CN116082536B - Polymer with bone targeting function, preparation method and application thereof, nano-carrier and preparation method thereof - Google Patents

Polymer with bone targeting function, preparation method and application thereof, nano-carrier and preparation method thereof Download PDF

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CN116082536B
CN116082536B CN202310124741.XA CN202310124741A CN116082536B CN 116082536 B CN116082536 B CN 116082536B CN 202310124741 A CN202310124741 A CN 202310124741A CN 116082536 B CN116082536 B CN 116082536B
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CN116082536A (en
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杨婵珍
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Guangzhou Huiyuan Pharmaceutical Technology Co ltd
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Abstract

The application discloses a polymer with a bone targeting function, a preparation method and application thereof, a nano carrier and a preparation method thereof, and relates to the technical field of pharmaceutical preparations; the polymer provided by the application has a bone targeting function, and the polymer is used as a stabilizer to prepare the nano-carrier, so that the stability of vitamin D 3 can be obviously improved through the inclusion effect of cyclodextrin on one hand, and on the other hand, the purpose of bone targeting delivery to improve the enrichment of the medicine at a bone part can be achieved through the strong affinity of bisphosphonate and intra-bone hydroxyapatite, the co-delivery of bisphosphonate, calcium and vitamin D 3 can be realized, and the patient compliance is improved.

Description

Polymer with bone targeting function, preparation method and application thereof, nano-carrier and preparation method thereof
Technical Field
The application relates to the technical field of pharmaceutical preparations, in particular to a polymer with a bone targeting function, a preparation method and application thereof, a nano carrier and a preparation method thereof.
Background
Osteoporosis is a disease of systemic bone metabolic disorder in which bone tissue microstructure is impaired, bone mineral components and bone matrix are continuously reduced in equal proportion, bone fragility is increased, and fracture risk is increased.
At present, the clinical medicines for treating osteoporosis are various in variety, and the common medicines are mainly calcium agents, estrogens, bisphosphonates, vitamins, active analogues thereof and the like. Wherein, calcium can promote bone formation, maintain bone strength, and relieve osteoporosis, but is insufficient to prevent bone loss, and excessive calcium supplement can also risk hypercalcemia. The bisphosphonate is an amino bisphosphonate bone resorption inhibitor, has strong affinity with the hydroxyapatite in bone, can enter into bone matrix hydroxyapatite crystal, can inhibit the activity of osteoclast, indirectly plays a role in inhibiting bone resorption through osteoblast, has the characteristics of strong bone resorption resisting activity and no bone mineralization inhibiting effect, and is a first-line osteoporosis treatment drug. However, bisphosphonate drugs have an early bone resorption inhibiting effect, resulting in hypocalcemia, especially in patients lacking vitamin D 3, which is extremely pronounced. Calcium required for new bone formation during treatment is supplemented from food or calcium agents, while vitamin D 3 can promote calcium absorption in the intestinal tract and increase blood calcium concentration, just against this side effect of bisphosphonates. Therefore, it is necessary for the osteoporosis patient to supplement the appropriate amount of vitamin D 3 and calcium while taking bisphosphonates. However, it should be noted that vitamin D 3 is a high active substance, the specification of the oral preparation containing vitamin D 3 is very small, and the uniformity of the content of vitamin D 3 in the preparation is difficult to ensure. Vitamin D 3 is unstable to light, oxygen, and high temperatures, and thus, preparing a qualified oral formulation containing vitamin D 3 is quite challenging.
