CN107867677A - One-dimensional calcium phosphate nano/micro materials and its preparation method and application - Google Patents
One-dimensional calcium phosphate nano/micro materials and its preparation method and application Download PDFInfo
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Abstract
The invention provides a kind of one-dimensional calcium phosphate nanometer material and its preparation method and application, one-dimensional calcium phosphate nano/the micro materials are made by water-soluble Ca salt and water-soluble phosphate by microwave radiation technology hydro-thermal reaction, one-dimensional calcium phosphate nano/the micro materials are club shaped structure, its length is 500 μm of 50nm, and draw ratio is more than 5：1, after it can be mixed with nucleic acid, by delivery of nucleic acids to intracellular, its good biocompatibility, efficiency gene transfection height, duration length.
Description
Technical field
The present invention relates to genophore technical field, more particularly to a kind of one-dimensional calcium phosphate nano/micro materials and its system
Preparation Method and application.
Background technology
The defects of gene therapy causes disease in target cell core by the way that foreign gene is imported into repair gene or suppression
System causes the deleterious gene of disease, so as to reach the purpose for the treatment of disease.Successful gene therapy carries dependent on efficient gene
Body, common carrier are divided into viral vector and non-viral vector.And viral vector because great potential safety hazard being present and
Limit its development.In recent years, the appearance of minicircle dna (Minicircle DNA, mcDNA), turn into one big in genophore
Bright spot, minicircle dna are the expression boxes of a ring-type, are by DNA recombinant techniques, the bacterial backbone DNA of standard plasmid is gone
Product after removing, a gene expression frame is comprised only without external bacterial backbone sequence, its advantage is effectively to extend target
Gene expression time in the cell, the gene dosage carried are big and almost without toxicity.But because exposed DNA is in physiological environment
In can be rapidly cleared in humans, it is impossible to be efficiently entering target cell, still need to suitable delivery system by minicircle dna be delivered to target tissue or
Organ.
Conventional Gene delivery systems include cationic-liposome, cationic polymer (polyethyleneimine PEI) etc..It is this kind of
Delivery system immunogenicity is relatively low, gene struck capacity is big, but transfection efficiency is low, greatly limit its clinical practice.Therefore,
Research and development are efficient, and the Gene delivery systems with good biocompatibility and with targeting are that solve genomic medicine clinic to answer
Key.
For calcium phosphate as genophore, biological safety is higher, not bright while good protection parcel DNA
Aobvious cytotoxicity.But generally there are particle diameter it is uncontrollable, it is easy reunite, the problem such as transfection efficiency is low.But calcium phosphate-gene is common
Precipitation method transfection efficiency is very low (about 10~20%), and the calcium phosphate granules particle diameter of preparation is big and is difficult to control, and stability is poor, easily
Reunite, it is difficult to for transfecting research in vivo.The phospholipid bilayer bag that the propositions such as Leaf Huang are prepared by reverse microemulsion method
The DNA/ calcium phosphate nanoparticles systems wrapped up in, but, cationic lipid that preparation process use cumbersome to the purification step of calcium phosphate
Plastid has higher cytotoxicity, and phospholipid material is expensive, is unfavorable for further clinical practice.
The content of the invention
In view of this, the present invention is prepared for one-dimensional calcium phosphate nano/micro materials by microwave attenuation materials method, and it has
Larger draw ratio (it is nanometer rods or the cuspidated spicule of tool), the one-dimensional calcium phosphate nano/micro materials and micro-loop
DNA mixing after, through intraperitoneal injection, can in mouse peritoneal high-efficiency transfection.Can be as the genophore of high-efficiency transfection.
In a first aspect, the invention provides a kind of preparation method of one-dimensional calcium phosphate nano/micro materials, including following step
Suddenly：
(1) water-soluble Ca salt and water-soluble phosphate difference is soluble in water, preparation obtains water-soluble Ca salt and water solubility
Phosphate solution, the water-soluble Ca salt and water-soluble phosphoric acid salting liquid are added in the first solvent, first is obtained after mixing
Mixed solution, wherein, first solvent is water, or the mixed solvent of water and alcohol, and the alcohol includes ethanol, ethylene glycol and third
One or more in triol；
(2) pH value for adjusting first mixed solution is 5-11, and the mixed solution after regulation pH is placed in into microwave hydrothermal
Microwave radiation technology hydro-thermal reaction is carried out in kettle, wherein, reaction temperature is 110-180 DEG C, reaction time 10-60min；
(3) products therefrom after reaction is centrifuged, and the solid content to isolating is washed, dried, and is obtained
One-dimensional calcium phosphate nano/micro materials.
