CN103789348B - A kind of novel hybride nano-calcium phosphate genes delivery system and preparation method thereof - Google Patents
A kind of novel hybride nano-calcium phosphate genes delivery system and preparation method thereof Download PDFInfo
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- 229910000389 calcium phosphate Inorganic materials 0.000 title claims abstract description 81
- 238000002360 preparation method Methods 0.000 title abstract description 22
- 229920001661 Chitosan Polymers 0.000 claims abstract description 45
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- 238000003381 deacetylation reaction Methods 0.000 claims description 13
- 238000006467 substitution reaction Methods 0.000 claims description 13
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- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 8
- 229910052791 calcium Inorganic materials 0.000 claims description 8
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Abstract
The invention discloses a kind of novel hybride nano-calcium phosphate genes delivery system with and preparation method thereof, this genes delivery system comprises calcium phosphate and PEG grafting cm-chitosan, calcium phosphate parcel gene forms kernel, PEG grafting cm-chitosan is coated on core surface, PEG chain segment forms wetting ability shell, thus forming the nanoparticle with PEGization nucleocapsid structure, the particle diameter of this nanoparticle is 60 ~ 100nm.Novel hybride nano-calcium phosphate genes delivery system of the present invention, in preparation process without the need to an organic solvent, surface catalyst, process is simply controlled, and Financial cost is low, reproducible, is suitable for scale operation, and toxicity is low, and transfection efficiency is high.In addition, the invention still further relates to the test kit of this genes delivery system of preparation, and the inside and outside gene transfection of genes delivery system of the present invention and the purposes of gene therapy.
Description
Technical field
The present invention relates to field of pharmaceutical preparations and biomedicine technical field, relate in particular to a kind of novel hybride nano-calcium phosphate genes delivery system and preparation method thereof.
Background technology
Along with the development of the subjects such as genetically engineered, nanotechnology, modern medicine technology, becoming a kind for the treatment of means having future from gene level disease therapy.Gene therapy is by nucleic acid (DNA, siRNA, miRNA) being led people's tissue and cell, and regulation and control relate to that disease occurs, the expression of key protein of development, thus reach the object of disease treatment.But nucleic acid molecule, because its molecular weight is large, negatively charged, hydrophilic feature, effectively can not enter cell; In addition, nucleic acid molecule is very easily by the nuclease degradation in plasma proteins.So nucleic acid molecule needs to carry the object that could realize drug treatment by carrier bag.
Being carried out expressing by nucleic acid into cells is the committed step realizing gene therapy, and successful gene therapy depends on efficient gene carrier.Common carrier is divided into viral vector and non-virus carrier, wherein, viral vector comprises retrovirus, adenovirus (AV), adeno-associated virus (AAV), hsv (HSV), vaccinia virus (VV) etc., but viral vector easily causes the Immunoreactivity of human body, in clinical application, there is larger potential safety hazard.Non-virus carrier mainly prepares by cationic polymers or lipid and nucleic acid the cation carrier obtained by electrostatic adhesion, compared with virus vector, non-virus carrier have safety, effectively, the advantage such as non-immunogenicity.Cation carrier effectively can wrap up nucleic acid, promotes that nucleic acid enters cell.But positively charged ion non-virus carrier has the cytotoxicity because cationic characteristic brings usually, and be easy to precipitate in blood plasma, thus suppress the drug treatment in its body.
Calcium phosphate precipitation is widely used in cell transfecting, and calcium phosphate has good biocompatibility, biological degradability, and effectively can wrap a year nucleic acid molecule.In addition, calcium phosphate has sensitivity to acid, and under lysosomal acid environment, calcium phosphate dissociates and obtains calcium ion, phosphate anion, increases lysosome osmotic pressure, promotes that lysosome membrane breaks, and impels nucleic acid molecule to realize lysosome and escapes.But the colloidal stability of calcium phosphate is poor, rapid precipitation after preparation, lacks the repeatability of preparation; Precipitation calcium phosphate particles and can cytotoxicity be caused.Colloidal stability difference prevents in the body of calcium phosphate and applies.
