CN103834040A - Dendrimer drug carrier G3.5 with pH and temperature supersensitivity and preparation method thereof - Google Patents

Abstract

The invention relates to a dendrimer drug carrier G3.5 with pH and temperature supersensitivity and a preparation method thereof. The objective of the invention is to allow dendrimer with a low generation number to have the characteristic of pH and temperature supersensitivity. According to the invention, after modification, G3.5 polyamide dendrimer with three hydroxyl groups at its tail end bonds with N-propylpiperazine whose tail end turns into a carboxyl structure, thereby obtaining G3.5PAMAM-Tris-NPPOBA. Compared with the prior art, the invention has the following advantages: the prepared drug carrier has dual pH and temperature sensitivity, is supersensitive to pH and has a wide LCST value adjustable range and a critical solution temperature close to human body temperature.

Description

A kind of pH and temperature supersensitivity dendritic macromole pharmaceutical carrier three generations half and preparation method thereof

Technical field:

The present invention relates to a kind of pharmaceutical product take specific physical character as feature, further relate to a kind of pH and temperature supersensitivity dendritic macromole pharmaceutical carrier three generations half and preparation method thereof.

Background technology:

Dendritic macromole is the synthetic macromolecule of height cladodification, and it has the molecule homogeneity of height, clear and definite molecular size and shape and many surface functional groups.These characteristics of dendritic macromole make it can change kernel type (as polyamide-based, polyethers), the type of number of branches and surface functional group.By finishing, make dendritic macromole there is equilibrium water dissolubility and stimulate susceptibility (as temperature sensitive, pH sensitivity, photaesthesia, reduction sensitivity etc.) thereby different functional groups can be connected to dendritic macromole surface.These stimulate responsive characteristic to make dendritic macromole in biomedicine, and pharmaceutics comprises that the aspects such as imaging, useful for drug delivery, organizational project obtain extensive concern.

Traditional temperature sensing polymer is mostly linear polymeric polymkeric substance, as PNIPAM (PNIPAAM), polymethyl acrylic acid N, N-dimethylamino ethyl ester (PDMAEMA), poly-N, N-diethyl acrylamide (PDEA) etc., and there is the temperature sensing material of bodily form three-dimensional structure, up to the present report seldom.And this type material of dendritic macromole, in order to increase its function, researchist has carried out a lot of trials both at home and abroad, because having numerous groups for modifying, its surface exists, the most frequently used method is exactly the group that it is carried out finishing and is introduced various needs, as at linear temperature sensitive high molecular polymers such as dendrimer surface grafting macromole PNIPAAM, but the dendritic macromole that the polymolecularity of the linear temperature sensitive high molecular polymer such as PNIPAAM causes the linear temperature sensitive high molecular polymers such as PNIPAAM to be modified has been lost the homogeneity of molecule.Therefore be different from the research that linear temperature sensitive polymer can have the temperature sensitive functional group of shape of molecule and homogeneity simultaneously and be subject to extensive concern.In recent years, the people such as Kenji Kono have synthesized the temperature sensing polymer with ball-like structure of series of new, these polymkeric substance are by introducing sec.-propyl amides structure at polyamide-based dendritic macromole PAMAM or PPI class dendritic macromole surface, sec.-propyl amide structure is the structural unit of temperature sensitive polymer PNIPAAM, obtain 4.5 generation NIPAAM-PAMAM, 5 generation NIPAAM-PAMAM and 5.5 generation NIPAAM-PAMAM, the LCST value of these derivative aqueous solution is respectively 41, 56 and 43 ℃, the advantage of these materials is that linear temperature sensing polymer is incomparable, it has three-dimensional spherical structure, controlled accurate molecular size, all belong to the characteristic of dendritic macromole molecule homogeneity etc., but the polymkeric substance obtaining obtains under high algebraic conditions, and the adjustable scope of its Kraft point (LCST value Lower Critical Solution Temperature) is little, and all higher than Human Physiology temperature, these have all affected the scope of its application.Therefore the dendritic macromole pharmaceutical carrier that, develop that a kind of cytotoxicity is low, the large and LCST value of LCST value variable range approaches human body temperature is necessary.

Summary of the invention

The object of this invention is to provide a kind of preparation method who the dendritic macromole PAMAM modifying with polyhydroxy amine-Tutofusin tris is carried out to finishing with the temperature sensitive group of N-propyl group piperazine, make it under low algebraically, there is the characteristic of pH and temperature hypersensitization.

