CN103789348A - Novel hybrid nanometer calcium phosphate gene delivery system and preparation method thereof - Google Patents

Abstract

Translated fromChinese

本发明公开了一种新型杂化纳米磷酸钙基因递送系统以及其制备方法，该基因递送系统包括磷酸钙及PEG接枝羧甲基壳聚糖，磷酸钙包裹基因形成内核，PEG接枝羧甲基壳聚糖包覆于内核表面，PEG链段形成亲水性外壳，从而形成具有PEG化核壳结构的纳米粒子，该纳米粒子的粒径为60～100nm。本发明的新型杂化纳米磷酸钙基因递送系统，在制备过程中无需使用有机溶剂、表面催化剂，过程简单可控，经济成本低，重复性好，适合于大规模生产，毒性低，转染效率高。另外，本发明还涉及制备该基因递送系统的试剂盒，以及本发明基因递送系统的体内外基因转染与基因治疗的用途。

The invention discloses a novel hybrid nano-calcium phosphate gene delivery system and its preparation method. The gene delivery system comprises calcium phosphate and PEG grafted with carboxymethyl chitosan, calcium phosphate wraps genes to form a core, and PEG is grafted with carboxymethyl chitosan. The base chitosan coats the surface of the inner core, and the PEG chain segment forms a hydrophilic shell, thereby forming nanoparticles with a PEGylated core-shell structure, and the particle diameter of the nanoparticles is 60-100nm. The novel hybrid nano calcium phosphate gene delivery system of the present invention does not need to use organic solvents and surface catalysts in the preparation process, the process is simple and controllable, the economic cost is low, the repeatability is good, it is suitable for large-scale production, the toxicity is low, and the transfection efficiency high. In addition, the present invention also relates to a kit for preparing the gene delivery system, and the use of the gene delivery system in vivo and in vitro for gene transfection and gene therapy.

Description

A kind of novel hybride nano-calcium phosphate genes delivery system and preparation method thereof

Technical field

The present invention relates to field of pharmaceutical preparations and biological medicine technology 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 that has future from gene level treatment disease.Gene therapy is that regulation and control relate to the expression of the key protein of disease generation, development by nucleic acid (DNA, siRNA, miRNA) being led to people's tissue and cell, thereby reaches the object of disease treatment.But nucleic acid molecule, due to large, the electronegative property of its molecular weight, hydrophilic feature, can not effectively enter cell; In addition, nucleic acid molecule is very easily by the nuclease degradation in plasma proteins.So nucleic acid molecule need to carry the object that could realize drug treatment by carrier bag.

It is the committed step that realizes gene therapy that nucleic acid transfered cell is expressed, and successfully 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, exist larger potential safety hazard.Non-virus carrier is mainly to prepare by cationic polymers or lipid and nucleic acid the cation carrier obtaining by electrostatic adhesion, compared with virus vector, non-virus carrier have safety, effectively, the advantage such as non-immunogenicity.Cation carrier can effectively wrap up nucleic acid, promotes that nucleic acid enters cell.But positively charged ion non-virus carrier has the cytotoxicity of bringing due to cationic characteristic conventionally, and be easy to precipitate in blood plasma, thereby suppress the drug treatment in its body.

Calcium phosphate precipitation method is widely used in cell transfecting, and calcium phosphate has good biocompatibility, biological degradability, and can effectively 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 preparing; Precipitation calcium phosphate particle and can cause cytotoxicity.Application in the poor body that has stoped calcium phosphate of colloidal stability.

Preparing stable calcium phosphate nano particle becomes and realizes safety in nucleic acid body, the effective effective way of sending.Leaf Huang etc. has prepared the calcium phosphate hybridized nanometer grain of liposome; the system of nucleic acid after intravenous injection that realized is sent; what they adopted is that reverse micelle of microemulsion has been prepared coated calcium phosphate nano grain (the Li J of stable lipid; Chen YC; Tseng YC; Mozumdar S; Huang L.Biodegradable calcium phosphate nanoparticle, with lipid coating for systemic siRNA delivery.Journal of controlled release.2010; 142:416-21.).Chinese patent application (200910264114.6) has been authorized " a kind of calcium phosphate composite nanoparticle of carrying genes and method for making thereof and purposes ", has also adopted micro emulsion legal system for calcium phosphate nano grain.But this preparation method's relative complex and used and have the organic solvent of genotoxic potential and tensio-active agent, 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 development to gene therapy is of great importance.