Prepare stable calcium phosphate nano particle and become the effective way realizing safety in nucleic acid body, effectively send.LeafHuang etc. have prepared the calcium phosphate hybridized nanometer grain of liposome; after achieving intravenous injection, the system of nucleic acid is sent; what they adopted is that reverse micelle of microemulsion has prepared the stable coated calcium phosphate nano grain of lipid (LiJ; ChenYC; TsengYC; MozumdarS, HuangL.Biodegradablecalciumphosphatenanoparticle, withlipidcoatingforsystemicsiRNAdelivery.Journalofcontro lledrelease.2010; 142:416-21.).Chinese patent application (200910264114.6) authorizes " a kind of calcium phosphate composite nanoparticle of carrying genes and method for making and purposes " thereof, also uses micro emulsion legal system for calcium phosphate nano grain.But this preparation method's relative complex and used the organic solvent and tensio-active agent that there are genotoxic potential, is unfavorable for expanding production.
So, we attempt developing a kind of novel hybridized nanometer calcium phosphate genophore, this carrier has that preparation is simple, Financial cost is low, easy to use, transfection efficiency is high, good biocompatibility, can be used for the comprehensive advantages such as body inner injecting and administering, and this is of great importance to the development of gene therapy.
Summary of the invention
The object of the invention is to develop a kind of novel genes delivery system, improve the inside and outside transfection efficiency of gene, promote the development of gene therapy.
For achieving the above object, the technical solution used in the present invention is as follows:
A kind of novel hybride nano-calcium phosphate genes delivery system, comprise calcium phosphate and PEG grafting cm-chitosan, it is characterized in that, calcium phosphate parcel gene forms kernel, PEG grafting cm-chitosan is coated on core surface, PEG chain segment forms wetting ability shell, thus form the nanoparticle with PEGization nucleocapsid structure, the viscosity-average molecular weight of wherein said PEG grafting cm-chitosan is 1 ~ 1,000,000, deacetylation is greater than 80%, degree of substitution by carboxymethyl is 20 ~ 90%, PEG molecular weight is 500 ~ 10000, PEG percentage of grafting is 8% ~ 60%, and the particle diameter of this nanoparticle is 60 ~ 100nm.
Preferably, the viscosity-average molecular weight of described PEG grafting cm-chitosan is 10 ~ 400,000, and deacetylation is greater than 80%, and degree of substitution by carboxymethyl is 40 ~ 70%, PEG molecular weight be 2000 ~ 6000, PEG percentage of grafting is 20% ~ 40%.
The present invention more provides a kind of method preparing novel hybride nano-calcium phosphate genes delivery system, comprises the following steps:
1) prepare reagent a, comprise calcium salt, buffer reagent, surplus is water;
2) prepare reagent b, comprise PEG grafting cm-chitosan, phosphoric acid salt, sodium-chlor, buffer reagent, surplus is water;
3) gene is mixed with reagent a, then mixes with reagent b equal-volume,
It is characterized in that,
In described reagent a, calcium salt is CaCl
2, Ca (NO
3)
2in the combination of one or both arbitrary proportions, buffer reagent is one or more the arbitrary proportion combination of Hepes, MOPS, PBS, PIPES, Tris;
In described b reagent, the viscosity-average molecular weight of PEG grafting cm-chitosan is 1 ~ 1,000,000, and deacetylation is greater than 80%, and degree of substitution by carboxymethyl is 20 ~ 90%, PEG molecular weight be 500 ~ 10000, PEG percentage of grafting is 8% ~ 60%; Phosphoric acid salt comprises Na
3pO
4, Na
2hPO
4, NaH
2pO
4, K
3pO
4, K
2hPO
4, KH
2pO
4, (NH4)
3pO
4, (NH4)
2hPO
4one or more arbitrary proportion combination; Buffer reagent is one or more the arbitrary proportion combination in Hepes, MOPS, PBS, PIPES, Tris.
Preferably, the calcium salt in reagent a is CaCl
2or Ca (NO
3)
2, buffer reagent is Hepes or Tris; The viscosity-average molecular weight of the PEG grafting cm-chitosan in reagent b is 10 ~ 400,000, and deacetylation is greater than 80%, and degree of substitution by carboxymethyl is 40 ~ 70%, PEG molecular weight be 2000 ~ 6000, PEG percentage of grafting is 20% ~ 40%; Phosphoric acid salt is Na
2hPO
4or NaH
2pO
4; Buffer reagent is Hepes or Tris.
