CN102532565B - Degradable imine polycation, synthesizing method thereof and nanoparticles - Google Patents

Abstract

The invention relates to a degradable imine polycation for conveying nucleic acid, a synthesizing method thereof and nanoparticles, belonging to the technical field of medicaments. The polycation returning to an initial state of a poly-ammonia molecule is constructed and degraded by using low-molecular-weight PEI (Polyetherimide) and glyoxal. The synthesizing method of the polycation comprises the following steps of: dropwise adding an aqueous solution of glyoxal of which the molar mass is equal to that of the PEI slowly into an ethanol solution of the PEI, and adding a dried molecular sieve simultaneously; and stirring at the temperature of 15-30 DEG C, and undergoing a condensation reaction on a primary amino radical and an aldehyde group. The degradable imine polycation is degradable, and releases low-molecular-weight PEI containing non-protonized amino in a degrading process; and moreover, degraded products do not generate acid groups like other degradable polymers. Escape of lysosomes is facilitated, and genes are released from cytoplasm with low surface charge. Specific for a proton sponge effect, the lysosomes are broken under the action of an osmotic pressure generated by the proton sponge effect. The amino bond of the polycation is taken as proton sponge.

Description

Degradable imines class polycation and synthetic method thereof, nano particle

Technical field

What the present invention relates to is a kind of synthetic method of technical field of pharmaceuticals, particularly a kind of degradable imines class polycation of the conveying that can be used for nucleic acid (DNA and RNA) and synthetic method thereof, nano particle.

Background technology

The key bottleneck that DNA and siRNA medicine move towards clinical application from concept is the research and development of delivery system in its body.In all kinds of genophores, the polycation carrier compare with virus and liposome vectors have gene support density and loading all big, preparation is simple, be convenient to advantages such as chemically modified, exist also that chemical toxicity is big, the shortcoming in body-internal-circulation cycle short (being unfavorable for target).Ultra high molecular weight polyethylene imines (PEI, 25kDa) because its amino density is big, be easy to the genetic stew of wrap negative charge and become higher genetic stew (DNA, the siRNA) delivery vehicles of transfection activity that is widely studied in vitro and in vivo the earliest, but the cytotoxicity that can't avoid its high component alkyl skeleton can't degrade and bring.Therefore design and make up degradable polycation and be and improve the genetic stew transfection activity, reduce its Cytotoxic Key Strategy.Such as, utilizing the polymine of small molecular weight is the known example of this area scientist by the connecting key structure degradable polyethylene imines class polycation gene carrier that can rupture under physiological condition.

The Robert J.Lee of Ohio State University etc. adopt linking agent dithio two (succinyl phosphorons amino propyl acid esters) the earliest (DSP) and two sulphur dipropyl two forminoethe (DTBP) as linking agent respectively with crosslinked high molecular degradable polyethylene imines class polycation [the Gosselin MA that obtains of lower molecular weight PEI (PEI 800Da), Guo W, effective conveying genophore that the polyethylene reversible crosslink of the low molecule of Lee RJ. forms.Bioconjugate chemistry, [J] 2001; 12:989-94.].These two contain the disulfide linkage derivative and have the transfection activity suitable with commercially available high molecular PEI (25KDa) in Chinese hamster (CHO) gonad cells, the introducing of disulfide linkage makes polymkeric substance to be reduced by the original reagent gsh of going back in the body, thereby disulfide linkage disconnects, be degraded into the little lower molecular weight PEI of cytotoxicity, but do not provide concrete cell toxicity data in the document.There is lot of documents to report lower molecular weight PEI (PEI 800Da) by containing the crosslinked polymkeric substance that forms of linking agent [the Kloeckner J that disulfide linkage, ester bond, amine ester bond etc. can oneself fractures subsequently, the degradable polycation gene carrier that Wagner E, Ogris M. utilize the polyamines oligopolymer to make up.[J]. European pharmaceutics .2006; 29:414-25. in publication].Though these researchs have shown notional feasibility, the PEI of small molecular weight and the lower molecular weight PEI that has various linking agents fracture fragments still are not accepted in the application of human body.