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 segment forms wetting ability shell, thereby 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 that 40～70%, PEG molecular weight is that 2000～6000, PEG percentage of grafting is 20%～40%.

The present invention more provides a kind of method of 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 CaCl₂, Ca (NO₃)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 that 20～90%, PEG molecular weight is that 500～10000, PEG percentage of grafting is 8%～60%; Phosphoric acid salt comprises Na₃pO₄, Na₂hPO₄, NaH₂pO₄, K₃pO₄, K₂hPO₄, KH₂pO₄, (NH4)₃pO₄, (NH4)₂hPO₄one 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 CaCl₂or Ca (NO₃)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 that 40～70%, PEG molecular weight is that 2000～6000, PEG percentage of grafting is 20%～40%; Phosphoric acid salt is Na₂hPO₄or NaH₂pO₄; Buffer reagent is Hepes or Tris.

Preferably, (μ is g) 10～200 μ g/mL with the ratio of reagent a volume (mL) to gene quality when gene mixes with reagent a.

More preferably, (μ is g) 40～120 μ g/mL with the ratio of reagent a volume (mL) to gene quality.

As preferred version, the calcium concentration in described a reagent is 20～1000mM, and buffer concentration is 5～200mM, and 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, and NaCl concentration is 50～500mM, and buffer concentration is 5～200mM, and pH is 6.0～10.0.

Further preferably, the calcium concentration in described a reagent is 100～300mM, and buffer concentration is 20～60mM, and 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, and NaCl concentration is 100～300mM, and buffer concentration is 20～60mM, and pH is 7.0～8.0.

On the other hand, the invention provides above-mentioned novel hybride nano-calcium phosphate genes delivery system for the purposes that in the gene transfection of inside and outside cell and human or animal body, genomic medicine is carried, wherein said gene is to appoint 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 its aqueous solution that comprises buffer reagent; And PEG grafting cm-chitosan, phosphoric acid salt, sodium-chlor or its mixed aqueous solution that comprises buffer reagent, it is characterized in that, calcium salt or its aqueous solution that comprises buffer reagent are to separate independent packaging with PEG grafting cm-chitosan, phosphoric acid salt, sodium-chlor or its mixed aqueous solution that comprises buffer reagent, and wherein calcium salt is CaCl₂, Ca (NO₃)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 that 20～90%, PEG molecular weight is that 500～10000, PEG percentage of grafting is 8%～60%; Phosphoric acid salt is Na₃pO₄, Na₂hPO₄, NaH₂pO₄, K₃pO₄, K₂hPO₄, KH₂pO₄, (NH4)₃pO₄, (NH4)₂hPO₄in 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, Financial cost is low, reproducible, is suitable for scale operation; Carrier good stability, security is good, and inside and outside toxicity is extremely low; Transfection efficiency is high, can effectively realize the gene delivery of inside and outside, realizes gene therapy.This carrier has advantages of that preparation is simple, Financial cost is low, easy to use and reliable, transfection efficiency is high, good biocompatibility, can be used for 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 treatment 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, can, by electrostatic interaction active adsorption in positive polarity calcium phosphate granules sub-surface, reach the object of stablizing calcium phosphate particle.In addition, PEGization also can, by shielding effect stabilized nanoscale carrier, extend cycling time, and reaching long circulating effect increases curative effect.Therefore, PEG grafting cm-chitosan has the potentiality of stablizing calcium phosphate and obtaining hybridized nanometer grain, sends thereby realize in the safe and effective body of nucleic acid, realizes the gene therapy of disease.In addition, by the nanometer precipitating action of PEG grafting cm-chitosan and calcium phosphate, self-assembly forms nanoparticle, has more simplified preparation production process.

The invention provides a kind of novel hybride nano-calcium phosphate genes delivery system, overcome the shortcoming of prior art, improved the inside and outside transfection efficiency of gene, promote the development of gene therapy.This carrier has advantages of that preparation is simple, Financial cost is low, easy to use and reliable, transfection efficiency is high, good biocompatibility, can be used for body inner injecting and administering.

On the one hand, novel hybride nano-calcium phosphate genes delivery system of the present invention comprises calcium phosphate, two kinds of components of PEG grafting cm-chitosan; Nucleocapsid structure, calcium phosphate forms kernel, and PEG grafting cm-chitosan is coated on core surface, and PEG segment forms wetting ability shell; Particle diameter is 60～100nm.