Preferably, the gene quality (μ g) when gene mixes with reagent a is 10 ~ 200 μ g/mL with the ratio of reagent a volume (mL).
More preferably, gene quality (μ g) is 40 ~ 120 μ g/mL with the ratio of reagent a volume (mL).
Preferably, the calcium concentration in described a reagent is 20 ~ 1000mM, and buffer concentration is 5 ~ 200mM, pH is 6.0 ~ 10.0; PEG grafting cm-chitosan concentration in described b reagent is 50 ~ 2000mg/L, and phosphate concn is 0.5 ~ 10mM, NaCl concentration is 50 ~ 500mM, and buffer concentration is 5 ~ 200mM, pH is 6.0 ~ 10.0.
Further preferably, the calcium concentration in described a reagent is 100 ~ 300mM, and buffer concentration is 20 ~ 60mM, pH is 7.0 ~ 8.0; PEG grafting cm-chitosan concentration in described b reagent is 200 ~ 800mg/L, and phosphate concn is 1.5 ~ 3.0mM, NaCl concentration is 100 ~ 300mM, and buffer concentration is 20 ~ 60mM, pH is 7.0 ~ 8.0.
On the other hand, the invention provides the purposes of above-mentioned novel hybride nano-calcium phosphate genes delivery system for genomic medicine conveying in the gene transfection of inside and outside cell and human or animal body, wherein said gene appoints one or more in DNA, siRNA, miRNA or shRNA.
The present invention also provides a kind of test kit, and for the preparation of above-mentioned novel hybride nano-calcium phosphate genes delivery system, described test kit comprises calcium salt or it comprises the aqueous solution of buffer reagent; And PEG grafting cm-chitosan, phosphoric acid salt, sodium-chlor or its comprise the mixed aqueous solution of buffer reagent, it is characterized in that, calcium salt or its aqueous solution comprising buffer reagent are separate independent packaging with PEG grafting cm-chitosan, phosphoric acid salt, sodium-chlor or its mixed aqueous solution comprising buffer reagent, and wherein calcium salt is CaCl
2, Ca (NO
3)
2in the combination of one or both arbitrary proportions; The viscosity-average molecular weight of PEG grafting cm-chitosan is 1 ~ 1,000,000, and deacetylation is greater than 80%, and degree of substitution by carboxymethyl is 20 ~ 90%, PEG molecular weight be 500 ~ 10000, PEG percentage of grafting is 8% ~ 60%; Phosphoric acid salt is Na
3pO
4, Na
2hPO
4, NaH
2pO
4, K
3pO
4, K
2hPO
4, KH
2pO
4, (NH4)
3pO
4, (NH4)
2hPO
4in one or more arbitrary proportion combination; Buffer reagent is one or more the arbitrary proportion combination in Hepes, MOPS, PBS, PIPES, Tris.
Compared with prior art, beneficial effect of the present invention is mainly reflected in:
Production process not with an organic solvent, surface catalyst, process is simply controlled, and Financial cost is low, reproducible, is suitable for scale operation; Vector stabilisation is good, and security is good, and inside and outside toxicity is extremely low; Transfection efficiency is high, effectively can realize the gene delivery of inside and outside, realize gene therapy.This carrier has that preparation is simple, Financial cost is low, easy to use and reliable, transfection efficiency is high, good biocompatibility, can be used for the advantage of body inner injecting and administering.
Accompanying drawing explanation
Fig. 1: the transmission electron microscope photo of novel hybride nano-calcium phosphate genes delivery system of the present invention.
Fig. 2: the cell transfecting effect of novel hybride nano-calcium phosphate genes delivery system of the present invention.
Fig. 3: cytotoxicity (mtt assay) result of novel hybride nano-calcium phosphate genes delivery system of the present invention.
Fig. 4: the result for the treatment of of the vivo gene therapy administration of novel hybride nano-calcium phosphate genes delivery system of the present invention.
Fig. 5: the immunotoxicity of novel hybride nano-calcium phosphate genes delivery system of the present invention.