In order to make the lower molecular weight PEI after the degraded not have the linking agent fragment, people such as the Sung Wan Kim of University of Utah have reported that the employing glutaraldehyde is crosslinked as linking agent and lower molecular weight PEI (1800Da), generate degradable polyethylene imines class polycation [the Kim YH that imine linkage connects, Park JH, Kim SW, et al. makes up degradable polyethylene imines class polycation gene carrier by acid-sensitive sense key.[J]. control releasing research 2005; 103:209-19.].The characteristics of this structure are: behind the cellular uptake, the polycation of cohesion gene disconnects with the imine linkage that lower molecular weight PEI directly links to each other under endocytosis body and lysosomal acidic conditions, generates the small component PEI of no cytotoxicity.But the paradigmatic structure of imine linkage can not guarantee that this polycation carries nucleic acid and enters before the cell and enough stablize, and its transfection activity is more undesirable.

In order to improve the stability of imine structure, Jin and du adopt and can form the oxalic dialdehyde of the imines that has the conjugated structure as linking agent with primary amine, with human body endogenous polyamino monomer---spermine and spermidine aggregate into polycation, stability and gene transfection activity [DuZ have preferably been shown, Jin T. is that basic building unit and degradable are the polycation gene carrier of human body endogenous polyamines monomer with human body endogenous polyamines monomer, be used for DNA (comprising dna vaccination) parcel and carry .Jin T, 2009.].Therefore, we are by identical connecting key, utilize lower molecular weight PEI to be basic building unit and the degradable polycation gene carrier for the PEI of initial low molecule.

Summary of the invention

The objective of the invention is to overcome deficiency of the prior art, a kind of degradable imines cationic synthetic method of birdsing of the same feather flock together is provided.The polycation that the present invention makes up is compared chemical stability and is greatly improved, it is outstanding that the polycation that the PEI of the low molecule that is polymerized with acid amides and ammonia ester structure makes up is compared degradation property, takes into account to arrive the stability before the target cell and enter biological responding after the target cell.

The objective of the invention is to be achieved through the following technical solutions:

The present invention relates to get back to the polycation of the initial state of many amino molecules with lower molecular weight PEI (polymine) with oxalic dialdehyde structure and degraded.Be specifically related to a kind of degradable imines class polycation, structural formula is:

Wherein, n 〉=3, m 〉=3.

The invention still further relates to a kind of above-mentioned degradable imines cationic synthetic method of birdsing of the same feather flock together, comprise the steps:

A, will with the oxalic dialdehyde of molar mass such as PEI the aqueous solution slowly be added drop-wise to the ethanolic soln of PEI, add dry molecular sieve simultaneously;

The condensation reaction of primary amino and aldehyde radical is carried out in b, stirring under 15～30 ℃, namely gets product.

Owing to there is in the reaction process water to generate, dewater so add molecular sieve in the reaction, be conducive to the carrying out that react and the growth of polymericular weight.

Preferably, PEI described in the step a is lower molecular weight PEI.

Preferably, the molecular weight of described lower molecular weight PEI is 800Da.

Preferably, primary amino described in the step b is specially with the condensation reaction of aldehyde radical: two or more primary amine of PEI and oxalic dialdehyde aldehyde radical generate the imines that has conjugated and be combined.

Preferably, to the further separation and purification of the described product that obtains, described purification procedures is as follows: place dialysis tubing dialysis back low-temperature freeze drying vacuum except anhydrating described product.

Preferably, the molecular weight of described dialysis tubing is 3500; The time of described dialysis is 12～38 hours.

The invention still further relates to a kind of nano particle (also being polyplex), described nano particle is got by the method preparation that comprises the steps:

A, aforesaid degradable imines class polycation is added ultrapure water or DEPC is configured to said polycation solution, plasmid is added ultrapure water or DEPC is configured to plasmid solution;

B, described said polycation solution is joined in the plasmid solution fast, mix;

C, mixing solutions is hatched under room temperature, namely get described nano particle.

Preferably, described plasmid is DNA or RNA.Be the polyplex that contains DNA or the polyplex that contains siRNA.

Compared with prior art, beneficial effect of the present invention is: the polycation gene carrier of structure not only can be degraded under physiological condition, and discharge the lower molecular weight PEI that contains unprotonated amino in degradation process, and their degraded product can not produce acidic-group as other degradable polymers.Their degradation product can produce more amino group, can cushion the acidity of endosome.This character can help the lysosome of escaping, and discharges gene in the tenuigenin of low surface charge.For the proton sponge effect, lysosome is because the osmotic pressure that proton sponge effect (absorption proton) produces breaks.The amino key of polycation plays the proton sponge effect.

Description of drawings

The synthetic route chart of Fig. 1: Polyimine-PEI.

Fig. 2: the gel electrophoresis figure of Polyimine-PEI/DNA mixture.