Described novel hybride nano-calcium phosphate genes delivery system can wrap the gene molecule carrying and comprise DNA, siRNA, miRNA, shRNA, and gene molecule is loaded in calcium phosphate kernel by bag.

On the other hand, the method for preparing novel hybride nano-calcium phosphate genes delivery system of the present invention comprises the steps:

1) prepare reagent a, the aqueous solution that comprises calcium salt, buffer reagent;

2) prepare reagent b, the aqueous solution that comprises 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 CaCl₂, Ca (NO₃)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 that 20～90%, PEG molecular weight is that 500～10000, PEG percentage of grafting is 8%～60%; Phosphoric acid salt, comprises Na₃pO₄, Na₂hPO₄, NaH₂pO₄, K₃pO₄, K₂hPO₄, KH₂pO₄, (NH4)₃pO₄, (NH4)₂hPO₄one or more arbitrary proportion combination; Buffer reagent is one or more the arbitrary proportion combination of Hepes, MOPS, PBS, PIPES, Tris.

As preferred version, calcium salt is CaCl₂or Ca (NO₃)₂, buffer reagent is Hepes or Tr is.

As preferred version, 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 that 40～70%, PEG molecular weight is that 2000～6000, PEG percentage of grafting is 20%～40%; Phosphoric acid salt is Na₂hPO₄or NaH₂pO₄; Buffer reagent is Hepes or Tris.

Further, in described a reagent, calcium concentration is 20～1000mM, and buffer concentration is 5～200mM, and 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, and NaCl concentration is 50～500mM, and buffer concentration is 5～200mM, and pH is 6.0～10.0.

Further, as preferred version, in described a reagent, calcium concentration is 100～300mM, and buffer concentration is 20～60mM, and 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, and NaCl concentration is 100～300mM, and buffer concentration is 20～60mM, and pH is 7.0～8.0.

In preparation method's step (3) of described novel hybride nano-calcium phosphate genes delivery system, (μ is g) 10～200 μ g/mL with the ratio of reagent a volume (mL) to gene quality when gene first mixes with reagent a; As preferred version, (μ is g) 40～120 μ g/mL with the ratio of reagent a volume (mL) to gene quality.

On the other hand, provide the purposes of described novel hybride nano-calcium phosphate genes delivery system, be gene transfection and the interior genomic medicine conveying of human or animal body of inside and outside cell; 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 of preparing for described novel hybride nano-calcium phosphate genes delivery system, two kinds of reagent of a, b that this test kit contains independent packaging: reagent a, the aqueous solution that comprises calcium salt, buffer reagent; Reagent b, the aqueous solution that comprises PEG grafting cm-chitosan, phosphoric acid salt, sodium-chlor, buffer reagent.

In a reagent of described test kit, calcium salt is for comprising CaCl₂, Ca (NO₃)₂the combination of one or both arbitrary proportions, buffer reagent is one or more the arbitrary proportion combination of Hepes, MOPS, PBS, PIPES, T ris.

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 that 20～90%, PEG molecular weight is that 500～10000, PEG percentage of grafting is 8%～60%; Phosphoric acid salt, comprises Na₃pO₄, Na₂hPO₄, NaH₂pO₄, K₃pO₄, K₂hPO₄, KH₂pO₄, (NH4)₃pO₄, (NH4)₂hPO₄one 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 CaCl₂or Ca (NO₃)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 that 40～70%, PEG molecular weight is that 2000～6000, PEG percentage of grafting is 20%～40%; Phosphoric acid salt is Na₂hPO₄or NaH₂pO₄; 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, and 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, and NaCl concentration is 50～500mM, and buffer concentration is 5～200mM, and 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, and 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, and NaCl concentration is 100～300mM, and buffer concentration is 20～60mM, and 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, Financial cost is low, reproducible, is suitable for scale operation; Carrier good stability, security is good, and inside and outside toxicity is extremely low; And transfection efficiency is high, effectively the gene delivery of inside and outside, 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) prepare reagent a, contain CaCl₂for the aqueous solution that 250mM, Tris are 20mM, salt acid for adjusting pH is 7.4;

2) prepare reagent b, the concentration that contains PEG grafting cm-chitosan is 200mg/L, Na₂hPO₄concentration is that 1.5mM, NaCl concentration are the aqueous solution that 280mM, Hepes concentration are 50mM, and it is 7.4 that NaOH regulates pH;

3) get 40 μ g gene DNA molecule and 1mL reagent mix, more even with reagent b short mix, prepare the gene transfection carrier of hybridized nanometer calcium phosphate.