Embodiment
Cm-chitosan is a kind of water soluble anion chitosan derivatives, has good biocompatibility and biological degradability, by electrostatic interaction active adsorption in positive polarity calcium phosphate granules sub-surface, can reach the object of stable calcium phosphate particles.In addition, PEGization also can pass through shielding effect stabilized nanoscale carrier, extends cycling time, and reaching long circulating effect increases curative effect.Therefore, PEG grafting cm-chitosan has stable calcium phosphate and obtains the potentiality of hybridized nanometer grain, thus sends in the safe and effective body realizing nucleic acid, realizes the gene therapy of disease.In addition, by the nanoprecipitation effect of PEG grafting cm-chitosan and calcium phosphate, self-assembly forms nanoparticle, more simplifies preparation production process.
The invention provides a kind of novel hybride nano-calcium phosphate genes delivery system, overcome the shortcoming of prior art, improve the inside and outside transfection efficiency of gene, promote the development of gene therapy.This carrier has that preparation is simple, Financial cost is low, easy to use and reliable, transfection efficiency is high, good biocompatibility, can be used for the advantage of body inner injecting and administering.
On the one hand, novel hybride nano-calcium phosphate genes delivery system of the present invention comprises calcium phosphate, PEG grafting cm-chitosan two kinds of components; Nucleocapsid structure, calcium phosphate forms kernel, and PEG grafting cm-chitosan is coated on core surface, and PEG chain segment forms wetting ability shell; Particle diameter is 60 ~ 100nm.
Described novel hybride nano-calcium phosphate genes delivery system can wrap the gene molecule carried and comprise DNA, siRNA, miRNA, shRNA, and gene molecule is loaded in calcium phosphate kernel by bag.
On the other hand, the method preparing novel hybride nano-calcium phosphate genes delivery system of the present invention comprises the steps:
1) prepare reagent a, comprise the aqueous solution of calcium salt, buffer reagent;
2) prepare reagent b, comprise the aqueous solution of PEG grafting cm-chitosan, phosphoric acid salt, sodium-chlor, buffer reagent.
3) gene first mixes with reagent a, then mixes with reagent b equal-volume.
In described a reagent, calcium salt is for comprising CaCl
2, Ca (NO
3)
2the combination of one or both arbitrary proportions, buffer reagent is one or more the arbitrary proportion combination of Hepes, MOPS, PBS, PIPES, Tris.
In described b reagent, the viscosity-average molecular weight of PEG grafting cm-chitosan is 1 ~ 1,000,000, and deacetylation is greater than 80%, and degree of substitution by carboxymethyl is 20 ~ 90%, PEG molecular weight be 500 ~ 10000, PEG percentage of grafting is 8% ~ 60%; Phosphoric acid salt, comprises Na
3pO
4, Na
2hPO
4, NaH
2pO
4, K
3pO
4, K
2hPO
4, KH
2pO
4, (NH4)
3pO
4, (NH4)
2hPO
4one or more arbitrary proportion combination; Buffer reagent is one or more the arbitrary proportion combination of Hepes, MOPS, PBS, PIPES, Tris.
Preferably, calcium salt is CaCl
2or Ca (NO
3)
2, buffer reagent is Hepes or Tris.
Preferably, the viscosity-average molecular weight of PEG grafting cm-chitosan is 10 ~ 400,000, and deacetylation is greater than 80%, and degree of substitution by carboxymethyl is 40 ~ 70%, PEG molecular weight be 2000 ~ 6000, PEG percentage of grafting is 20% ~ 40%; Phosphoric acid salt is Na
2hPO
4or NaH
2pO
4; Buffer reagent is Hepes or Tris.
Further, in described a reagent, calcium concentration is 20 ~ 1000mM, and buffer concentration is 5 ~ 200mM, pH is 6.0 ~ 10.0; In described b reagent, PEG grafting cm-chitosan concentration is 50 ~ 2000mg/L, and phosphate concn is 0.5 ~ 10mM, NaCl concentration is 50 ~ 500mM, and buffer concentration is 5 ~ 200mM, pH is 6.0 ~ 10.0.
Further, preferably, in described a reagent, calcium concentration is 100 ~ 300mM, and buffer concentration is 20 ~ 60mM, pH is 7.0 ~ 8.0; In described b reagent, PEG grafting cm-chitosan concentration is 200 ~ 800mg/L, and phosphate concn is 1.5 ~ 3.0mM, NaCl concentration is 100 ~ 300mM, and buffer concentration is 20 ~ 60mM, pH is 7.0 ~ 8.0.