Preferably, the water-soluble Ca salt is calcium chloride and/or its hydrate, calcium nitrate and/or its hydrate (Ca
(NO3)2·4H2), and/or calcium acetate and/or its hydrate (Ca (CH O3COO)2·H2O), it is but unlimited.
Preferably, the water-soluble phosphate is ammonium phosphate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, sodium phosphate, biphosphate
Sodium, disodium hydrogen phosphate, potassium phosphate, potassium dihydrogen phosphate or dipotassium hydrogen phosphate etc.；, but it is not limited to Soluble Inorganic Phosphorus listed above
Hydrochlorate.
That is, (NH is included but is not limited to4)3PO4, (NH4)2HPO4, (NH4)H2PO4, Na3PO4, Na2HPO4, NaH2PO4,
K3PO4, K2HPO4, KH2PO4Or K2HPO4。
Preferably, the Ca of water-soluble Ca salt and the water-soluble phosphoric acid salting liquid：P mol ratios are (0.5-5)：1.
It is further preferred that the Ca of the water-soluble Ca salt and water-soluble phosphoric acid salting liquid：P mol ratios are (0.5-2)：1.
Further preferably into (0.5-1.5)：1.
Preferably, first solvent is the mixed solvent of water and alcohol.
It is further preferred that in first solvent, the volume ratio of water and ethanol is (0.1-10)：1.
It is further preferred that in first solvent, the volume ratio of water and ethanol is (0.5-3)：1.
Preferably, the pH value of the first mixed solution is 7-11.
Preferably, the regulation pH is using acid or alkali.
It is further preferred that the acid that the regulation pH is used is hydrochloric acid, nitric acid or acetic acid；The alkali that the regulation pH is used for
Ammoniacal liquor, NaOH or KOH.
Preferably, in step (2), the reaction temperature is 120-160 DEG C, for example, 130,140,150 DEG C.
In step (2), the reaction time can be 20,30,45,45,50 or 60min.
Preferably, the one-dimensional calcium phosphate nano/micro materials are club shaped structure.
Preferably, a diameter of 10-1000nm of the one-dimensional calcium phosphate nano/micro materials.
Preferably, the length of the one-dimensional calcium phosphate nano/micro materials is 50nm-500 μm.More preferably
100nm-100μm；Either 350-30 μm.More preferably 150-350nm.
Preferably, the draw ratio of the one-dimensional calcium phosphate nano/micro materials is more than 5：1.Further preferably greater than 10：
1。
One-dimensional calcium phosphate nano/the micro materials can be that the smooth Nano/micron rod in both ends or both ends are equal
For the Nano/micron rod structure (now be alternatively referred to as acicular texture) at tip, it can also be that one end is smooth, the other end is sophisticated
Structure.
It is further preferred that the one-dimensional calcium phosphate nano/micro materials are the Nano/micron rod knot that both ends are tip
Structure.Draw ratio is more than 20 in such cases：1.More preferably (20-50)：1.
Further, the part of cell-specific identification is also modified with the one-dimensional calcium phosphate nano/micro materials.
The part of the cell-specific identification includes but is not limited to such as lactose or gala carbohydrate ligands, hyaluronic acid part, leaf
Sour part, transferrins part, lipopolysaccharides part, polypeptide ligand (such as arginine-glycine-aspartic acid series polypeptide) etc.
Modification.It is understood that the cell type of the ligands specific targeting includes but is not limited to various tumour cells, is immunized carefully
Born of the same parents' (such as macrophage, DC cells, T cell, B cell, NK cells), normal tissue cell (such as stem cell, liver cell, muscle
Cell, epithelial cell etc.).The part can be by the one-dimensional calcium phosphate nano/micro materials when as genophore
The destination of delivering selects.The part of the cell-specific identification, can prepare to obtain described one in microwave radiation technology hydro-thermal method
After tieing up calcium phosphate nano/micro materials, the part in modification, cell-specific can also be added in above-mentioned first mixed solution
The part of identification.But if selection adds the part in preparation process, therefore, to assure that is added in first mixed solution
After part, ligand modified one-dimensional calcium phosphate material structure (not changing its one-dimentional structure) can be still obtained.Preferably, it is being made
After the one-dimensional calcium phosphate nano/micro materials, then modify above-mentioned part.