At present, compound preparations of bisphosphonate and vitamin D 3 are sold in the market, but research on the combination of bisphosphonate, vitamin D 3 and calcium is lacking, and the bisphosphonate, the vitamin D and the calcium are prepared into a stable compound preparation, so that side effects generated by taking the bisphosphonate can be relieved, the absorption of calcium can be promoted, the osteoporosis symptom can be prevented and treated, the compound preparation can be conveniently used by patients, the toxic and side effects are small, the safety is high, and the compound preparation has broad market prospect and huge market development potential. Pierce et al in 1986 have clearly proposed the concept of bone targeting, i.e. the coupling of drugs to bone targeting carriers, which makes the compound molecules have a tendency to deposit on bone and blend into hydroxyapatite crystals, have binding capacity with bone calcium, and increase the drug concentration in local bone tissue. Bisphosphonates are also commonly used in the study of bone targeting vectors because of their bone philic bisphosphonate head group. Therefore, if the bone targeting effect of the bisphosphonate is used in researching the combination of the bisphosphonate, the vitamin D 3 and the calcium agent, the application prospect of the preparation is further increased.
Disclosure of Invention
In order to overcome the defects of the prior art, the application provides a polymer with a bone targeting function, a preparation method and application thereof, a nano-carrier and a preparation method thereof, the polymer provided by the application has the bone targeting function, and the nano-carrier is prepared by taking the polymer as a stabilizer, wherein the nano-carrier can obviously improve the stability of vitamin D 3 through the inclusion effect of cyclodextrin on one hand, and can achieve the purpose of bone targeting delivery through the strong affinity of bisphosphonate and intraosseous hydroxyapatite to improve the enrichment of medicines at bone parts on the other hand, so that the co-delivery of bisphosphonate, calcium and vitamin D 3 can be realized, and the patient compliance is improved.
In order to achieve the above purpose, the present application proposes the following technical solutions:
In a first aspect, the present application provides a polymer having a bone targeting function, the polymer having a structural formula as shown in formula I:
Wherein n <25 and n is an integer.
As a preferred embodiment, the polymer of the application has a molecular weight of <10kDa.
The polymer (sodium hyaluronate-alendronate sodium graft polymer) provided by the application has a bone targeting function.
In a second aspect, the present application provides a method for preparing a polymer having a bone targeting function according to the first aspect, the method comprising:
(1) Dissolving hyaluronic acid, 1-ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide in a phosphate buffer salt solution, carrying out ice bath reaction, and transferring to room temperature for reaction to obtain a solution A;
(2) Dissolving bisphosphonate in sodium hydroxide solution, adding water, adjusting pH to be alkaline after the bisphosphonate is completely dissolved, and placing the solution on ice bath for stirring to obtain solution B;
(3) And (3) dropwise adding the solution A into the solution B, stirring the reaction solution in an ice bath, transferring the reaction solution into a constant-temperature water bath, stirring the reaction solution, and freeze-drying, separating and purifying the reaction solution after the reaction is finished to obtain the polymer.
As a preferred embodiment, the polymer of the application has a molecular weight of <10kDa. Because the molecular weight of the polymer is too large to be beneficial to the reaction, the application directly selects the hyaluronic acid with the molecular weight smaller than 10kDa as a reaction raw material, the hyaluronic acid is a polymer composed of disaccharides, the molecular weight of the disaccharides is about 400, and if the molecular weight of the hyaluronic acid is controlled to be less than 10kDa, n is less than 25.
As a preferred technical scheme, the separation and purification steps include:
re-dissolving the freeze-dried reaction solution in acetone, centrifuging to remove suspended matters in the solution, and drying with nitrogen to obtain light yellow oily matter;
Re-dissolving the light yellow oily matter in water, and performing pre-freezing and freeze-drying treatment after dialysis to obtain the polymer.
As a preferable technical scheme, in the step (1), the temperature of the ice bath reaction is 0-5 ℃, preferably 0-3 ℃, and more preferably 0 ℃;
The reaction time of the ice bath is 20-60 min, preferably 25-40 min, more preferably 30min;
The reaction time at room temperature is 20 to 60 minutes, preferably 25 to 40 minutes, more preferably 30 minutes.
Preferably, the alkaline pH is 7 to 10, preferably 7 to 9, more preferably 7 to 8.