In preparation method's step (3) of described novel hybride nano-calcium phosphate genes delivery system, when gene first mixes with reagent a, gene quality (μ g) is 10 ~ 200 μ g/mL with the ratio of reagent a volume (mL); Preferably, gene quality (μ g) is 40 ~ 120 μ g/mL with the ratio of reagent a volume (mL).
On the other hand, provide the purposes of described novel hybride nano-calcium phosphate genes delivery system, be genomic medicine conveying in the gene transfection of inside and outside cell and human or animal body; Wherein said gene be in DNA, siRNA, miRNA, shRNA appoint one or more.
On the other hand, the invention provides a kind of test kit prepared for described novel hybride nano-calcium phosphate genes delivery system, this test kit contains a, b two kinds of reagent of independent packaging: reagent a, comprises the aqueous solution of calcium salt, buffer reagent; Reagent b, comprises the aqueous solution of PEG grafting cm-chitosan, phosphoric acid salt, sodium-chlor, buffer reagent.
In a reagent of described test kit, calcium salt is for comprising CaCl
2, Ca (NO
3)
2the combination of one or both arbitrary proportions, buffer reagent is one or more the arbitrary proportion combination of Hepes, MOPS, PBS, PIPES, Tris.
In the b reagent of described test kit, the viscosity-average molecular weight of PEG grafting cm-chitosan is 1 ~ 1,000,000, and deacetylation is greater than 80%, and degree of substitution by carboxymethyl is 20 ~ 90%, PEG molecular weight be 500 ~ 10000, PEG percentage of grafting is 8% ~ 60%; Phosphoric acid salt, comprises Na
3pO
4, Na
2hPO
4, NaH
2pO
4, K
3pO
4, K
2hPO
4, KH
2pO
4, (NH4)
3pO
4, (NH4)
2hPO
4one or more arbitrary proportion combination; Buffer reagent is one or more the arbitrary proportion combination of Hepes, MOPS, PBS, PIPES, Tris.
As the preferred version of described test kit, calcium salt is CaCl
2or Ca (NO
3)
2, buffer reagent is Hepes or Tris.
More preferably, the viscosity-average molecular weight of the PEG grafting cm-chitosan in described test kit is 10 ~ 400,000, and deacetylation is greater than 80%, and degree of substitution by carboxymethyl is 40 ~ 70%, PEG molecular weight be 2000 ~ 6000, PEG percentage of grafting is 20% ~ 40%; Phosphoric acid salt is Na
2hPO
4or NaH
2pO
4; Buffer reagent is Hepes or Tris.
Can be enriched material at the reagent of described test kit, rear use to be prepared, also can plug and play.In one embodiment, the calcium concentration in a reagent is 20 ~ 1000mM, and buffer concentration is 5 ~ 200mM, pH is 6.0 ~ 10.0; PEG grafting cm-chitosan concentration in b reagent is 50 ~ 2000mg/L, and phosphate concn is 0.5 ~ 10mM, NaCl concentration is 50 ~ 500mM, and buffer concentration is 5 ~ 200mM, pH is 6.0 ~ 10.0.
As the preferred version of this embodiment, the calcium concentration in a reagent is 100 ~ 300mM, and buffer concentration is 20 ~ 60mM, pH is 7.0 ~ 8.0; PEG grafting cm-chitosan concentration in b reagent is 200 ~ 800mg/L, and phosphate concn is 1.5 ~ 3.0mM, NaCl concentration is 100 ~ 300mM, and buffer concentration is 20 ~ 60mM, pH is 7.0 ~ 8.0.
Novel hybride nano-calcium phosphate genes delivery system of the present invention compared with prior art, production process not with an organic solvent, surface catalyst, process is simply controlled, and Financial cost is low, reproducible, is suitable for scale operation; Vector stabilisation is good, and security is good, and inside and outside toxicity is extremely low; And transfection efficiency is high, the gene delivery of the effective inside and outside of energy, realizes gene therapy.
The invention will be further elaborated by the following examples.
Embodiment 1: a kind of preparation of novel hybride nano-calcium phosphate genes delivery system.