Ultimate principle of the present invention is as follows:

The Thermo-sensitive of dendritic macromole depends on hydrophobicity and the tightness degree of surface functional group, the pH of solution and carrier concn.Thereby the hydrophobicity that increases functional group has increased the temperature sensitivity that molecular interaction causes pH to guide.In like manner, thus increasing the number of surface functional group also can increase molecular interaction and obtain lower Kraft point (LCST value Lower Critical Solution Temperature).

The present invention reacts with Tutofusin tris by the end ester group of dendritic macromole PAMAM, PAMAM surface group is become to hydroxyl, and surface group number increases to original three times, then use N-propyl group piperazine analogue N-propyl group piperazine-4-ketobutyric acid (NPPOBA) to carry out finishing to PAMAM-Tris, the novel dendritic polymers obtaining has temperature, pH Dual Sensitive performance, can improve the solubleness of medicine as hydrophobic drug carrier, can be used as intellectual drug carrier and be applied to biomedical sector.

The PAMAM derivative with temperature sensitive character is made into the phosphate buffered saline buffer of different pH values (pH6,7,8,9), at 500nm place, gradient increased temperature detects with ultraviolet spectrophotometer, in testing process, every 5min raises 1 ℃, record the temperature-light transmittance curve of polymkeric substance, when transmittance is 50%, corresponding temperature is its LCST value, and when detection, selected suitable concentration is 1mg/mL.

The PAMAM derivative of temperature sensitive character shows temperature sensitive characteristic in the aqueous solution, along with the progressively rising of temperature, and the water-soluble reduction of polymkeric substance, and its water-soluble adjusting with pH changes.

Invention is achieved in that

(1) in G3.5 generation, has the preparation of the PAMAM dendritic polymkeric substance of ester group end.

Take quadrol as core, repeat with methyl acrylate that Michael addition reaction completely and amidate action obtain until G3.5 generation has the PAMAM dendritic polymkeric substance of ester group end.Detailed process is as follows:

Take quadrol and methyl acrylate as raw material, mol ratio is according to 1:4-5, ice bath, and temperature remains on 0 ℃; The methanol solution that slowly drips methyl acrylate, in the methanol solution of quadrol, drips and finishes, and repeatedly vacuumizes, and nitrogen protection, recovers room temperature, then at 35 ℃, reacts 6h; After reaction finishes, revolve to steam and remove solvent methanol, obtain flaxen thick liquid G0.5PAMAM;

With G0.5PAMAM: quadrol=1:4-8(mol ratio); The methanol solution of quadrol is joined in the there-necked flask with magnetic agitation, thermometer, and ice bath, slowly drips the methanol solution of G0.5PAMAM; Drip and finish, repeatedly vacuumize, nitrogen protection is reacted 24h at 35 ℃, obtains product G1.0PAMAM;

With G1.0PAMAM: methyl acrylate=1:16(mol ratio); The methanol solution of methyl acrylate is joined in the there-necked flask with magnetic agitation, thermometer, and ice bath, slowly drips the methanol solution of G1.0PAMAM; Drip and finish, repeatedly vacuumize, nitrogen protection is reacted 24h at 35 ℃; Revolve to steam and remove unreacted methyl acrylate and solvent methanol, obtain lurid thick liquid G1.5PAMAM;

With G1.5PAMAM: quadrol=1:80(mol ratio); The methanol solution of quadrol is joined in the there-necked flask with magnetic agitation, thermometer, and ice bath, slowly drips the methanol solution of G1.5PAMAM; Drip and finish, repeatedly vacuumize, nitrogen protection is reacted 72h at 35 ℃; React complete, adopt vacuum rotary evaporator at 35 ℃, to steam except methyl alcohol, obtain product G2.0PAMAM;

With G2.0PAMAM: methyl acrylate=1:24(mol ratio); The methanol solution of methyl acrylate is joined in the there-necked flask with magnetic agitation, thermometer, and ice bath, slowly drips the methanol solution of G2.0PAMAM; Drip and finish, repeatedly vacuumize, nitrogen protection is reacted 72h at 35 ℃; Revolve to steam and remove unreacted methyl acrylate and solvent methanol, obtain lurid thick liquid G2.5PAMAM;