As a preferable technical scheme, in the step (3), the temperature of the ice bath stirring is 0-5 ℃, preferably 0-3 ℃, and more preferably 0 ℃; the stirring time of the ice bath is 0.5-3 h, preferably 0.5-2 h, more preferably 1h; the temperature of the constant-temperature water bath is 35-40 ℃, preferably 37 ℃; the stirring time of the constant-temperature water bath is more than or equal to 24 hours.
In a preferred technical scheme, in the step (3), the freeze-drying temperature is-90 to-70 ℃, preferably-80 ℃.
In the present application, the dialysis step may employ dialysis means commonly used in the art, specifically, for example:
Redissolving the pale yellow oil in water, filling a dialysis bag (mw=5000 daltons) and dialyzing in a large amount of water for 5 days, wherein water is changed 3-5 times per day; pre-freezing the dialyzate at-80 ℃ overnight, and freeze-drying to obtain white flocculent polymer.
In the present application, the water is preferably ultrapure water.
In a third aspect, the present application provides the use of a polymer having bone targeting function as described in the first aspect in the manufacture of a medicament for the treatment of osteoporosis.
In a fourth aspect, the present application also provides a nanocarrier for treating osteoporosis, the nanocarrier comprising an inner core and a stabilizer coated on the surface of the inner core;
Wherein the inner core takes calcium ions as a metal center, and cyclodextrin containing Lewis acid end groups and containing vitamin D 3 is taken as an organic ligand;
the stabilizer is the polymer of the first aspect.
Referring specifically to fig. 1, in fig. 1, CD is cyclodextrin. According to the application, the polymer is used as a stabilizer to prepare the nano-carrier, the stability of vitamin D 3 can be obviously improved through the inclusion effect of cyclodextrin on one hand, and on the other hand, the purpose of improving the enrichment of the medicine at a bone part can be achieved through the strong affinity of bisphosphonate and intraosseous hydroxyapatite to achieve bone targeted delivery, so that the co-delivery of bisphosphonate, calcium and vitamin D3 can be realized, and the patient compliance can be improved.
As described above, the nanocarriers of the present application can be used for preparing related drugs for treating osteoporosis.
In a fifth aspect, the present application also provides a method for preparing a nanocarrier for treating osteoporosis according to the fourth aspect, the method comprising:
(1) Dissolving water-in-oil surfactant and cyclohexane in hexanol to prepare an oil phase;
(2) Dissolving a calcium source in water to obtain a calcium solution;
(3) Dissolving vitamin D 3 in ethanol, dissolving cyclodextrin containing Lewis acid end groups in water, slowly dripping vitamin D 3 ethanol solution into the cyclodextrin solution, and uniformly stirring to obtain VD 3 -CD inclusion solution;
(4) Dissolving a polymer in ultrapure water to obtain a polymer solution;
(5) Dropwise adding a calcium solution and a VD 3 -CD inclusion solution into the oil phase, stirring for reaction, then dropwise adding a polymer solution, and continuing stirring for reaction to obtain nano-carrier microemulsion;
(6) Adding ethanol into the nano-carrier microemulsion, stirring, demulsifying, centrifuging, washing, purifying, and dispersing with purified water to obtain a nano-carrier solution;
(7) And freeze-drying the nano-carrier solution to obtain the nano-carrier.
As a preferable technical scheme, the water-in-oil type surfactant is at least one selected from sorbitan stearate, glyceryl monostearate, polyoxyethylene castor oil and triton; preferably triton X-100.
Preferably, the calcium source is at least one selected from calcium chloride, tricalcium phosphate, calcium lactate, calcium acetate, monocalcium phosphate and calcium hydrogen phosphate, preferably calcium chloride.
As a preferred technical scheme, the cyclodextrin containing the lewis acid end group is at least one selected from carboxymethyl betacyclodextrin, sulfobutyl betacyclodextrin and phosphobetacyclodextrin, preferably carboxymethyl betacyclodextrin.