1) reagent a is prepared, containing CaCl
2for 250mM, Tris are the aqueous solution of 20mM, salt acid for adjusting pH is 7.4;
2) prepare reagent b, the concentration containing PEG grafting cm-chitosan is 200mg/L, Na
2hPO
4the aqueous solution of concentration is 1.5mM, NaCl concentration to be 280mM, Hepes concentration be 50mM, NaOH regulates pH to be 7.4;
3) 40 μ g gene DNA molecule and 1mL reagent mix are got, more even with reagent b short mix, namely prepare the Gene transfer vector of hybridized nanometer calcium phosphate.
Embodiment 2: a kind of preparation of novel hybride nano-calcium phosphate genes delivery system.
1) reagent a is prepared, containing CaCl
2for 250mM, Tris are the aqueous solution of 20mM, salt acid for adjusting pH is 7.4;
2) prepare reagent b, the concentration containing PEG grafting cm-chitosan is 200mg/L, Na
2hPO
4the aqueous solution of concentration is 1.5mM, NaCl concentration to be 280mM, Hepes concentration be 50mM, NaOH regulates pH to be 7.4;
3) 120 μ g gene DNA molecule and 1mL reagent mix are got, more even with reagent b short mix, namely prepare the Gene transfer vector of hybridized nanometer calcium phosphate.
Embodiment 3: a kind of preparation of novel hybride nano-calcium phosphate genes delivery system.
1) reagent a is prepared, containing CaCl
2for 250mM, Tris are the aqueous solution of 20mM, salt acid for adjusting pH is 7.4;
2) prepare reagent b, the concentration containing PEG grafting cm-chitosan is 200mg/L, Na
2hPO
4the aqueous solution of concentration is 1.5mM, NaCl concentration to be 280mM, Hepes concentration be 50mM, NaOH regulates pH to be 7.4;
3) 80 μ g gene DNA molecule and 1mL reagent mix are got, more even with reagent b short mix, namely prepare the Gene transfer vector of hybridized nanometer calcium phosphate.
Embodiment 4: a kind of preparation of novel hybride nano-calcium phosphate genes delivery system.
1) reagent a is prepared, containing CaCl
2for 250mM, Tris are the aqueous solution of 20mM, salt acid for adjusting pH is 7.4;
2) prepare reagent b, the concentration containing PEG grafting cm-chitosan is 800mg/L, Na
2hPO
4the aqueous solution of concentration is 1.5mM, NaCl concentration to be 280mM, Hepes concentration be 50mM, NaOH regulates pH to be 7.4;
3) 80 μ g gene DNA molecule and 1mL reagent mix are got, more even with reagent b short mix, namely prepare the Gene transfer vector of hybridized nanometer calcium phosphate.
Embodiment 5: a kind of preparation of novel hybride nano-calcium phosphate genes delivery system.
1) reagent a is prepared, containing CaCl
2for 250mM, Tris are the aqueous solution of 20mM, salt acid for adjusting pH is 7.4;
2) prepare reagent b, the concentration containing PEG grafting cm-chitosan is 400mg/L, Na
2hPO
4the aqueous solution of concentration is 1.5mM, NaCl concentration to be 280mM, Hepes concentration be 50mM, NaOH regulates pH to be 7.4;
3) 80 μ g gene siRNA molecule and 1mL reagent mix are got, more even with reagent b short mix, namely prepare the Gene transfer vector of hybridized nanometer calcium phosphate.
Embodiment 6: a kind of preparation of novel hybride nano-calcium phosphate genes delivery system.
1) reagent a is prepared, containing CaCl
2for 250mM, Tris are the aqueous solution of 20mM, salt acid for adjusting pH is 7.4;
2) prepare reagent b, the concentration containing PEG grafting cm-chitosan is 800mg/L, Na
2hPO
4the aqueous solution of concentration is 1.5mM, NaCl concentration to be 280mM, Hepes concentration be 50mM, NaOH regulates pH to be 7.4;
3) 80 μ g gene miRNA molecule and 1mL reagent mix are got, more even with reagent b short mix, namely prepare the Gene transfer vector of hybridized nanometer calcium phosphate.
As shown in Figure 1, novel hybride nano-calcium phosphate genes delivery system of the present invention is equally distributed spherical, and particle diameter is 60 ~ 100nm.
Embodiment 7: a kind of cell transfecting of novel hybride nano-calcium phosphate genes delivery system.