Embodiment 2: a kind of preparation of novel hybride nano-calcium phosphate genes delivery system.

1) prepare reagent a, contain CaCl₂for the aqueous solution that 250mM, Tris are 20mM, salt acid for adjusting pH is 7.4;

2) prepare reagent b, the concentration that contains PEG grafting cm-chitosan is 200mg/L, Na₂hPO₄concentration is that 1.5mM, NaCl concentration are the aqueous solution that 280mM, Hepes concentration are 50mM, and it is 7.4 that NaOH regulates pH;

3) get 120 μ g gene DNA molecule and 1mL reagent mix, more even with reagent b short mix, prepare the gene transfection carrier of hybridized nanometer calcium phosphate.

Embodiment 3: a kind of preparation of novel hybride nano-calcium phosphate genes delivery system.

1) prepare reagent a, contain CaCl₂for the aqueous solution that 250mM, Tris are 20mM, salt acid for adjusting pH is 7.4;

2) prepare reagent b, the concentration that contains PEG grafting cm-chitosan is 200mg/L, Na₂hPO₄concentration is that 1.5mM, NaCl concentration are the aqueous solution that 280mM, Hepes concentration are 50mM, and it is 7.4 that NaOH regulates pH;

3) get 80 μ g gene DNA molecule and 1mL reagent mix, more even with reagent b short mix, prepare the gene transfection carrier of hybridized nanometer calcium phosphate.

Embodiment 4: a kind of preparation of novel hybride nano-calcium phosphate genes delivery system.

1) prepare reagent a, contain CaCl₂for the aqueous solution that 250mM, Tris are 20mM, salt acid for adjusting pH is 7.4;

2) prepare reagent b, the concentration that contains PEG grafting cm-chitosan is 800mg/L, Na₂hPO₄concentration is that 1.5mM, NaCl concentration are the aqueous solution that 280mM, Hepes concentration are 50mM, and it is 7.4 that NaOH regulates pH;

3) get 80 μ g gene DNA molecule and 1mL reagent mix, more even with reagent b short mix, prepare the gene transfection carrier of hybridized nanometer calcium phosphate.

Embodiment 5: a kind of preparation of novel hybride nano-calcium phosphate genes delivery system.

1) prepare reagent a, contain CaCl₂for the aqueous solution that 250mM, Tris are 20mM, salt acid for adjusting pH is 7.4;

2) prepare reagent b, the concentration that contains PEG grafting cm-chitosan is 400mg/L, Na₂hPO₄concentration is that 1.5mM, NaCl concentration are the aqueous solution that 280mM, Hepes concentration are 50mM, and it is 7.4 that NaOH regulates pH;

3) get 80 μ g gene siRNA molecule and 1mL reagent mix, more even with reagent b short mix, prepare the gene transfection carrier of hybridized nanometer calcium phosphate.

Embodiment 6: a kind of preparation of novel hybride nano-calcium phosphate genes delivery system.

1) prepare reagent a, contain CaCl₂for the aqueous solution that 250mM, Tris are 20mM, salt acid for adjusting pH is 7.4;

2) prepare reagent b, the concentration that contains PEG grafting cm-chitosan is 800mg/L, Na₂hPO₄concentration is that 1.5mM, NaCl concentration are the aqueous solution that 280mM, Hepes concentration are 50mM, and it is 7.4 that NaOH regulates pH;

3) get 80 μ g gene miRNA molecule and 1mL reagent mix, more even with reagent b short mix, prepare the gene transfection carrier 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 cell and be plated on 24 orifice plates, after growth 24h, cell degree of converging is 50～60%.Be loaded with the novel hybride nano-calcium phosphate genes delivery system cell of the present invention administration of the siRNA of target hTERT gene, every hole siRNA concentration is 0.5 μ g.After transfection 48h, extract RNA, RT-PCR measures the expression of hTERT gene.Lipofectamine2000 is with identical siRNA concentration transfectional cell, in contrast.

As shown in Figure 2, be loaded with the efficiency gene transfection of novel hybride nano-calcium phosphate genes delivery system of the present invention of target hTERT gene siRNA suitable with commercial preparation Lipofectamine2000.

Embodiment 8:MTT method is measured the cytotoxicity of novel hybride nano-calcium phosphate genes delivery system of the present invention.