With G2.5PAMAM: quadrol=1:160(mol ratio); The methanol solution of quadrol is joined in the there-necked flask with magnetic agitation, thermometer, and ice bath, slowly drips the methanol solution of G2.5PAMAM; Drip and finish, repeatedly vacuumize, nitrogen protection is reacted 6 days at 35 ℃.React complete, adopt vacuum rotary evaporator at 35 ℃, to steam except methyl alcohol, obtain product G3.0PAMAM;

With G3.0PAMAM: methyl acrylate=1:48(mol ratio); The methanol solution of methyl acrylate is joined in the there-necked flask with magnetic agitation, thermometer, and ice bath, slowly drips the methanol solution of G3.0PAMAM; Drip and finish, repeatedly vacuumize, nitrogen protection is reacted 48h at 35 ℃.Revolve to steam and remove unreacted methyl acrylate and solvent methanol, obtain lurid thick liquid G3.5PAMAM.

(2) with Tutofusin tris (Tris), G3.5 is modified for PAMAM, make its end trihydroxy-.

G3.5PAMAM, Tutofusin tris, Anhydrous potassium carbonate feed intake according to mol ratio 1:48:48;

Get in the anhydrous DMSO solution or the round-bottomed flask of DMF solution under ice-water bath (3～5 ℃ of temperature) of Tutofusin tris (Tris) and Anhydrous potassium carbonate, G3.5PAMAM0.2876g is dissolved in a small amount of anhydrous DMSO or dry DMF in constant pressure funnel, repeatedly vacuumize for three times, after nitrogen protection, under the at the uniform velocity stirring of magnetic stirring apparatus, DMSO solution or the DMF solution of G3.5PAMAM slowly splash in round-bottomed flask, after dropwising, take off constant pressure funnel, reaction unit is moved into thermostatical oil bath, temperature is controlled at 50 ℃, successive reaction 72h;

After reaction finishes, with Rotary Evaporators except desolventizing DMSO, obtain faint yellow opaque thick liquid, being dissolved in stirring and dissolving in a small amount of distilled water is settled solution, then splash in a large amount of acetone and produce white precipitate, abandoning supernatant, carries out removing water-soluble impurity three times repeatedly, the product obtaining carries out lyophilize after adding dialysis tubing (molecular weight cut-off is 3500) dialysis 24h, obtains G3.5PAMAM-Tris.

(3) end becomes the preparation of the N-propyl group piperazine of carboxyl structure.

Utilize acid anhydrides to modify N-propyl group piperazine, make its end become carboxyl structure; The purified product that obtains; As follows.

Concrete preparation method is:

Methylene dichloride is added in container, under agitation in container, slowly add Succinic anhydried, make while stirring Succinic anhydried dissolve;

N-propyl group piperazine, with methylene dichloride dissolving, then packs in constant pressure funnel; At 15 ℃, the aminated compounds that methylene dichloride is dissolved is slowly added drop-wise in there-necked flask, dropwises, and at this temperature, reacts 5h;

After reaction finishes, rotary evaporation steams unnecessary methylene dichloride, and solution becomes reddish-brown viscous liquid, adds a small amount of ether after taking-up, leave standstill after for some time, have pink solid to separate out, filter, washed with dichloromethane twice for filter cake, uses dehydrated alcohol recrystallization, 80 vacuum-drying 24h.

(4) after modifying three hydroxyls of end 3.5 generation polyamide dendroid macromole and the end N-propyl group piperazine that becomes carboxyl structure be good for and close, obtain G3.5PAMAM-Tris-NPPOBA.

Detailed process is:

To under the DMSO solution of N-propyl group piperazine-4-ketobutyric acid (NPPOBA) or DMF solution and 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDCHCl) and DMAP (DMAP) mixed room temperature, stir 1h, NPPOBA is activated, until solution has activated from the colourless reddish-brown that becomes;

Then get the DMSO solution of quantitative G3.5PAMAM-Tris or DMF solution (reaction mol ratio: G3.5PAMAM-Tris/NPPOBA/EDCHCl/DMAP=1:192:192) in constant pressure funnel, repeatedly vacuumize for three times, after nitrogen protection, under the at the uniform velocity stirring of magnetic stirring apparatus, DMSO solution or the DMF solution of G3.5PAMAM-Tris slowly splash in round-bottomed flask, after dropwising, take off constant pressure funnel, at room temperature more than successive reaction 72h;

Remove most of solvent DMSO or DMF with Rotary Evaporators, the product obtaining dissolves with a small amount of distilled water, it is the brown-red solution of clarification lower than certain temperature, during higher than this temperature, solution becomes muddy, be 40 ℃ with ultracentrifuge in temperature by muddy liquid, centrifugal under rotating speed 5000rpm condition, gained for throw out a large amount of distill water dialysis 24h of dialysis tubing (molecular weight cut-off is 3500) obtain target product.