Get growth logarithmic phase HepG2 plating cells in 24 orifice plates, after growth 24h, cell confluency degree is 50 ~ 60%.Be loaded with the novel hybride nano-calcium phosphate genes delivery system cell administration of the present invention of the siRNA of target hTERT gene, every hole siRNA concentration is 0.5 μ g.After transfection 48h, extract the expression that RNA, RT-PCR measure hTERT gene.Lipofectamine2000 with identical siRNA concentration transfectional cell, in contrast.
As shown in Figure 2, the efficiency gene transfection being loaded with the novel hybride nano-calcium phosphate genes delivery system of the present invention of target hTERT gene siRNA is suitable with commercial preparation Lipofectamine2000.
Embodiment 8:MTT method measures the cytotoxicity of novel hybride nano-calcium phosphate genes delivery system of the present invention.
Growth logarithmic phase HepG2 plating cells is in 96 orifice plates, and after growth 24h, cell confluency degree is 70 ~ 80%.Suck nutrient solution, be loaded with the novel hybride nano-calcium phosphate genes delivery system of the present invention of luciferase plasmids pGL3 by concentration gradient administration.After 48h, add MTT solution (5mg/mLinpH7.4PBS) 10 μ L, 37 DEG C, continue to hatch 4h.Abandoning supernatant, adds 150 μ LDMSO and dissolves the crystallization of hepatic first a ceremonial jade-ladle, used in libation, measure absorbancy with enzyme-linked immunoassay instrument in 570nm.Calculate cells survival rate.
As shown in Figure 3, at mrna concentration 0.5 ~ 50 μ g/mL, cells survival rate, all 100%, shows the hypotoxicity of novel hybride nano-calcium phosphate genes delivery system of the present invention to experimental result.
Embodiment 9: novel hybride nano-calcium phosphate genes delivery system of the present invention vivo gene therapy effect.
With 5 × 10
6hepG2 cell is inoculated in nude mice oxter, obtains tumour nude mice model.When gross tumor volume reaches 100mm
3time, nude mice is divided into 3 groups at random, often organizes 5, and each group tail intravenously administrable respectively: the novel hybride nano-calcium phosphate genes delivery system of the present invention carrying therapeutic gene, carries the novel hybride nano-calcium phosphate genes delivery system of the present invention of non-treatment gene, PBS.Measure gross tumor volume over time.Result is as accompanying drawing 4.
Experimental result shows, intravenous injection is loaded with the novel hybride nano-calcium phosphate genes delivery system of the present invention of therapeutic gene, can the growth of effective Tumor suppression, thus reaches the object of systematic treating tumour.
Embodiment 10: the immunotoxicity of novel hybride nano-calcium phosphate genes delivery system of the present invention is investigated.
Healthy nude mice is divided into two groups at random, often organizes 5, respectively administration novel hybride nano-calcium phosphate of the present invention genes delivery system and PBS.After administration 4h, nude mice afterbody gets blood, the concentration of the immune factors such as IFN-γ, IL-2 and IL-6 in euzymelinked immunosorbent assay (ELISA) measurement blood plasma.
As shown in Figure 5, compared with control group, intravenous injection novel hybride nano-calcium phosphate of the present invention genes delivery system does not cause the increase of immune factor to immunotoxicity result, and the low immunotoxicity of this carrier is described.
In conjunction with specific embodiments embodiments of the present invention are described in detail above, but the invention is not restricted to above-mentioned embodiment, in the ken that art those of ordinary skill possesses, can also make a variety of changes under the prerequisite not departing from present inventive concept.
Claims (8)
1. a novel hybride nano-calcium phosphate genes delivery system, comprise calcium phosphate and PEG grafting cm-chitosan, it is characterized in that, calcium phosphate parcel gene forms kernel, PEG grafting cm-chitosan is coated on core surface, PEG chain segment forms wetting ability shell, thus form the nanoparticle with PEGization nucleocapsid structure, the viscosity-average molecular weight of wherein said PEG grafting cm-chitosan is 1 ~ 1,000,000, deacetylation is greater than 80%, degree of substitution by carboxymethyl is 20 ~ 90%, PEG molecular weight is 500 ~ 10000, PEG percentage of grafting is 8% ~ 60%, and the particle diameter of this nanoparticle is 60 ~ 100nm.