Fig. 3: the grain-size graph of Polyimine-PEI/DNA mixture.

Fig. 4: the Zeta potential figure of Pol yimine-PEI/DNA mixture.

The active figure of the COS-7 cell transfecting of Fig. 5: Polyimine-PEI.

The active figure of the Raw264.7 cell transfecting of Fig. 6: Polyimine-PEI.

The COS-7 cell MTT toxicity figure of Fig. 7: Polyimine-PEI.

The Raw264.7 cell MTT toxicity figure of Fig. 8: Polyimine-PEI.

Embodiment

Below in conjunction with accompanying drawing embodiments of the invention are elaborated: present embodiment is being to implement under the prerequisite with the technical solution of the present invention, has provided detailed embodiment and concrete operating process.But protection scope of the present invention is not limited to following embodiment.

The present invention comprising the physico-chemical property characterizing method of polycation and genetic stew formation nano particle (also being polyplex): gel electrophoresis, dynamic light scattering and Zeta potential; Polycation and genetic stew form the transfection activity of nano particle (also being polyplex) and the DNA plasmid of toxicity test is luciferase plasmids; Polycation and genetic stew form the transfection activity of nano particle (also being polyplex) and the cell of toxicity test is Cos-7 and Raw264.7 cell.

Embodiment 1

The synthetic method of Polyimine-PEI

As shown in Figure 1, the synthetic route of polycation.Entire reaction is carried out in the environment of anhydrous and oxygen-free.Take by weighing a certain amount of PEI800Da (can be other lower molecular weight PEI), add an amount of new dissolve with ethanol that steams (adding an amount of dry molecular sieve simultaneously), then according to suitable stoichiometric ratio weighing oxalic dialdehyde (with the oxalic dialdehyde of molar masss such as PEI the aqueous solution, wherein, the oxalic dialdehyde mass percentage content is 40%), under 0 ℃, oxalic dialdehyde slowly is added drop-wise in the said mixture, add dry molecular sieve simultaneously.Then 15～30 ℃ (preferred room temperature, namely 25 ℃) reaction down (this reaction is the condensation reaction of primary amino with aldehyde radical, is specially: two or more primary amine of PEI and oxalic dialdehyde the aldehyde radical generation imines that has a conjugated be combined) spend the night.Filter, with dialysis tubing (selecting the dialysis tubing of Mw=3500 in the present embodiment) 12～38 (the selecting 24 hours in the present embodiment) of dialysing in deionized water, the low-temperature freeze drying vacuum obtains end product after removing and anhydrating with filtrate.

Embodiment 2

Polyimine-PEI and plasmid form the preparation of mixture polyplex

The preparation of mixture.Detect polycation (Polylink SP) as genophore the quality different with DNA than under the situation by the compound DNA of electrostatic interaction.Take by weighing quantitative polycationic polymer, add the solution that ultrapure water is configured to 2mg/mL, aseptic filter with 0.22 μ m filters then, the concentration dilution of plasmid becomes 1mg/mL, the complex solution of configuration different mass ratio, need to keep the concentration of plasmid solution constant, dilute the concentration of said polycation solution then according to different mass ratioes, said polycation solution after noting keeping diluting and the volume of plasmid solution equate, at last said polycation solution is joined in the plasmid solution fast and mix, hatch 30min under the room temperature, so just obtain the mixture of a series of mass ratioes.

Embodiment 3

Polyimine-PEI and plasmid form the gel electrophoresis of mixture

Configuration quality is than 1.0% agarose solution, heating for dissolving in microwave oven, get 40mL solution, pour in the beaker of special-purpose EB pollution, add the EB solution of about 40 μ L, pour in the mould after stirring evenly, plug comb, after about 30min, glue has just solidified, add an amount of TAE damping fluid in the electrophoresis chamber, sepharose is put into electrophoresis chamber wait for upward sample.Dispose the complex solution of different mass ratio then, incubated at room 30min.The Marker of last sample selects the plasmid maker of 1000-10000kb for use, gets the sample-loading buffer of 1 μ L during last sample earlier, adds the sample sample of 5 μ L, after mixing, joins in the gel pore.Add the voltage of 160 volts of voltages, blue tetrabromophenol sulfonphthalein can move to after the bottom of glue fast, and about 20min takes pictures with the ultraviolet gel imaging system.The gel electrophoresis result of mixture, as shown in Figure 2; As seen from the figure: band from left to right is respectively naked DNA and marker, and polymkeric substance and plasmid mass ratio are respectively 0.5,1,3,5,7,10,15,20,50,70 and 100.When the mixture of Polyimine-MSP and DNA is before mass ratio 5, because wrapping DNA, carrier therefore can't not block the migration of DNA, so demonstrate bright band.And because some plasmid is wrapped up by polymer, so band is the hangover form, and brightness is not as naked DNA.When matter than 〉=5 the time because carrier wraps DNA fully, so DNA is non-migratory when electrophoresis, do not have band in the swimming lane, show that polymkeric substance has the ability of very strong complex gene material when mass ratio 5 is above.