Growth logarithmic phase HepG2 cell is plated on 96 orifice plates, and after growth 24h, cell degree of converging 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/mL in pH7.4PBS) 10 μ L, 37 ℃, continue to hatch 4h.Abandoning supernatant, adds 150 μ L DMSO to dissolve the crystallization of hepatic first a ceremonial jade-ladle, used in libation, measures 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 result for the treatment of.

With 5 × 10₆hepG2 cell is inoculated in nude mice oxter, obtains tumour nude mice model.When gross tumor volume reaches 100mm³time, nude mice is divided into 3 groups at random, and 5 every group, each group tail intravenously administrable respectively: carry the novel hybride nano-calcium phosphate genes delivery system of the present invention of therapeutic gene, carry the novel hybride nano-calcium phosphate genes delivery system of the present invention of non-therapeutic gene, PBS.Measure gross tumor volume over time.Result is as accompanyingdrawing 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 effectively suppress the growth of tumour, thereby 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, and 5 every group, administration novel hybride nano-calcium phosphate of the present invention genes delivery system and PBS respectively.After administration 4h, nude mice afterbody is got blood, and euzymelinked immunosorbent assay (ELISA) is measured the concentration of the immune factor such as IFN-γ, IL-2 and IL-6 in 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 possessing at affiliated technical field those of ordinary skill, can also under the prerequisite that does not depart from aim of the present invention, make a variety of changes.

Claims (10)