2. novel hybride nano-calcium phosphate genes delivery system as claimed in claim 1, it is characterized in that, the viscosity-average molecular weight of described PEG grafting cm-chitosan is 10 ~ 400,000, deacetylation is greater than 80%, degree of substitution by carboxymethyl is 40 ~ 70%, PEG molecular weight is 2000 ~ 6000, PEG percentage of grafting is 20% ~ 40%.
3. prepare a method for novel hybride nano-calcium phosphate genes delivery system, comprise the following steps:
1) prepare reagent a, comprise calcium salt, buffer reagent, surplus is water;
2) prepare reagent b, comprise PEG grafting cm-chitosan, phosphoric acid salt, sodium-chlor, buffer reagent, surplus is water;
3) gene is mixed with reagent a, then mixes with reagent b equal-volume,
It is characterized in that,
In described reagent a, calcium salt is CaCl
2, Ca (NO
3)
2in the combination of one or both arbitrary proportions, buffer reagent is one or more the arbitrary proportion combination of Hepes, MOPS, PBS, PIPES, Tris;
In described b reagent, the viscosity-average molecular weight of PEG grafting cm-chitosan is 1 ~ 1,000,000, and deacetylation is greater than 80%, and degree of substitution by carboxymethyl is 20 ~ 90%, PEG molecular weight be 500 ~ 10000, PEG percentage of grafting is 8% ~ 60%; Phosphoric acid salt comprises Na
3pO
4, Na
2hPO
4, NaH
2pO
4, K
3pO
4, K
2hPO
4, KH
2pO
4, (NH4)
3pO
4, (NH4)
2hPO
4one or more arbitrary proportion combination; Buffer reagent is one or more the arbitrary proportion combination in Hepes, MOPS, PBS, PIPES, Tris.
4. prepare the method for novel hybride nano-calcium phosphate genes delivery system as claimed in claim 3, it is characterized in that, the calcium salt in reagent a is CaCl
2or Ca (NO
3)
2, buffer reagent is Hepes or Tris; The viscosity-average molecular weight of the PEG grafting cm-chitosan in reagent b is 10 ~ 400,000, and deacetylation is greater than 80%, and degree of substitution by carboxymethyl is 40 ~ 70%, PEG molecular weight be 2000 ~ 6000, PEG percentage of grafting is 20% ~ 40%; Phosphoric acid salt is Na
2hPO
4or NaH
2pO
4; Buffer reagent is Hepes or Tris.
5. prepare the method for novel hybride nano-calcium phosphate genes delivery system as claimed in claim 3, it is characterized in that, the ratio of gene quality when gene mixes with reagent a and reagent a volume is 10 ~ 200 μ g/mL.
6. prepare the method for novel hybride nano-calcium phosphate genes delivery system as claimed in claim 5, it is characterized in that, the ratio of gene quality and reagent a volume is 40 ~ 120 μ g/mL.
7. the method preparing novel hybride nano-calcium phosphate genes delivery system according to any one of claim 3 to 6, is characterized in that, the calcium concentration in described a reagent is 20 ~ 1000mM, and buffer concentration is 5 ~ 200mM, pH is 6.0 ~ 10.0; PEG grafting cm-chitosan concentration in described b reagent is 50 ~ 2000mg/L, and phosphate concn is 0.5 ~ 10mM, NaCl concentration is 50 ~ 500mM, and buffer concentration is 5 ~ 200mM, pH is 6.0 ~ 10.0.
8. prepare the method for novel hybride nano-calcium phosphate genes delivery system as claimed in claim 7, it is characterized in that, the calcium concentration in described a reagent is 100 ~ 300mM, and buffer concentration is 20 ~ 60mM, pH is 7.0 ~ 8.0; PEG grafting cm-chitosan concentration in described b reagent is 200 ~ 800mg/L, and phosphate concn is 1.5 ~ 3.0mM, NaCl concentration is 100 ~ 300mM, and buffer concentration is 20 ~ 60mM, pH is 7.0 ~ 8.0.
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| PEG-grafted chitosan as aninjectable thermosensitive hydrogel for sustained protein release;Narayan Bhattarai,et al;《Journal of Controlled Release》;20050418;全文 * |
| 壳聚糖的功能化修饰及其在药物释放系统上的应用研究;邵晓红;《中国优秀硕士学位论文全文数据库》;20140115;全文 * |
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