Embodiment 4

Polyimine-PEI and plasmid form the Zeta potential of mixture

Carry out the dilution of said polycation solution according to mass ratio, the mass ratio of required mensuration is 0.5,1,5,8,10,20,30.The medium that is used for dilution is physiological saline or PBS damping fluid preferably.Adopt zetasizer 2000 to measure the surface charge of mixture, the sample determination time is set to automatically, each sample determination 3 times, the mapping of averaging.The zeta-potential measurement result of mixture as shown in Figure 3.As seen from the figure: along with the increase of polymkeric substance and DNA plasmid mass ratio, Zeta potential also increases thereupon, and from mass ratio 5, Zeta potential just is stabilized in 10-15mV, illustrates that the nano particle of polymkeric substance and plasmid formation is more stable.

Embodiment 5

Polyimine-PEI and plasmid form the particle diameter of mixture

The sample that the mensuration of mixture particle diameter needs is generally 1ml most, each 500 μ L of plasmid solution and said polycation solution, and the concentration of plasmid is 20 μ g/mL, carries out the dilution of said polycation solution according to mass ratio, the mass ratio of required mensuration is 0.5,1,5,8,10,20,30.At ambient temperature, with the hydrated radius of Nano-S laser particle analyzer mensuration mixture, refracting medium is set to water, and specific refractory power is 1.33, and viscosity is 0.8872cP, each sample determination 3 times, the mapping of averaging.The particle size determination result of mixture, as shown in Figure 4.As shown in Figure 4: the particle diameter of two kinds of carriers and plasmid composite all can be stabilized in below the 200nm.Mass ratio is that 0.5 o'clock polymer does not wrap DNA, and DNA and polymer are loose condition (of surface), causes particle diameter bigger than normal approaching, and very unstable, data are bigger than normal.When ratio is between 1 to 20, polymkeric substance energy stable existence, size is all between 100nm-200nm.

Embodiment 6

Polyimine-PEI and plasmid form Cos-7 and the experiment of Raw264.7 cell transfecting of mixture

At first, in 48 porocyte culture plates, add the cell suspension of 0.5mL, density is 5.0-10*10 ⁴/ mL, overnight incubation.During 48 orifice plate transfections, what every hole added plasmid fixes the plasmid of every hole 500ng, volume 25 μ L most, polymkeric substance is configured to the solution of 2mg/mL, and with the filter membrane sterile filtration of 0.22 μ m, according to the testing sample that arranges and the mass ratio of plasmid, be diluted to required ratio, the cumulative volume of said polycation solution is 25 μ L, then said polycation solution is joined in the middle of the solution of plasmid, mixing is hatched 30min fast.The volume that adds the polycation mixture in every hole like this is 50 μ L, and is for 1/10th of cumulative volume (500 μ L), up to specification.Each mass ratio is done three multiple holes.Positive controls PEI25kDa, result with its best plasmid during than 2 (N/P=5) makes three control wells, hatch during this period of time in, from incubator, take out cell, remove the substratum of serum, wash one time with the PBS solution of 200 μ L, substratum changes the substratum of the serum-free of 250 μ L into again, and the mixture that will hatch is sequentially added in the cell then.After 4 hours, remove the substratum of serum-free, every hole adds and contains 10% foetal calf serum and 1% pair of anti-ideal culture medium, cultivates 48 hours again, detects transfection results.

Investigated a series of quality than the transfection activity under the condition with the reporter gene luciferase, the result is shown in Fig. 5,6.Positive controls be set be mass ratio and be 3 PEI25kDa (the N/P ratio is 15) and mass ratio and be 2 Lipfectamine2000, as can be seen, the synthetic polycationic polymer of we oneself design in mass ratio 10-20 scope in, the result of transfection efficiency and control group is suitable, increase along with polycationic polymer and plasmid mass ratio, the transfection activity that we conceive synthetic polycationic polymer ourselves further increases, when mass ratio reaches 100: 1, than high two orders of magnitude of transfection efficiency of control group.Above-mentioned experiment shows that the polycation carrier that we make up has good transfection activity.