As a preferred embodiment, the molar ratio of cyclodextrin containing lewis acid end groups to vitamin D 3 is greater than 300:1, preferably 1000:1.
In the present application, the above washing purification step may employ washing purification means commonly used in the art, specifically, for example:
washing and purifying with ethanol/tetrahydrofuran solution for 2 times; preferably, the volume ratio of ethanol/tetrahydrofuran solution is 1:1.
In a preferred technical scheme, in the step (7), the freeze-drying temperature is-90 to-70 ℃, preferably-80 ℃.
Compared with the prior art, the application has the following beneficial effects:
The polymer provided by the application has a bone targeting function, and the polymer is used as a stabilizer to prepare the nano-carrier, so that the stability of vitamin D 3 can be obviously improved through the inclusion effect of cyclodextrin on one hand, and on the other hand, the purpose of bone targeting delivery to improve the enrichment of the medicine at a bone part can be achieved through the strong affinity of bisphosphonate and intra-bone hydroxyapatite, the co-delivery of bisphosphonate, calcium and vitamin D 3 can be realized, and the patient compliance is improved.
Additional aspects and advantages of the application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the application.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and constitute a part of this specification, are incorporated in and constitute a part of this specification and do not limit the application in any way, and in which:
FIG. 1 is a schematic diagram of the ALN-HA-CaCD according to the present application;
FIG. 2 is a schematic diagram showing the results of in vitro hydroxyapatite powder (HAP) adsorption experiments of ALN-HA-CaCD and CaCD in example 3;
FIG. 3 is a schematic diagram showing the results of in vitro bone tissue adsorption experiments of ALN-HA-CaCD and CaCD in example 4.
Detailed Description
The technical solutions in the embodiments will be clearly and completely described below with reference to the embodiments of the present application and the accompanying drawings. It will be apparent that the embodiments described below are only some, but not all, embodiments of the application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
It should be understood that the terms "comprises" and "comprising," when used in this specification and the appended claims, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.
It is also to be understood that the terminology used in the description of the embodiments of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of embodiments of the application. As used in the specification of the embodiments of the application and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
In the following examples, (ALN-HA) is a polymer with bone targeting function, and (ALN-HA-CaCD) is a nanocarrier.
Example 1
Synthesis of sodium hyaluronate-alendronate sodium graft Polymer (ALN-HA)
(1) Hyaluronic acid (1 mmol, molecular weight <10 kDa), 1-ethyl- (3-dimethylaminopropyl) carbodiimide (1.5 mol) and N-hydroxysuccinimide (1.5 mol) were dissolved in 15mL PBS (0.02M, pH 5.7), reacted for 30 minutes in an ice bath, and then transferred to room temperature for 30 minutes to obtain solution A.
(2) Sodium alendronate (1 mol) was dissolved in 2M NaOH (10 mL), ultrapure water (8 mL) was added thereto, and after the sodium alendronate was completely dissolved, 1M NaOH was adjusted to pH 8, and the mixture was stirred on an ice bath to obtain solution B.
(3) Solution A was slowly added dropwise to solution B, during which time the pH was maintained at 7-8 with 1M NaOH. After the addition, the reaction solution is placed in an ice bath for stirring for 1h, and then is transferred to a 37 ℃ water bath for stirring overnight (more than or equal to 24 h). After the reaction is finished, the reaction solution is placed at the temperature of minus 80 ℃ for pre-freezing overnight, and is treated after freeze-drying.
(4) The lyophilized reaction solution was redissolved in acetone, and after the suspension in the solution was removed by centrifugation (5,000 rpm,15 min), the yellowish oily substance was obtained by blow-drying with nitrogen. The pale yellow oil was redissolved in a small amount of ultrapure water, filled into dialysis bags (mw=5000 daltons) and dialyzed in a large amount of ultrapure water for 5 days, during which water was changed 3-5 times per day. Pre-freezing the dialyzate at-80 ℃ overnight, and freeze-drying to obtain white flocculent ALN-HA pure product.