Translated fromChinese

1.一种新型杂化纳米磷酸钙基因递送系统，包括磷酸钙及PEG接枝羧甲基壳聚糖，其特征在于，磷酸钙包裹基因形成内核，PEG接枝羧甲基壳聚糖包覆于内核表面，PEG链段形成亲水性外壳，从而形成具有PEG化核壳结构的纳米粒子，其中所述PEG接枝羧甲基壳聚糖的粘均分子量为1～100万，脱乙酰度大于80%，羧甲基取代度为20～90%，PEG分子量为500～10000，PEG接枝率为8%～60%，并且该纳米粒子的粒径为60～100nm。1. A novel hybrid nano-calcium phosphate gene delivery system, comprising calcium phosphate and PEG-grafted carboxymethyl chitosan, characterized in that the calcium phosphate-wrapped gene forms a core, and the PEG-grafted carboxymethyl-chitosan is coated On the surface of the inner core, the PEG segment forms a hydrophilic shell, thereby forming nanoparticles with a PEGylated core-shell structure, wherein the viscosity-average molecular weight of the PEG-grafted carboxymethyl chitosan is 1 to 1 million, and the degree of deacetylation is greater than 80%, the degree of carboxymethyl substitution is 20-90%, the molecular weight of PEG is 500-10000, the grafting rate of PEG is 8%-60%, and the particle size of the nanoparticles is 60-100nm.2.如权利要求1所述的新型杂化纳米磷酸钙基因递送系统，其特征在于，所述PEG接枝羧甲基壳聚糖的粘均分子量为10～40万，脱乙酰度大于80%，羧甲基取代度为40～70%，PEG分子量为2000～6000，PEG接枝率为20%～40%。2. The novel hybrid nano-calcium phosphate gene delivery system as claimed in claim 1, wherein the viscosity-average molecular weight of the PEG-grafted carboxymethyl chitosan is 100,000 to 400,000, and the degree of deacetylation is greater than 80%. , the degree of carboxymethyl substitution is 40-70%, the molecular weight of PEG is 2000-6000, and the grafting rate of PEG is 20%-40%.3.一种制备新型杂化纳米磷酸钙基因递送系统的方法，包括以下步骤：3. A method for preparing a novel hybrid nano calcium phosphate gene delivery system, comprising the following steps:1）制备试剂a，包含钙盐、缓冲剂，余量为水；1) Prepare reagent a, including calcium salt, buffer, and water as the balance;2）制备试剂b，包含PEG接枝羧甲基壳聚糖、磷酸盐、氯化钠、缓冲剂，余量为水；2) Preparation of reagent b, including PEG-grafted carboxymethyl chitosan, phosphate, sodium chloride, buffer, and water as the balance;3）将基因与试剂a混合，再与试剂b等体积混合均匀，3) Mix the gene with reagent a, and then mix it with reagent b in equal volume,其特征在于，It is characterized in that,在所述试剂a中，钙盐为CaCl₂、Ca(NO₃)₂中的一种或两种任意比例的组合，缓冲剂为Hepes、MOPS、PBS、PIPES、Tris的一种或两种以上的任意比例组合；In the reagent a, the calcium salt is one of CaCl₂ , Ca(NO₃ )₂ or a combination of two in any ratio, and the buffer is one or more of Hepes, MOPS, PBS, PIPES, and Tris Any combination of proportions;在所述b试剂中，PEG接枝羧甲基壳聚糖的粘均分子量为1～100万，脱乙酰度大于80%，羧甲基取代度为20～90%，PEG分子量为500～10000，PEG接枝率为8%～60%；磷酸盐包括Na₃PO₄、Na₂HPO₄、NaH₂PO₄、K₃PO₄、K₂HPO₄、KH₂PO₄、(NH4)₃PO₄、(NH4)₂HPO₄的一种或两种以上的任意比例组合；缓冲剂为Hepes、MOPS、PBS、PIPES、Tris中的一种或两种以上的任意比例组合。In the b reagent, the viscosity-average molecular weight of PEG-grafted carboxymethyl chitosan is 1-1 million, the degree of deacetylation is greater than 80%, the degree of carboxymethyl substitution is 20-90%, and the molecular weight of PEG is 500-10000 , PEG grafting rate is 8% to 60%; phosphates include Na₃ PO₄ , Na₂ HPO₄ , NaH₂ PO₄ , K₃ PO₄ , K₂ HPO₄ , KH₂ PO₄ , (NH4)₃ PO_4. One of (NH4)₂ HPO₄ or a combination of two or more in any ratio; the buffer is one of Hepes, MOPS, PBS, PIPES, Tris or a combination of two or more in any ratio.4.如权利要求3所述的制备新型杂化纳米磷酸钙基因递送系统的方法，其特征在于，试剂a中的钙盐为CaCl₂或Ca(NO₃)₂，缓冲剂为Hepes或Tris；试剂b中的PEG接枝羧甲基壳聚糖的粘均分子量为10～40万，脱乙酰度大于80%，羧甲基取代度为40～70%，PEG分子量为2000～6000，PEG接枝率为20%～40%；磷酸盐为Na₂HPO₄或NaH₂PO₄；缓冲剂为Hepes或Tris。4. the method for preparing novel hybrid nano calcium phosphate gene delivery system as claimed in claim 3, is characterized in that, the calcium salt in reagent a is CaCl₂ or Ca(NO₃ )₂ , buffering agent is Hepes or Tris; The viscosity-average molecular weight of PEG-grafted carboxymethyl chitosan in reagent b is 100,000-400,000, the degree of deacetylation is greater than 80%, the degree of carboxymethyl substitution is 40-70%, the molecular weight of PEG is 2000-6000, and the PEG-grafted The branch rate is 20% to 40%; the phosphate is Na₂ HPO₄ or NaH₂ PO₄ ; the buffer is Hepes or Tris.5.