In an embodiment of the present invention, target protein and/or polypeptide can be made into the finite concentration aqueous solution, will prepared
Good one-dimensional calcium phosphate nano/micro materials are scattered in above-mentioned protein/polypeptide solution, and after being sufficiently stirred overnight, centrifugation obtains
Surface modification has the one-dimensional calcium phosphate material of protein/polypeptide.
In another embodiment of the present invention, by one-dimensional calcium phosphate nano/micro materials first in multiamino compound (as gathered
Aziridine) react in solution overnight, process is attracted by positive and negative charge, makes a large amount of amino of calcium phosphate surface distributed, then pass through
The amino of connection is with the part (RGD cyclic peptide and/or folate molecule) with carboxyl in coupling agent 1- (3- dimethylamino-propyls) -3- second
Covalent coupling reaches the purpose of the upper part of grafting in the presence of base carbodiimide (EDC) etc..
The preparation method for one-dimensional calcium phosphate nano/micro materials that first aspect present invention provides, its preparation flow is simple,
Yield is higher, and without using the bar such as raw material, the ratio by adjusting reaction temperature, time, system pH, calcium source and phosphorus source of costliness
Part can control the pattern of gained calcium phosphate nano/micro materials, size etc..
One-dimensional calcium phosphate nano/the micro materials of gained, there is higher draw ratio, biocompatibility is good, can conduct
Genophore, DNA- calcium phosphate solutions directly are mixed to get with DNA (such as minicircle dna), when DNA- calcium phosphate solutions are through abdominal cavity
It is injected in mouse peritoneal, because it is the one-dimentional structure compared with high length-diameter ratio, the one-dimensional calcium phosphate material can be stimulated easily
Mesothelial cell, cell membrane passage during division or hyperplasia increase, and DNA is rapidly introduced into cell,
Moreover, cell understands multiple fission when being upset, at this moment nucleus nuclear membrane is opened, and nucleus is divided into two, and DNA is entered
Cell examines existing high-efficiency transfection.
Second aspect, the invention provides the one-dimensional calcium phosphate as made from the preparation method described in first aspect present invention to receive
Rice/micro materials.
The third aspect, the invention provides a kind of pharmaceutical composition (or be genophore compound, gene delivery system
System), including above-mentioned one-dimensional calcium phosphate nano/micro materials and nucleic acid.Described in the one-dimensional calcium phosphate nano/micro materials load
Nucleic acid, the load can be blending or combine.
Preferably, the mass ratio of the one-dimensional calcium phosphate nano/micro materials and nucleic acid is (1-200)：1.
It is further preferred that the mass ratio of the one-dimensional calcium phosphate nano/micro materials and nucleic acid is (10-150)：1.
It is further preferred that the mass ratio of the one-dimensional calcium phosphate nano/micro materials and nucleic acid is (15-100)：1.
It is highly preferred that the mass ratio of the one-dimensional calcium phosphate nano/micro materials and nucleic acid is 100：1.
Preferably, the nucleic acid includes the one or more in DNA fragmentation and RNA fragments, but not limited to this.
As described in the present invention, " DNA fragmentation " includes one kind of DNA fragmentation that is artificial synthesized and being purified in biological sample
It is or a variety of；" RNA fragments " includes the one or more of RNA fragments that are artificial synthesized and being purified in biological sample.Specifically, institute
It can be DNA, minicircle dna or DNA fragmentation etc. to state DNA fragmentation that is artificial synthesized or being purified in biological sample；Specifically,
RNA fragments that are described artificial synthesized or being purified in biological sample can be microRNA (micro rna), siRNA (small interference
RNA), shRNA etc..
It is further preferred that the nucleic acid is minicircle dna, but not limited to this.