Can be the polyamide-based dendritic macromole that kernel and methyl acrylate form with quadrol, also can use PPI, polyethers dendritic macromole.

The present invention is with respect to prior art, and tool has the following advantages:

The product that the method for the invention obtains has pH and temperature dual susceptibility, and for pH hypersensitization, LCST value adjustable extent is very extensive.There is the Kraft point of the human body temperature of approaching simultaneously.

The product that the method for the invention obtains is the dendritic macromole of low algebraically, increases low algebraically modified surface group by surface hydroxylation, has overcome the problem such as loaded down with trivial details synthetic and toxicity of high algebraically dendritic macromole, is a kind of novel intellectual drug carrier.When kernel bag carries hydrophobic drug, after undergoing phase transition by adjusting ambient condition polymkeric substance by drug release out.

Accompanying drawing explanation:

Fig. 1 is the nuclear magnetic spectrum of NPPOBA.

Fig. 2 is the nuclear-magnetism figure of G3.5PAMAM-Tris-NPPOBA.

Fig. 3 is the infared spectrum of G3.5PAMAM-Tris-NPPOBA; X-coordinate represents wave number, and unit is cm ^-1.

Fig. 4 is that G3.5PAMAM-Tris-NPPOBA is respectively the LCST value under 6.0,7.0,8.0,9.0 at pH; X-coordinate representation temperature, unit is degree Celsius; Ordinate zou represents solid rate, and unit is per-cent.

Fig. 5 is that G3.5PAMAM-Tris-NPPOBA is respectively the LCST value under 6.6,6.8,7.0,7.2,7.4,7.6,7.8 at pH; X-coordinate representation temperature, unit is degree Celsius; Ordinate zou represents solid rate, and unit is per-cent.

Embodiment:

(1) in G3.5 generation, has the preparation of the PAMAM dendritic polymkeric substance of ester group end.Repeatedly carry out by Michael addition and amidation two-step reaction as raw material adopts the method for dispersing take quadrol and methyl acrylate, obtain the polyamide dendroid macromole of low algebraically.

Embodiment 1-1-1: with quadrol (EDA): methyl acrylate=1:4(mol ratio).Ice bath, temperature remains on 0 ℃.The methanol solution that slowly drips methyl acrylate, in the methanol solution of quadrol, drips and finishes, and repeatedly vacuumizes, and nitrogen protection, recovers room temperature, then at 35 ℃, reacts 6h.After reaction finishes, revolve to steam and remove solvent methanol, obtain flaxen thick liquid G0.5PAMAM.

Embodiment 1-1-2: with quadrol (EDA): methyl acrylate=1:5(mol ratio).Ice bath, temperature remains on 0 ℃.The methanol solution that slowly drips methyl acrylate, in the methanol solution of quadrol, drips and finishes, and repeatedly vacuumizes, and nitrogen protection, recovers room temperature, then at 35 ℃, reacts 6h.After reaction finishes, revolve to steam and remove unreacted methyl acrylate and solvent methanol, obtain flaxen thick liquid G0.5PAMAM.

Embodiment 1-2-1: with G0.5PAMAM: quadrol=1:4(mol ratio).The methanol solution of quadrol is joined in the there-necked flask with magnetic agitation, thermometer, and ice bath, slowly drips the methanol solution of G0.5PAMAM.Drip and finish, repeatedly vacuumize, nitrogen protection is reacted 24h at 35 ℃, obtains product G1.0PAMAM.

Embodiment 1-2-2: with G0.5PAMAM: quadrol=1:8(mol ratio).The methanol solution of quadrol is joined in the there-necked flask with magnetic agitation, thermometer, and ice bath, slowly drips the methanol solution of G0.5PAMAM.Drip and finish, repeatedly vacuumize, nitrogen protection is reacted 24h at 35 ℃, obtains product G1.0PAMAM.

Embodiment 1-3: with G1.0PAMAM: methyl acrylate=1:16(mol ratio).The methanol solution of methyl acrylate is joined in the there-necked flask with magnetic agitation, thermometer, and ice bath, slowly drips the methanol solution of G1.0PAMAM.Drip and finish, repeatedly vacuumize, nitrogen protection is reacted 24h at 35 ℃.Revolve to steam and remove unreacted methyl acrylate and solvent methanol, obtain lurid thick liquid G1.5PAMAM.