Embodiment 7

The Cos-7 of Polyimine-PEI and Raw264.7 cytotoxicity experiment

Adopt mtt assay to measure the toxicity of polycation, with PEI25kDa and Lipofectamine2000 as positive controls, the negative control group of the cell that is left intact.At first, inoculating cell with the COS-7 cell dissociation, is diluted to the cell suspension that density is 1.0-5.0*104/mL, and every hole adds 100 μ L, overnight incubation in 96 orifice plates.The said polycation solution of second step with 2mg/mL is diluted to different concentration gradients, and final volume is 100 μ L, and positive controls PEI25kDa is the concentration gradient consistent with the testing sample group with the Lipofectamine2000 dilution.Take out cell, remove the substratum of serum, wash one time with the PBS of 100 μ L, again the substratum of the no phenol red serum-free of adding.Add polycation macromolecular solution and positive controls PEI25kDa and Lipofectamine2000 then, add the water of 100 μ L in the negative control group.After 4 hours, remove nutrient solution and polycation macromolecular solution, every hole adds the no phenol red serum free medium of 100 μ L, adds the MTT solution of 25 μ L under the lucifuge condition again, places cell culture incubator to cultivate 6 hours.Microscopically is observed the crystallization situation of viable cell, after the crystallization, then adds 100 μ LDMSO and places 10min microplate reader 570nm place measurement result fully, and 630nm does contrast in the place.As a result, shown in Fig. 7,8.In the middle of the Cos-7 cell, toxicity and the Lipofectamine2000 of polymkeric substance are suitable, and in the middle of the Raw264.7 cell, the toxicity of polycationic polymer is much smaller than control group PEI25kDa and Lipofectamine2000.Even when concentration 150 μ g/mL, cell survival rate still is higher than 90%.

Claims (9)

1. degradable imines class polycation is characterized in that structural formula is:

Wherein, n 〉=3, m 〉=3; The described degradable imines cationic synthetic method of birdsing of the same feather flock together comprises the steps:

A, will with the oxalic dialdehyde of molar mass such as polymine the aqueous solution slowly be added drop-wise to the ethanolic soln of polymine, add dry molecular sieve simultaneously;

The condensation reaction of primary amino and aldehyde radical is carried out in b, stirring under 15～30 ℃, namely gets product.

2. a degradable imines as claimed in claim 1 cationic synthetic method of birdsing of the same feather flock together is characterized in that, comprises the steps:

A, will with the oxalic dialdehyde of molar mass such as polymine the aqueous solution slowly be added drop-wise to the ethanolic soln of polymine, add dry molecular sieve simultaneously;

The condensation reaction of primary amino and aldehyde radical is carried out in b, stirring under 15～30 ℃, namely gets product.

3. the degradable imines according to claim 2 cationic synthetic method of birdsing of the same feather flock together is characterized in that polymine described in the step a is the low molecular weight polyethylene imines.

4. the degradable imines according to claim 3 cationic synthetic method of birdsing of the same feather flock together is characterized in that the molecular weight of described low molecular weight polyethylene imines is 800Da.

5. the degradable imines according to claim 2 cationic synthetic method of birdsing of the same feather flock together, it is characterized in that primary amino described in the step b is specially with the condensation reaction of aldehyde radical: two or more primary amine of polymine and oxalic dialdehyde aldehyde radical generate the imines that has conjugated and be combined.

6. the degradable imines according to claim 2 cationic synthetic method of birdsing of the same feather flock together, it is characterized in that, to the further separation and purification of the described product that obtains, described purification procedures is as follows: place dialysis tubing dialysis back low-temperature freeze drying vacuum except anhydrating described product.

7. the degradable imines according to claim 6 cationic synthetic method of birdsing of the same feather flock together is characterized in that the molecular weight of described dialysis tubing is 3500; The time of described dialysis is 12～38 hours.

8. a nano particle is characterized in that, described nano particle is got by the method preparation that comprises the steps:

A, the described degradable imines of claim 1 class polycation is added ultrapure water or the diethyl pyrocarbonate is configured to said polycation solution, plasmid is added ultrapure water or the diethyl pyrocarbonate is configured to plasmid solution;

B, described said polycation solution is joined in the plasmid solution fast, mix;

C, mixing solutions is hatched under room temperature, namely get described nano particle.

9. nano particle according to claim 8 is characterized in that, described plasmid is DNA or RNA.

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