30mL buffer solution containing 0.5% Tween 80 and having pH =7.4PBS, and stirred at 37 ℃ in the dark;
c. at the scheduled time point, 5mL of solution was aspirated, and 5mL of fresh PBS was replenished, and the test results showed significant environmental responsiveness (see figure 4 for details).
Experiment: experiment for inhibiting tumor cell growth in mice
Performing experiments on HELA cell strains by adopting a tetrazolium salt reduction Method (MTT), selecting HELA cells for conventional culture, performing conventional culture until the cells are expanded to a certain number and have good growth state, digesting the HELA cells in logarithmic growth phase with 0.25% pancreatin for 2min, centrifuging at l000rpm for 5min, discarding supernatant, and resuspending cell precipitates with a fresh serum-free DMEM medium to prepare a cell suspension of 1 × 106 cells/mL. Then, the cell suspension was aspirated by a syringe, vacuoles were removed, and the cell suspension was injected into the subcutaneous tissue of the left axilla of BALB/C female nude mice weighing about 20-22g, each of which was inoculated with 100. Mu.L of about 1X 105 cells, for a total of about 120 cells. The growth of the tumor of the nude mice is observed, the nude mice are weighed regularly, and the tumor size is measured. (animals normally consumed their diets during the experiment.
When the tumor volume was 100 to 150mm3, the nude mice were randomly divided into 5 groups of 10 nude mice (n = 4) each, negative control group (saline group), tumor-targeting vector HA-GTS-Man (example 1), tumor-targeting vector HA-GTS-Man (example 2), tumor-targeting vector HA-GTS-Man (example 3), and tumor-targeting vector HA-GTS-Man (example 4), administered every other day, mice were normally bred after administration, the survival status of mice was observed every day, the body weight of mice was recorded every two days, the length and the short diameter of the tumor of mice were measured with a vernier caliper, and the tumor volume (V) and the relative tumor volume were calculated according to the formulas.
The administration is carried out once every other day, the mice are normally raised after the administration, the survival condition of the mice is observed every day, the weight of the mice is recorded every two days, the length and the short diameter of the tumor of the mice are measured by a vernier caliper, and the tumor volume (V) and the relative tumor volume are calculated according to a formula.
Calculating the tumor inhibition rate of each administration group:
Tumor inhibiton ratio％＝(1─Vtest/Vcontrol)×100％
wherein Vtest is the relative tumor volume of the administration group at day 12, vcontrol is the relative tumor volume of the physiological saline group at day 12, and the tumor inhibition rate is shown in Table 1.
TABLE 1
| The tumor inhibition rate% | |
| Example 1 | 57.75 |
| Example 2 | 58.11 |
| Example 3 | 57.98 |
| Example 4 | 59.10 |
| |
0 |
As can be seen from Table 1, the tumor targeting vector HA-GTS-Man HAs better quality, and the tumor targeting vector HA-GTS-Man of the embodiment 4 HAs better quality, so that the effect of the targeting drug carrier on treating the tumor is better;
as can be seen from fig. 4, in the buffer solution of PBS with pH =7.4, the n value is lower than 0.5, indicating that the release mechanism is mainly Fick diffusion;
in pH5.5, 0.5-n-0.89 were constructed, indicating that the release mechanism was mainly non-Fick diffusion, and that the drug was released at a constant rate (or ratio).
As can be seen from fig. 5:
(a) The HA-GTS-Man prepared by the method HAs a spherical shape, and the average particle size is less than 20nm;
(b) Inside it, there is a quantum dot N-CQDs core (about 4 nm) as a "nucleation template".
The embodiments of the present invention are preferred embodiments of the present invention, and the scope of the present invention is not limited by these embodiments, so: equivalent changes made according to the structure, shape and principle of the invention shall be covered by the protection scope of the invention.
Claims (7)
1. A targeting type drug carrier based on nano hydroxyapatite is characterized in that: the molecular formula of the targeting drug carrier is
Man, structural formula:
the preparation method of the targeted drug carrier comprises the following steps:
s1, dissolving citric acid in water to obtain CQDs, and uniformly mixing the CQDs with anhydrous ethylenediamine to obtain an ethylenediamine modified carbon quantum point N-CQDs;
s2, adding N-CQDs and Ca (NO) into the ethylene diamine modified carbon quantum dots 3 ) 2 ·4H 2 Mixing O to form solution A, adding Na 2 HPO 4 Dissolving in pure water to form solution B; dripping the B liquid into the A liquid, stirring, precipitating, centrifuging, drying and calcining to form mesoporous sphere-like hydroxyapatite HA;
s3, ultrasonically dispersing the mesoporous quasi-spherical hydroxyapatite HA into toluene, and adding 3- (2,3-glycidoxy) propyltrimethoxysilane GTS to form HA-GTS;
s4, ultrasonically dispersing HA-GTS in an organic solvent, adding mannose to modify HA-GTS, and uniformly stirring to form a tumor targeting vector HA-GTS-Man;
adding Ca (NO) in the step S2 3 ) 2 ·4H 2 O, adjusting the pH of the solution to pH =8-13;
the calcining temperature in the step S2 is 350-650 ℃, and the calcining time is 3-8h.
2. The targeted drug carrier of claim 1, wherein: after the CQDs and the anhydrous ethylenediamine are uniformly mixed in the step S1, a hydrothermal reaction is carried out at 70 ℃ for 24-72h.
3. The targeted drug carrier of claim 1, wherein: adding Ca (NO) in the step S2 3 ) 2 ·4H 2 Before O, N-CQDs were diluted with water.
4. The targeted drug carrier of claim 1, wherein: and in the step S2, the solution B is dropwise added to the solution A, and stirring is required to be carried out while dropwise adding.
5. The targeted drug carrier of claim 1, wherein: and (3) adding 3- (2,3-glycidoxy) propyl trimethoxysilane GTS in the step S3, and reacting at 80 ℃ for 2-6h.
6. The targeted drug carrier of claim 1, wherein: and after adding mannose in the step S4, reacting at 90 ℃ under the protection of nitrogen.
7. A method for constructing the targeted drug carrier according to any one of claims 1 to 6, wherein: the method comprises the following steps:
(1) Adding curcumin solution into tumor targeting carrier HA-GTS-Man, and performing ultrasonic treatment for 5-20min to form a premix;
(2) Stirring the premix in the dark, centrifugally washing and collecting supernatant;
(3) And detecting the absorbance of the supernatant.
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