Embodiment 1-4: with G1.5PAMAM: quadrol=1:80(mol ratio).The methanol solution of quadrol is joined in the there-necked flask with magnetic agitation, thermometer, and ice bath, slowly drips the methanol solution of G1.5PAMAM.Drip and finish, repeatedly vacuumize, nitrogen protection is reacted 72h at 35 ℃.React complete, adopt vacuum rotary evaporator at 35 ℃, to steam except methyl alcohol, obtain product G2.0PAMAM.

Embodiment 1-5: with G2.0PAMAM: methyl acrylate=1:24(mol ratio).The methanol solution of methyl acrylate is joined in the there-necked flask with magnetic agitation, thermometer, and ice bath, slowly drips the methanol solution of G2.0PAMAM.Drip and finish, repeatedly vacuumize, nitrogen protection is reacted 72h at 35 ℃.Revolve to steam and remove unreacted methyl acrylate and solvent methanol, obtain lurid thick liquid G2.5PAMAM.

Embodiment 1-6: with G2.5PAMAM: quadrol=1:160(mol ratio).The methanol solution of quadrol is joined in the there-necked flask with magnetic agitation, thermometer, and ice bath, slowly drips the methanol solution of G2.5PAMAM.Drip and finish, repeatedly vacuumize, nitrogen protection is reacted 6 days at 35 ℃.React complete, adopt vacuum rotary evaporator at 35 ℃, to steam except methyl alcohol, obtain product G3.0PAMAM.

Embodiment 1-7: with G3.0PAMAM: methyl acrylate=1:48(mol ratio).The methanol solution of methyl acrylate is joined in the there-necked flask with magnetic agitation, thermometer, and ice bath, slowly drips the methanol solution of G3.0PAMAM.Drip and finish, repeatedly vacuumize, nitrogen protection is reacted 48h at 35 ℃.Revolve to steam and remove unreacted methyl acrylate and solvent methanol, obtain lurid thick liquid G3.5PAMAM.

(2) with Tutofusin tris (Tris), G3.5 is modified for PAMAM, make its end trihydroxy-.

Embodiment 2-1: with G3.5PAMAM: Tutofusin tris: Carbon Dioxide nak response mol ratio=1:48:48.In the round-bottomed flask of the anhydrous DMSO solution of getting excessive Tutofusin tris (Tris) 1.0164g and Anhydrous potassium carbonate 1.1592g under ice-water bath (3～5 ℃ of temperature); quantitative G3.5PAMAM1.0510g is dissolved in a small amount of anhydrous DMSO in constant pressure funnel; repeatedly vacuumize for three times; after nitrogen protection; under the at the uniform velocity stirring of magnetic stirring apparatus; the DMSO solution of G3.5PAMAM slowly splashes in round-bottomed flask; after dropwising, take off constant pressure funnel; reaction unit is moved into thermostatical oil bath; temperature is controlled at 50 ℃, successive reaction 72h.After reaction finishes, with Rotary Evaporators except desolventizing DMSO, obtain faint yellow opaque thick liquid, being dissolved in stirring and dissolving in a small amount of distilled water is settled solution, then splash in a large amount of acetone and produce white precipitate, abandoning supernatant, carries out removing water-soluble impurity three times repeatedly, the product obtaining carries out lyophilize after adding dialysis tubing (molecular weight cut-off is 3500) dialysis 24h, obtains G3.5PAMAM-Tris.

Embodiment 2-2: with G3.5PAMAM: Tutofusin tris: Carbon Dioxide nak response mol ratio=1:48:48.In the round-bottomed flask of the anhydrous DMF solution of getting excessive Tutofusin tris (Tris) 1.0164g and Anhydrous potassium carbonate 1.1592g under ice-water bath (3～5 ℃ of temperature); quantitative G3.5PAMAM1.0510g is dissolved in a small amount of dry DMF in constant pressure funnel; repeatedly vacuumize for three times; after nitrogen protection; under the at the uniform velocity stirring of magnetic stirring apparatus; the DMF solution of G3.5PAMAM slowly splashes in round-bottomed flask; after dropwising, take off constant pressure funnel; reaction unit is moved into thermostatical oil bath; temperature is controlled at 50 ℃, successive reaction 72h.After reaction finishes, remove solvent DMF with Rotary Evaporators, obtain faint yellow opaque thick liquid, being dissolved in stirring and dissolving in a small amount of distilled water is settled solution, then splash in a large amount of acetone and produce white precipitate, abandoning supernatant, carries out removing water-soluble impurity three times repeatedly, the product obtaining carries out lyophilize after adding dialysis tubing (molecular weight cut-off is 3500) dialysis 24h, obtains G3.5PAMAM-Tris.