Preferably, described pharmaceutical composition also includes at least one of bio-pharmaceutical and chemicals, wherein, the life
The one or more that thing medicine includes but is not limited in polypeptide, albumen and vaccine；The chemicals includes but is not limited to anticancer
One or more in medicine, developer and tracer.The tracer can be fluorescent dye, lymphatic tracer etc..
Fourth aspect, the invention provides the preparation method of aforementioned pharmaceutical compositions, comprise the following steps：
After one-dimensional calcium phosphate nano/micro materials solution and nucleic acid solution are mixed, the drug regimen is made through being incubated
Thing.Wherein, the one-dimensional calcium phosphate nano/micro materials load the nucleic acid, and the load can be blending or combine.
Preferably, the mass ratio of the one-dimensional calcium phosphate nano/micro materials and nucleic acid is (1-200)：1.
It is further preferred that the mass ratio of the one-dimensional calcium phosphate nano/micro materials and nucleic acid is (10-150)：1.
It is further preferred that the mass ratio of the one-dimensional calcium phosphate nano/micro materials and nucleic acid is (15-100)：1.
It is highly preferred that the mass ratio of the one-dimensional calcium phosphate nano/micro materials and nucleic acid is 100：1.
Preferably, when the one-dimensional calcium phosphate nano/micro materials solution and nucleic acid solution mix, life can also be added
At least one of thing medicine and chemicals, wherein, the bio-pharmaceutical includes but is not limited in polypeptide, albumen and vaccine
It is one or more；The one or more that the chemicals includes but is not limited in cancer therapy drug, developer and tracer.
It is further preferred that the one-dimensional calcium phosphate nano/micro materials solution is one-dimensional calcium phosphate nano/micro materials
Aqueous solution, but not limited to this.Specifically, the aqueous solution of including but not limited to one-dimensional calcium phosphate nano/micro materials, one-dimensional
The normal saline solution of calcium phosphate nano/micro materials, the glucose solution of one-dimensional calcium phosphate nano/micro materials, one-dimensional phosphorus
Phosphate buffer of sour calcium Nano/micron material etc..
It is further preferred that the nucleic acid solution is the aqueous solution of nucleic acid, but not limited to this.Specifically, the water of nucleic acid
It is that solution includes but is not limited to the aqueous solution of nucleic acid, the normal saline solution of nucleic acid, the glucose solution of nucleic acid, the phosphoric acid of nucleic acid
Salt buffer etc..
As described in the present invention, the incubation is directly reacted at room temperature, described to be incubated temperature without heating or cooling
Spend for 20-37 DEG C.
Preferably, the time of the incubation is 10-100min.
Preferably, described pharmaceutical composition can pass through dilution, aqueous solution injection is made in concentration, for gene therapy.
Preferably, also freeze drying powder injection can be made, for gene therapy by the technique such as lyophilized in described pharmaceutical composition.
5th aspect, the invention provides one-dimensional calcium phosphate nano/micro materials as described in the first aspect of the invention or
Application of the pharmaceutical composition in delivering nucleic acid medicine is prepared as described in third aspect present invention.
One-dimensional calcium phosphate nano/the micro materials or described pharmaceutical composition are when as delivering nucleic acid medicine, its poison
Property it is small, biocompatibility is high, and delivery efficiency, the expression efficiency of nucleic acid are higher.
6th aspect, present invention also offers a kind of delivery nucleic acid molecules to the method for cell, it includes making the cell
With the pharmaceutical composition thereof described in third aspect present invention.
Preferably, the cell in vivo, but not limited to this.
It is further preferred that the mode for making the cell and the pharmaceutical composition thereof described in third aspect present invention
For：Being subcutaneously injected using in-vitro transfection or in vivo by the way of (as be injected intraperitoneally) or intramuscular injection makes the cell and the present invention the
Pharmaceutical composition thereof described in three aspects.
Wherein, using intraperitoneal injection mode by described pharmaceutical composition injection as in Mice Body when, can in ascites and
The DNA expression products of high concentration are detected in blood.
When preferably, using the way of contact subcutaneously or intramuscularly injected in vivo, described pharmaceutical composition according to not less than
The usage amount of 0.1-10 μ g nucleic acid/kg the weight of animals is applied, but is not particularly limited.