Example 2
Construction of vitamin D 3 -loaded cyclodextrin-calcium coordination nanoparticle (ALN-HA-CaCD)
(1) Dissolving triton X-100 and cyclohexane in hexanol to prepare an oil phase;
(2) 0.3g of calcium chloride was dissolved in 2ml of ultrapure water to obtain a calcium chloride solution;
(3) Dissolving vitamin D 3 mug in 0.5ml of ethanol, dissolving carboxymethyl cyclodextrin 0.3g in 3ml of ultrapure water, slowly dripping the vitamin D 3 ethanol solution into the cyclodextrin solution, and stirring to obtain VD 3 -CD inclusion solution;
(4) Dissolving ALN-HA0.01g in 5ml of ultrapure water to obtain an ALN-HA solution;
(5) Sequentially dripping 500 mu l of calcium chloride solution and 500 mu l of VD 3 -CD inclusion solution into 5ml of the oil phase prepared in the step (1), stirring and reacting for 12 hours, dripping 250 mu l of ALN-HA solution, and continuously stirring and reacting for 12 hours to obtain ALN-HA-CaCD nanoparticle microemulsion;
(6) Adding 10ml of ethanol into the microemulsion, slowly stirring for demulsification, centrifuging at 20000rpm/15min, removing supernatant, washing the lower layer solid with ethanol/tetrahydrofuran solution (v/v, 1:1), purifying for 2 times, and dispersing with purified water after purification to obtain ALN-HA-CaCD nanoparticle solution.
(7) And (3) placing the ALN-HA-CaCD nano particle solution in an ultralow temperature refrigerator at the temperature of minus 80 ℃ for pre-freezing overnight, transferring to a freeze dryer for freeze drying, and finally obtaining fluffy ALN-HA-CaCD nano particle powder.
Example 3
In vitro hydroxyapatite powder (HAP) adsorption simulation experiments
CaCD nanoparticles without ALN-HA and ALN-HA-CaCD nanoparticles were prepared by the method of example 2, and the purified nanoparticles were resuspended in physiological saline as a dispersion medium. Mixing 1mL of nanoparticle heavy suspension with 50mg of hydroxyapatite powder (HAP) in an EP tube, and placing inVortex at room temperature at minimum rotational speed on a vortex finder. After the set time point, one tube per group was removed, and centrifuged at 3000rpm for 5 minutes to completely remove HAP from the solution. 600uL of supernatant was carefully aspirated and stored at 4deg.C until assayed.
And (3) measuring the concentration of vitamin D 3 in the supernatant by a standard curve method, measuring the absorbance As of the obtained supernatant by using 265nm As a detection wavelength, and substituting As into a corresponding linear equation of the prepared standard curve to obtain the concentration Cs of vitamin D 3 in the supernatant after the incubation experiment. And (3) taking the nanoparticle suspension, adding physiological saline for injection for dilution, measuring absorbance Ai, and carrying into a linear equation to obtain the concentration Ci of vitamin D 3 in the nanoparticle suspension before incubation experiments. The absorption curve is calculated and plotted by the following equation.
Absorption (%) = (Ci-Cs)/ci×100%
The adsorption results of CaCD nanoparticles and ALN-HA-CaCD nanoparticles with hydroxyapatite powder are shown in FIG. 2. As can be seen from FIG. 2, ALN-HA-CaCD HAs a strong adsorbability with hydroxyapatite powder. The data show that the AP-CPNP can be adsorbed on HAP quickly, adsorption happens in 5 minutes, and the adsorption reaches equilibrium in 10 minutes, and CaCD nano particles without ALN-HA have no obvious bone adsorption performance, so that the synthesized ALN-HA-CaCD nano particles have certain bone targeting performance, namely the polymer provided by the application HAs certain bone targeting performance.