(3) with Succinic anhydried, N-propyl group piperazine is modified, made end become carboxyl structure.

Embodiment 3: 100mL methylene dichloride is added in 250mL there-necked flask, under agitation slowly add 10.07g(0.10mol in there-necked flask) Succinic anhydried, make while stirring Succinic anhydried dissolve.Take 0.10mol N-propyl group piperazine, with the dissolving of 60mL methylene dichloride, then pack in 100mL constant pressure funnel.At 15 ℃, the aminated compounds that methylene dichloride is dissolved is slowly added drop-wise in there-necked flask, dropwises, and at this temperature, reacts 5h.After reaction finishes, rotary evaporation steams unnecessary methylene dichloride, and solution becomes reddish-brown viscous liquid, adds a small amount of ether after taking-up, leave standstill after for some time, have pink solid to separate out, filter, washed with dichloromethane twice for filter cake, uses dehydrated alcohol recrystallization, 80 vacuum-drying 24h.

(4) after modifying three hydroxyls of end 3.5 generation polyamide dendroid macromole and the end N-propyl group piperazine that becomes carboxyl structure be good for and close, obtain G3.5PAMAM-Tris-NPPOBA.

Embodiment 4-1:. will stir 1h under the DMSO solution of N-propyl group piperazine-4-ketobutyric acid (NPPOBA) and 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDCHCl) and DMAP (DMAP) mixed room temperature, NPPOBA is activated, until solution has activated from the colourless reddish-brown that becomes.Then get the DMSO solution (reaction mol ratio: G3.5PAMAM-Tris/NPPOBA/EDCHCl/DMAP=1:192:192) of quantitative G3.5PAMAM-Tris in constant pressure funnel; repeatedly vacuumize for three times; after nitrogen protection; under the at the uniform velocity stirring of magnetic stirring apparatus; the DMSO solution of G3.5PAMAM-Tris slowly splashes in round-bottomed flask; after dropwising, take off constant pressure funnel, at room temperature successive reaction 72h.Remove most of solvent DMSO with Rotary Evaporators, the product obtaining dissolves with a small amount of distilled water, it is the brown-red solution of clarification lower than certain temperature, during higher than this temperature, solution becomes muddy, be 40 ℃ with ultracentrifuge in temperature by muddy liquid, centrifugal under rotating speed 5000rpm condition, gained for throw out a large amount of distill water dialysis 24h of dialysis tubing (molecular weight cut-off is 3500) obtain target product.

Embodiment 4-2:. will stir 1h under the DMSO solution of N-propyl group piperazine-4-ketobutyric acid (NPPOBA) and 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDCHCl) and DMAP (DMAP) mixed room temperature, NPPOBA is activated, until solution has activated from the colourless reddish-brown that becomes.Then get the DMSO solution (reaction mol ratio: G3.5PAMAM-Tris/NPPOBA/EDCHCl/DMAP=1:192:192) of quantitative G3.5PAMAM-Tris in constant pressure funnel; repeatedly vacuumize for three times; after nitrogen protection; under the at the uniform velocity stirring of magnetic stirring apparatus; the DMSO solution of G3.5PAMAM-Tris slowly splashes in round-bottomed flask; after dropwising, take off constant pressure funnel, at room temperature successive reaction 90h.Remove most of solvent DMSO with Rotary Evaporators, the product obtaining dissolves with a small amount of distilled water, it is the brown-red solution of clarification lower than certain temperature, during higher than this temperature, solution becomes muddy, be 40 ℃ with ultracentrifuge in temperature by muddy liquid, centrifugal under rotating speed 5000rpm condition, gained for throw out a large amount of distill water dialysis 24h of dialysis tubing (molecular weight cut-off is 3500) obtain target product.