Beneficial effects of the present invention include the following aspects：
1) unique structure of the one-dimensional calcium phosphate nano/micro materials, biocompatibility is good, as genophore
When transfection efficiency is high, small toxicity；
2) stabilization for the pharmaceutical composition that the one-dimensional calcium phosphate nano/micro materials are formed with nucleic acid (such as minicircle dna)
Property is good, can efficiently, long-term expression minicircle dna；
3) one-dimensional calcium phosphate nano/micro materials of the present invention, the preparation method of pharmaceutical composition are simple and easy to do, controllable
Degree is high.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is the required accompanying drawing used in technology description to be briefly described.Specific embodiment described herein is only explaining this
Invention, is not intended to limit the present invention.
Fig. 1 is the XRD results for one-dimensional calcium phosphate nano/micro materials that different experiments group of the present invention provides；
Fig. 2 is the Electronic Speculum for one-dimensional calcium phosphate nano/micro materials that different experiments group of the present invention and comparative example provide
Photo, and its transfection results of the minicircle dna with fluorescein mediated after intraperitoneal injection；
Fig. 3 is that the agarose gel electrophoresis of one-dimensional calcium phosphate nano/micro materials and DNA made from the embodiment of the present invention is real
The result tested；
Fig. 4 be different quality than one-dimensional calcium phosphate nano/micro materials/DNA luciferase is transfected to mouse peritoneal
Influential effect figure；
Fig. 5 is that one-dimensional calcium phosphate nano/micro materials mediate minicircle dna to transfect CD3/ in abdominal cavity in the embodiment of the present invention
The transfection results of the double targeting antibodies of Cmet, left figure is antibody concentration in serum；Right figure is antibody concentration in abdominal cavity.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Site preparation describes, it is clear that described embodiment is only part of the embodiment of the present invention, rather than whole embodiments.It is based on
Embodiment in the present invention, those of ordinary skill in the art are obtained every other under the premise of creative work is not made
Embodiment, belong to the scope of protection of the invention.
Prepare embodiment：The preparation of one-dimensional calcium phosphate nano/micro materials
The invention provides the preparation method of one-dimensional calcium phosphate nano/micro materials, comprise the following steps：
(1) by 3mLNaH2PO4The CaCl of solution (0.05mol/L) and 3mL2Solution (0.1mol/L) be added to 15mL go from
The in the mixed solvent of sub- water and 15mL absolute ethyl alcohol composition, at room temperature uniformly mixing；Then the pH value of solution after mixing is adjusted
To 7.0 ± 0.5, reaction solution A is obtained.(2) reaction solution A is poured into microwave hydrothermal reaction kettle, microwave heats at 110 DEG C
10min, reactor is taken out when naturally cooling to less than 60 DEG C etc. system；(3) product in reactor is centrifuged under 12000g
10min, and being washed 3 times using absolute ethyl alcohol to the solid content isolated, is finally freeze-dried, and obtains calcium phosphate nano/micro-
Rice material, sample 1 is named as by this sample.
It should be noted that according to different needs, thus it is possible to vary pH, reaction temperature and the time of solution A, water-soluble Ca salt
With the Ca of water-soluble phosphoric acid salting liquid：P mol ratios, water system volume etc., obtain the one-dimensional calcium phosphate sample of different-grain diameter.This implementation
Example provides calcium phosphate sample prepared by 1~10 group of different experimental conditions altogether, specifically matches as shown in table 1 below：
Table 1
| Experimental group | The pH of solution A | T/℃ | t/min |
| 1 | 7.0 | 110 | 10 |
| 2 | 11.0 | 110 | 10 |
| 3 | 5.0 | 110 | 10 |
| 4 | 6.0 | 110 | 10 |
| 5 | 8.0 | 110 | 10 |
| 6 | 9.0 | 110 | 10 |
| 7 | 10.0 | 110 | 10 |
| 8 | 7.0 | 110 | 30 |
| 9 | 10.0 | 150 | 30 |
| 10 | 10.0 | 180 | 10 |
Calcium phosphate sample characterization is tested：
Calcium phosphate sample made from each experimental group of embodiment 1 is placed on XRD testers, carries out XRD powder diffraction tables
Sign, as a result as shown in Figure 1.From Fig. 1 XRD can be seen that reaction solution pH value be 7,11,6 when, the crystalline substance of gained reaction product
Body phase is hydroxyapatite (P63/m, card numbering 25-0166), and reacting solution pH value be 5 when, the crystal phase of reaction product
All it is the common crystal structure of calcium phosphate for brushite (la (9), card numbering 11-0293).