Example 4 in vitro bone tissue adsorption simulation experiment
Taking SD milk mice (within 24 hours of birth), dissecting after sacrifice to obtain skull, taking required skull fragments before cleaning with normal saline, peeling off periosteum and other connective tissues on the surface of the skull under a microscope, only leaving bone tissue hydroxyapatite parts, cutting into skull fragments with uniform size after cleaning with normal saline, and drying in a 60 ℃ oven for later use.
CaCD nanoparticles and ALN-HA-CaCD nanoparticles which take coumarin as model medicaments are prepared by adopting the method in the embodiment 2, respectively, caCD nanoparticles and ALN-HA-CaCD nanoparticles with the same mass are weighed, and physiological saline with the same volume is used for resuspension, so that nanoparticle suspension with uniform dispersion is obtained. Putting a certain amount of skull fragments into 2 groups of nanoparticle suspensions respectively, placing on a shaking table, incubating the skull fragments with the nanoparticles at 37 ℃ and 100rpm, and taking out one skull fragment from each group of nanoparticle suspensions at 10min, 20min, 30min, 60min, 120min and 240 min. After washing the skull fragments with normal saline three times and drying at room temperature in dark, the conditions of the skull fragments emitting fluorescence are observed by an inverted fluorescence microscope, and the average fluorescence intensity is calculated according to the following consensus.
Average fluorescence intensity = sum of fluorescence intensities of the regions/area of the regions
The contribution of ALN bone targeting head group to the targeting capability of the nanoparticle bone can be evaluated by observing the adsorption condition between the two groups of nanoparticles and the skull. As can be seen from fig. 3, the CaCD groups began to adsorb slightly from the skull at 20-30 minutes, but the adsorption did not increase over time, probably due to the weak adsorption between exposed-COOH and hydroxyapatite on CaCD. Compared with CaCD groups, the adsorption of the ALN-HA-CaCD group is obviously enhanced, the adsorption can be seen after the adsorption is incubated with the skull for 10 minutes, the adsorption amount of the nanoparticles on the bone tissue is gradually increased along with the time, and the adsorption maximum value is reached after 60 minutes. Therefore, the ALN bone targeting head group plays an important role in targeting the ALN-HA-CaCD nanoparticle to bone tissues.
Example 5
Determination of vitamin D 3 content and content uniformity in ALN-HA-CaCD nanoparticle
The ALN-HA-CaCD prepared in example 2 was subjected to vitamin D 3 content, content uniformity and detection, and the results are shown in Table 1.
Table 1: detection result of content and content uniformity of vitamin D 3 in ALN-HA-CaCD nano-particles
According to the test results in Table 1, the content of vitamin D 3 is within the quality control limit (90% -120%), and the content uniformity meets the requirements.
Example 6
Stability test of vitamin D 3 in ALN-HA-CaCD nanoparticles
The ALN-HA-CaCD nanoparticle prepared in example 2 and the commercially available pediatric calcium carbonate D 3 particle were placed under 40 ℃/75% RH conditions for 1 month, 3 months and 6 months, respectively; the results are shown in Table 2.
Table 2: accelerated stability test results (40 ℃ C./75% RH)
Note that: "N/A" is indicated as undetected.
According to the test results of Table 2, it can be seen that the content of vitamin D 3 in ALN-HA-CaCD nanoparticles is within the control limit (90% -120%) after the nanoparticles are placed under 40 ℃/75% RH conditions for 1,3 and 6 months, while the content of vitamin D 3 in the commercially available pediatric calcium carbonate D 3 particles is obviously reduced along with the prolonged placement time, the content of vitamin D 3 in the commercially available pediatric calcium carbonate D 3 particles is lower than the lower quality control limit after the nanoparticles are placed for 6 months, and the content of the impurity A exceeds the control standard (less than or equal to 4.0%) after the impurities A are placed for 6 months. In addition, the related substances of vitamin D 3 in the ALN-HA-CaCD nano-particles are far smaller than the control limit, and the related substances of the commercial pediatric calcium carbonate D 3 particles are obviously increased, so that the prepared ALN-HA-CaCD nano-particles have good stability under the acceleration condition and are superior to the commercial granules.