Embodiment 4-3: will stir 1h under the DMF solution of N-propyl group piperazine-4-ketobutyric acid (NPPOBA) and 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDCHCl) and DMAP (DMAP) mixed room temperature, NPPOBA is activated, until solution has activated from the colourless reddish-brown that becomes.Then get the DMF solution (reaction mol ratio: G3.5PAMAM-Tris/NPPOBA/EDCHCl/DMAP=1:192:192) of quantitative G3.5PAMAM-Tris in constant pressure funnel; repeatedly vacuumize for three times; after nitrogen protection; under the at the uniform velocity stirring of magnetic stirring apparatus; the DMSO solution of G3.5PAMAM-Tris slowly splashes in round-bottomed flask; after dropwising, take off constant pressure funnel, at room temperature successive reaction 72h.Remove most of solvent DMF with Rotary Evaporators, the product obtaining dissolves with a small amount of distilled water, it is the brown-red solution of clarification lower than certain temperature, during higher than this temperature, solution becomes muddy, be 40 ℃ with ultracentrifuge in temperature by muddy liquid, centrifugal under rotating speed 5000rpm condition, gained for throw out a large amount of distill water dialysis 24h of dialysis tubing (molecular weight cut-off is 3500) obtain target product.

Claims (8)

1. a pH and temperature supersensitivity dendritic macromole pharmaceutical carrier three generations half, it is characterized in that, carrier is G3.5PAMAM-Tris-NPPOBA, after modifying three hydroxyls of end 3.5 generation polyamide dendroid macromole and the end N-propyl group piperazine that becomes carboxyl structure be good for the product closing.

2. a kind of pH and temperature supersensitivity dendritic macromole pharmaceutical carrier three generations half preparation method described in claim 1, it is characterized in that, reaction process is: after modifying three hydroxyls of end 3.5 generation polyamide dendroid macromole and the end N-propyl group piperazine that becomes carboxyl structure be good for and close.

3. a kind of pH and temperature supersensitivity dendritic macromole pharmaceutical carrier three generations half preparation method described in claim 2, is characterized in that, reaction process is:

To under the DMSO solution of N-propyl group piperazine-4-ketobutyric acid (NPPOBA) or DMF solution and 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDCHCl) and DMAP (DMAP) mixed room temperature, stir 1h, NPPOBA is activated, until solution has activated from the colourless reddish-brown that becomes;

Then get the DMSO solution of quantitative G3.5PAMAM-Tris or DMF solution (reaction mol ratio: G3.5PAMAM-Tris/NPPOBA/EDCHCl/DMAP=1:192:192:192) in constant pressure funnel, repeatedly vacuumize for three times, after nitrogen protection, under the at the uniform velocity stirring of magnetic stirring apparatus, DMSO solution or the DMF solution of G3.5PAMAM-Tris slowly splash in round-bottomed flask, after dropwising, take off constant pressure funnel, at room temperature more than successive reaction 72h;

Remove most of solvent DMSO or DMF with Rotary Evaporators, the product obtaining dissolves with a small amount of distilled water, it is the brown-red solution of clarification lower than certain temperature, during higher than this temperature, solution becomes muddy, be 40 ℃ with ultracentrifuge in temperature by muddy liquid, centrifugal under rotating speed 5000rpm condition, gained for throw out a large amount of distill water dialysis 24h of dialysis tubing (molecular weight cut-off is 3500) obtain target product.

4. according to a kind of pH described in claim 2 or 3 and temperature supersensitivity dendritic macromole pharmaceutical carrier three generations's half preparation method, it is characterized in that, the preparation method of the triatomic 3.5 generation polyamide dendroid macromole G3.5PAMAM-Tris of described raw material end is as follows:

With Tutofusin tris, Tris modifies G3.5PAMAM, makes its end three hydroxyls again.

5. a kind of pH and temperature supersensitivity dendritic macromole pharmaceutical carrier three generations half preparation method according to claim 4, it is characterized in that, the preparation method of the triatomic 3.5 generation polyamide dendroid macromole G3.5PAMAM-Tris of described raw material end is as follows:

G3.5PAMAM, Tutofusin tris, Anhydrous potassium carbonate feed intake according to mol ratio 1:48:48;

Get in the anhydrous DMSO solution or the round-bottomed flask of DMF solution under ice-water bath (3～5 ℃ of temperature) of Tutofusin tris (Tris) and Anhydrous potassium carbonate, G3.5PAMAM is dissolved in a small amount of anhydrous DMSO or dry DMF in constant pressure funnel, repeatedly vacuumize for three times, after nitrogen protection, under the at the uniform velocity stirring of magnetic stirring apparatus, DMSO solution or the DMF solution of G3.5PAMAM slowly splash in round-bottomed flask, after dropwising, take off constant pressure funnel, reaction unit is moved into thermostatical oil bath, temperature is controlled at 50 ℃, successive reaction 72h;