By calcium phosphate sample dispersion made from each experimental group in aqueous, ultrasonic 10min is completely dispersed with up to sample,
Then drop on copper mesh, each sample is obtained under 110V voltages with FEI Tecnai G2F20S-Twin models electron microscopes
Electromicroscopic photograph, as a result as shown in Figure 2.
The cytoactive experiment of calcium phosphate sample：By one-dimensional phosphorus made from the experimental group 1 of the embodiment of the present invention in above-mentioned table 1
After sour calcium sample is incubated 24 hours altogether with 293T cells, its cytotoxicity is detected with CCK8 kits.The results show, in phosphorus
The concentration of sour calcium sample is 1 × 10-5During~1mg/mL, for the survival rate of 283T cells to 90-100%, this illustrates calcium phosphate sample
To 283T cells without overt toxicity, cellular morphology and normal proliferation.
Agarose gel electrophoresis is tested：Each test group DNA containing 0.5 μ g is controlled, according to calcium phosphate sample and DNA
Different quality ratio (0.25:1,0.5:1,1:1,2:Isosorbide-5-Nitrae:1), DNA solution is mixed with calcium phosphate sample (sample 1), to total
The μ L of volume 20, (while being used as control using single DNA solution) add the Ago-Gel containing DNA dyestuffs (1%, w/v).Again
After adding the uniform mixing of 2 μ L loading buffer (sample-loading buffer), it is added in well.Electrophoresis process is in Tris- vinegar
Carried out in acid buffering solution (TAE), electrophoresis is carried out 40 minutes under 80V voltages, then with gel imager under ultraviolet irradiation
Imaging, as a result as shown in Figure 3.
DNA is electronegative macromolecular, can be moved under electric field action to positive pole.In Fig. 3 Ago-Gel experiment, hair
After existing DNA is incubated with calcium phosphate, DNA can be moved under electric field action, this movement and fluent work of explanation calcium phosphate to DNA
With this experiment proves do not have chemical interactions between DNA and calcium phosphate.
Calcium phosphate sample mediation minicircle dna abdominal cavity transfection (luciferase)：
By 2mg each group calcium phosphate sample dispersion in 200 μ L ultra-pure waters, ultrasound 10 minutes, calcium phosphate solution is formed；Will
Minicircle dnas of the 20 μ g with luciferase (luciferase) expressing gene is dispersed in 200 μ L water, forms minicircle dna solution
B；
Above-mentioned minicircle dna solution B is added in above-mentioned calcium phosphate solution, forms medicinal composition solution C；By drug regimen
Thing solution C is injected into the age in 6-8 weeks, the Mice Body that body weight is 20g through abdominal cavity, and IVIS small animal imaging instrument is used after 24 hours
Expression of the luciferase in Mice Body is observed, ROI (region are represented as a result as shown in Fig. 2 in figure, at red circle
Of interest, area-of-interest).
Comparative example 1：(1) 200mg egg yolk lecithins are dissolved in 15ml absolute ethyl alcohols, obtain egg yolk lecithin solution；
By 3mL NaH2PO4Solution (0.05mol/L) and 3ml CaCl2Solution (0.1mol/L) is added to 15mL deionized waters and 15mL
The in the mixed solvent of absolute ethyl alcohol composition, uniformly mixing, obtains the first mixed solution at room temperature；(2) by above-mentioned yolk lecithin
Lipoprotein solution is added in above-mentioned first mixed solution B, obtains the second mixed solution；The second obtained mixed solution is placed in microwave
Hydro-thermal reaction is answered in kettle, and microwave heats 10min at 110 DEG C, and reactor is taken out when naturally cooling to less than 60 DEG C etc. system；
(3) product in reactor is centrifuged into 10min under 12000g, and 3 will be washed using absolute ethyl alcohol to the solid content isolated
It is secondary, finally it is freeze-dried, obtains the calcium phosphate nanoparticles of lecithin fat modification, this sample is named as to 1 sample.