The foregoing has described in detail the technical solutions provided by the embodiments of the present application, and specific examples have been applied to illustrate the principles and implementations of the embodiments of the present application, where the above description of the embodiments is only suitable for helping to understand the principles of the embodiments of the present application; meanwhile, as for those skilled in the art, according to the embodiments of the present application, there are variations in the specific embodiments and the application scope, and the present description should not be construed as limiting the present application.

Claims (8)

1. A nanocarrier for treating osteoporosis, wherein the nanocarrier comprises an inner core and a stabilizer coated on the surface of the inner core;
Wherein the inner core takes calcium ions as a metal center, and cyclodextrin containing Lewis acid end groups and containing vitamin D 3 is taken as an organic ligand;
the preparation method of the stabilizer comprises the following steps:
(1) Dissolving hyaluronic acid, 1-ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide in a phosphate buffer salt solution, carrying out ice bath reaction, and transferring to room temperature for reaction to obtain a solution A;
(2) Dissolving alendronate sodium in sodium hydroxide solution, adding water, adjusting pH to alkaline after the alendronate sodium is completely dissolved, and stirring on ice bath to obtain solution B;
(3) Dropwise adding the solution A into the solution B, stirring the solution in an ice bath after the dropwise adding, transferring the solution to a constant-temperature water bath for stirring, and freeze-drying, separating and purifying the solution after the reaction is finished to obtain the stabilizer;
Wherein the molecular weight of the stabilizer is <10kDa.
2. A method of preparing nanocarriers for the treatment of osteoporosis according to claim 1, comprising:
(1) Dissolving triton and cyclohexane in hexanol to prepare an oil phase;
(2) Dissolving a calcium source in water to obtain a calcium solution;
(3) Dissolving vitamin D 3 in ethanol, dissolving cyclodextrin containing Lewis acid end groups in water, slowly dripping vitamin D 3 ethanol solution into the cyclodextrin solution, and uniformly stirring to obtain VD 3 -CD inclusion solution;
(4) Dissolving a stabilizer in ultrapure water to obtain a polymer solution;
(5) Dropwise adding a calcium solution and a VD 3 -CD inclusion solution into the oil phase, stirring for reaction, then dropwise adding a polymer solution, and continuing stirring for reaction to obtain nano-carrier microemulsion;
(6) Adding ethanol into the nano-carrier microemulsion, stirring, demulsifying, centrifuging, washing, purifying, and dispersing with purified water to obtain a nano-carrier solution;
(7) And freeze-drying the nano-carrier solution to obtain the nano-carrier.
3. The method of claim 2, wherein the triton is triton X-100.
4. The method according to claim 2, wherein the calcium source is at least one selected from the group consisting of calcium chloride, tricalcium phosphate, calcium lactate, calcium acetate, monocalcium phosphate, and calcium hydrogen phosphate;
and/or the cyclodextrin containing the Lewis acid end group is selected from at least one of carboxymethyl betacyclodextrin, sulfobutyl betacyclodextrin and phosphobetacyclodextrin.
5. The method of claim 4, wherein the calcium source is calcium chloride.
6. The process of claim 4, wherein the cyclodextrin having a Lewis acid end group is carboxymethyl betacyclodextrin.
7. The method of claim 2, wherein the molar ratio of cyclodextrin containing lewis acid end groups to vitamin D 3 is greater than 300:1.
8. The method of claim 7, wherein the molar ratio of cyclodextrin containing lewis acid end groups to vitamin D3 is 1000:1.
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