如权利要求3所述的制备新型杂化纳米磷酸钙基因递送系统的方法，其特征在于，基因与试剂a混合时的基因质量（μg）与试剂a体积（mL）的比例为10～200μg/mL。5. The method for preparing a novel hybrid nano-calcium phosphate gene delivery system as claimed in claim 3, wherein the ratio of the gene mass (μg) to the reagent a volume (mL) when the gene is mixed with the reagent a is 10- 200 μg/mL.6.如权利要求5所述的制备新型杂化纳米磷酸钙基因递送系统的方法，其特征在于，基因质量（μg）与试剂a体积（mL）的比例为40～120μg/mL。6. The method for preparing a novel hybrid nano-calcium phosphate gene delivery system according to claim 5, wherein the ratio of gene mass (μg) to reagent a volume (mL) is 40-120 μg/mL.7.如权利要求3至6中任一项所述的制备新型杂化纳米磷酸钙基因递送系统的方法，其特征在于，所述a试剂中的钙盐浓度为20～1000mM，缓冲剂浓度为5～200mM，pH为6.0～10.0；所述b试剂中的PEG接枝羧甲基壳聚糖浓度为50～2000mg/L，磷酸盐浓度为0.5～10mM，NaCl浓度为50～500mM，缓冲剂浓度为5～200mM，pH为6.0～10.0。7. the method for preparing novel hybrid nanometer calcium phosphate gene delivery system as described in any one in claim 3 to 6, is characterized in that, the calcium salt concentration in described a reagent is 20～1000mM, and buffer concentration is 5-200mM, pH 6.0-10.0; the concentration of PEG-grafted carboxymethyl chitosan in the b reagent is 50-2000mg/L, the concentration of phosphate is 0.5-10mM, the concentration of NaCl is 50-500mM, the buffer The concentration is 5-200mM, and the pH is 6.0-10.0.8.如权利要求7所述的制备新型杂化纳米磷酸钙基因递送系统的方法，其特征在于，所述a试剂中的钙盐浓度为100～300mM，缓冲剂浓度为20～60mM，pH为7.0～8.0；所述b试剂中的PEG接枝羧甲基壳聚糖浓度为200～800mg/L，磷酸盐浓度为1.5～3.0mM，NaCl浓度为100～300mM，缓冲剂浓度为20～60mM，pH为7.0～8.0。8. The method for preparing a novel hybrid nano-calcium phosphate gene delivery system as claimed in claim 7, wherein the calcium salt concentration in the reagent a is 100 to 300 mM, the buffer concentration is 20 to 60 mM, and the pH is 7.0 to 8.0; the concentration of PEG-grafted carboxymethyl chitosan in the b reagent is 200 to 800 mg/L, the concentration of phosphate is 1.5 to 3.0 mM, the concentration of NaCl is 100 to 300 mM, and the concentration of buffer is 20 to 60 mM , pH is 7.0-8.0.9.如权利要求1或2所述的新型杂化纳米磷酸钙基因递送系统用于体内外细胞的基因转染以及人或动物体内基因药物输送的用途，其中所述基因是DNA、siRNA、miRNA或shRNA中的任一或多种。9. The novel hybrid nano-calcium phosphate gene delivery system as claimed in claim 1 or 2 is used for the gene transfection of cells in vivo and in vitro and the purposes of gene drug delivery in human or animal body, wherein said gene is DNA, siRNA, miRNA Or any one or more of shRNA.10.一种试剂盒，用于制备如权利要求1或2所述的新型杂化纳米磷酸钙基因递送系统，所述试剂盒包含钙盐或其包含缓冲剂的水溶液；以及PEG接枝羧甲基壳聚糖、磷酸盐、氯化钠或者其包含缓冲剂的混合水溶液，其特征在于，钙盐或其包含缓冲剂的水溶液与PEG接枝羧甲基壳聚糖、磷酸盐、氯化钠或者其包含缓冲剂的混合水溶液是分开独立包装的，其中钙盐为CaCl₂、_Ca(NO3)2中的一种或两种任意比例的组合；PEG接枝羧甲基壳聚糖的粘均分子量为1～100万，脱乙酰度大于80%，羧甲基取代度为20～90%，PEG分子量为500～10000，PEG接枝率为8%～60%；磷酸盐为Na₃PO₄、Na₂HPO₄、NaH₂PO₄、K₃PO₄、K₂HPO₄、KH₂PO₄、(NH4)₃PO₄、(NH4)₂HPO₄中的一种或两种以上的任意比例组合；缓冲剂为Hepes、MOPS、PBS、PIPES、Tris中的一种或两种以上的任意比例组合。10. A test kit, for preparing the novel hybrid nanometer calcium phosphate gene delivery system as claimed in claim 1 or 2, said test kit comprises calcium salt or its aqueous solution comprising buffer; and PEG grafted carboxymethyl Chitosan, phosphate, sodium chloride or its mixed aqueous solution comprising a buffer, is characterized in that calcium salt or its aqueous solution comprising a buffer and PEG grafted carboxymethyl chitosan, phosphate, sodium chloride Or its mixed aqueous solution containing a buffer is packaged separately and independently, wherein the calcium salt is one of CaCl₂ ,_{Ca(NO3) 2} or a combination of two in any ratio; the viscosity average of PEG grafted carboxymethyl chitosan The molecular weight is 1-1 million, the degree of deacetylation is greater than 80%, the degree of carboxymethyl substitution is 20-90%, the molecular weight of PEG is 500-10000, the grafting rate of PEG is 8%-60%; the phosphate is Na₃ PO₄ , Na₂ HPO₄ , NaH₂ PO₄ , K₃ PO₄ , K₂ HPO₄ , KH₂ PO₄ , (NH4)₃ PO₄ , (NH4)₂ HPO₄ , any ratio of two or more Combination; the buffer is one of Hepes, MOPS, PBS, PIPES, Tris or a combination of two or more in any ratio.

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