Comparative example 2：(1) by 0.111g adenosine disodium triphosphates (ATP) and 0.111gCaCl2It is dissolved in 50ml steamings
In distilled water, solution ph is adjusted to 7.0 by magnetic agitation to abundant dissolving；(2) solution of pH after jump is placed in microwave hydrothermal reaction
Answer in kettle, microwave heats 10min at 110 DEG C, and reactor is taken out when naturally cooling to less than 60 DEG C etc. system；(3) will reaction
Product in kettle centrifuges 10min under 12000g, and the solid content that isolated will be washed 3 times using absolute ethyl alcohol, last cold
It is lyophilized dry, the calcium phosphate nanoparticles using atriphos as phosphorus source are obtained, this sample is named as to 2 samples.
Fig. 2 is the electromicroscopic photograph of calcium phosphate sample and each calcium phosphate sample made from the embodiment of the present invention and comparative example
The result of product mediation minicircle dna abdominal cavity transfection luciferase.It can be seen from Fig. 2 that the calcium phosphate obtained by method provided by the invention
Sample is one-dimentional structure, wherein, sample 1,2 is that both ends are that sophisticated 1-dimention nano/micron bar structure (now can also claim
For acicular texture), sample 1,2 diameters are respectively 10nm, 20nm, and length is about 150nm, 300nm.Sample 3,10 is that both ends are smooth
Nano/micron rod.A diameter of 600nm of sample 3, length are about 20 μm；A diameter of 30nm of sample 10, length are about
400nm.And each one-dimensional calcium phosphate nano/micro materials can preferably mediate minicircle dna made from the embodiment of the present invention
Abdominal cavity transfects.And sample made from comparative example 1,2 is circular granular, diameter respectively may be about 100,150nm；This be probably by
It can preferentially may be chelated in being added during calcium phosphate crystal with the organic matter of calcium ion chelating, calcium ion with organic matter,
Calcium phosphate crystal crystallization process of long-range order in growth course is interrupted, and forms unformed spherical calcium phosphate granules.When
When they are respectively used to minicircle dna of the transfection in vivo with luciferase (luciferase) expressing gene, not in mouse peritoneal
It was observed that the luciferase of expression, the calcium phosphate material of this explanation circular granular can not play a part of stimulating cell well,
And then DNA effective transfection can not be realized.
Different quality than one-dimensional calcium phosphate nano/micro materials/DNA to mouse peritoneal transfect luciferase effect shadow
Ring
By 0.3,0.6,1mg each group calcium phosphate sample be dispersed in respectively in 200 μ L ultra-pure waters, ultrasound 10 minutes, formed
Calcium phosphate solution；Minicircle dnas of the 20 μ g with luciferase (luciferase) expressing gene is dispersed in 200 μ L water, shape
Into minicircle dna solution；Above-mentioned minicircle dna solution is added separately in above-mentioned each calcium phosphate solution, it is molten to form pharmaceutical composition
Liquid；Medicinal composition solution is expelled to the age in 6-8 weeks, the Balb/C Mice Bodies that body weight is about 20g through abdominal cavity, 24 hours
Expression with IVIS small animal imagings instrument observation luciferase in Mice Body afterwards, as a result as shown in Figure 4.
As seen from Figure 4, the dosage of calcium phosphate has considerable influence to internal transfection, every same mouse injection
20 μ g DNA, calcium phosphate dosage is bigger, and transfection is stronger.This is probably because substantial amounts of calcium phosphate is easier to stimulate mesothelium
The stress reaction of cell, be advantageous to DNA phagocytosiss.
Calcium phosphate sample mediation minicircle dna abdominal cavity transfection (the double targeting antibodies of AntiCD3 McAb/anti- Cmet)：By 2mg each group phosphoric acid
Calcium sample dispersion ultrasound 10 minutes, forms calcium phosphate solution in 200 μ L ultra-pure waters；20 μ g are double with AntiCD3 McAb/anti- Cmet
The minicircle dna of targeting antibodies expressing gene is dispersed in 200 μ L ultra-pure water, forms minicircle dna solution C；
Above-mentioned minicircle dna solution C is added in above-mentioned calcium phosphate solution, forms medicinal composition solution D；By the medicine group
Polymer solution D through abdominal cavity be injected into body weight be 20g Mice Body in, mouse is put to death after 24 hours, monitor mouse ascites and
The double targeting antibodies concentration expressed in serum, as a result as shown in Figure 5.Wherein, left figure is antibody concentration in serum in Fig. 5；Right figure
For antibody concentration in abdominal cavity.
Fig. 5 result shows：By calcium phosphate mediation, intraperitoneal injection minicircle dna can express target protein in abdominal cavity,
The double targeting antibodies of AntiCD3 McAb/anti- Cmet of high concentration can be such as detected in serum and ascites, and the antibody concentration in serum is higher than
Ascites.
Embodiment described above only expresses the several embodiments of the present invention, and its description is more specific and detailed, but simultaneously
Therefore the limitation to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that for one of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the guarantor of the present invention
Protect scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (10)
1. a kind of preparation method of one-dimensional calcium phosphate nano/micro materials, it is characterised in that comprise the following steps：
(1) water-soluble Ca salt and water-soluble phosphate difference is soluble in water, preparation obtains water-soluble Ca salt and water-soluble phosphoric acid
Salting liquid, the water-soluble Ca salt and water-soluble phosphoric acid salting liquid are added in the first solvent, and the first mixing is obtained after mixing
Solution, wherein, first solvent is water or the mixed solvent of water and alcohol, and the alcohol includes ethanol, ethylene glycol and glycerine
In one or more；
(2) pH value for adjusting first mixed solution is 5-11, and the mixed solution after regulation pH is placed in microwave hydrothermal kettle
Microwave radiation technology hydro-thermal reaction is carried out, wherein, reaction temperature is 110-180 DEG C, reaction time 10-60min；
(3) products therefrom after reaction is centrifuged, and the solid content to isolating is washed, dried, and is obtained one-dimensional
Calcium phosphate nano/micro materials.
2. a kind of one-dimensional calcium phosphate nano/micro materials, it is characterised in that the one-dimensional calcium phosphate nano/micro materials are rod
Shape structure, its length are 50nm-500 μm, and draw ratio is more than 5：1.
3. one-dimensional calcium phosphate nano/micro materials as claimed in claim 2, it is characterised in that the one-dimensional calcium phosphate nano/
Micro materials are the Nano/micron rod structure that both ends are tip.
4. a kind of pharmaceutical composition, it is characterised in that received including the one-dimensional calcium phosphate described in nucleic acid and claim any one of 1-2
One-dimensional calcium phosphate nano/micro materials described in obtained by the preparation method of rice/micro materials or claim any one of 3-4.
5. pharmaceutical composition as claimed in claim 4, it is characterised in that the one-dimensional calcium phosphate nano/micro materials and core
The mass ratio (1-200) of acid：1.
6. pharmaceutical composition as claimed in claim 4, it is characterised in that on the one-dimensional calcium phosphate nano/micro materials also
The part of cell-specific identification is modified with, the part of the cell-specific identification includes lactose part, gala carbohydrate ligands, hyalomitome
Sour part, the one or more of folate ligand, transferrins part and lipopolysaccharides part.
7. pharmaceutical composition as claimed in claim 4, it is characterised in that described pharmaceutical composition also includes bio-pharmaceutical and change
At least one of medicine is learned, wherein, the bio-pharmaceutical includes the one or more in polypeptide, albumen and vaccine；Describedization
Learning medicine includes the one or more in cancer therapy drug, developer and tracer.
8. the preparation method of a kind of pharmaceutical composition as described in claim any one of 4-7, it is characterised in that by one-dimensional phosphoric acid
After calcium Nano/micron material solution and nucleic acid solution mixing, described pharmaceutical composition is made through being incubated.
9. as described in the one-dimensional calcium phosphate nano/micro materials or claim any one of 4-7 as described in claim any one of 2-3
Application of the pharmaceutical composition in delivering nucleic acid medicine is prepared.
10. a kind of nucleic acid molecules of delivering are to the method for cell, it is characterised in that including making the cell and claim 4-7 institutes
Pharmaceutical composition thereof obtained by the preparation method for the pharmaceutical composition described in pharmaceutical composition or claim 8 stated.
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