After reaction finishes, with Rotary Evaporators except desolventizing DMSO, obtain faint yellow opaque thick liquid, being dissolved in stirring and dissolving in a small amount of distilled water is settled solution, then splash in a large amount of acetone and produce white precipitate, abandoning supernatant, carries out removing water-soluble impurity three times repeatedly, the product obtaining carries out lyophilize after adding dialysis tubing (molecular weight cut-off is 3500) dialysis 24h, obtains G3.5PAMAM-Tris.

6. a kind of pH and temperature supersensitivity dendritic macromole pharmaceutical carrier three generations half preparation method according to claim 4, is characterized in that, the preparation method of 3.5 generation polyamide dendroid macromole G3.5PAMAM is as follows:

Take quadrol and methyl acrylate as raw material, mol ratio is according to 1:4-5, ice bath, and temperature remains on 0 ℃; The methanol solution that slowly drips methyl acrylate, in the methanol solution of quadrol, drips and finishes, and repeatedly vacuumizes, and nitrogen protection, recovers room temperature, then at 35 ℃, reacts 6h; After reaction finishes, revolve to steam and remove solvent methanol, obtain flaxen thick liquid G0.5PAMAM;

With G0.5PAMAM: quadrol=1:4-8(mol ratio); The methanol solution of quadrol is joined in the there-necked flask with magnetic agitation, thermometer, and ice bath, slowly drips the methanol solution of G0.5PAMAM; Drip and finish, repeatedly vacuumize, nitrogen protection is reacted 24h at 35 ℃, obtains product G1.0PAMAM;

With G1.0PAMAM: methyl acrylate=1:16(mol ratio); The methanol solution of methyl acrylate is joined in the there-necked flask with magnetic agitation, thermometer, and ice bath, slowly drips the methanol solution of G1.0PAMAM; Drip and finish, repeatedly vacuumize, nitrogen protection is reacted 24h at 35 ℃; Revolve to steam and remove unreacted methyl acrylate and solvent methanol, obtain lurid thick liquid G1.5PAMAM;

With G1.5PAMAM: quadrol=1:80(mol ratio); The methanol solution of quadrol is joined in the there-necked flask with magnetic agitation, thermometer, and ice bath, slowly drips the methanol solution of G1.5PAMAM; Drip and finish, repeatedly vacuumize, nitrogen protection is reacted 72h at 35 ℃; React complete, adopt vacuum rotary evaporator at 35 ℃, to steam except methyl alcohol, obtain product G2.0PAMAM;

With G2.0PAMAM: methyl acrylate=1:24(mol ratio); The methanol solution of methyl acrylate is joined in the there-necked flask with magnetic agitation, thermometer, and ice bath, slowly drips the methanol solution of G2.0PAMAM; Drip and finish, repeatedly vacuumize, nitrogen protection is reacted 72h at 35 ℃; Revolve to steam and remove unreacted methyl acrylate and solvent methanol, obtain lurid thick liquid G2.5PAMAM;

With G2.5PAMAM: quadrol=1:160(mol ratio); The methanol solution of quadrol is joined in the there-necked flask with magnetic agitation, thermometer, and ice bath, slowly drips the methanol solution of G2.5PAMAM; Drip and finish, repeatedly vacuumize, nitrogen protection is reacted 6 days at 35 ℃.React complete, adopt vacuum rotary evaporator at 35 ℃, to steam except methyl alcohol, obtain product G3.0PAMAM;

With G3.0PAMAM: methyl acrylate=1:48(mol ratio); The methanol solution of methyl acrylate is joined in the there-necked flask with magnetic agitation, thermometer, and ice bath, slowly drips the methanol solution of G3.0PAMAM; Drip and finish, repeatedly vacuumize, nitrogen protection is reacted 48h at 35 ℃.Revolve to steam and remove unreacted methyl acrylate and solvent methanol, obtain lurid thick liquid G3.5PAMAM.

7. according to claims 1 to 3 a kind of pH and temperature supersensitivity dendritic macromole pharmaceutical carrier three generations's half described in any one preparation method, it is characterized in that, the preparation method that described end becomes the N-propyl group piperazine of carboxyl structure is:

Utilize acid anhydrides to modify N-propyl group piperazine, make its end become carboxyl structure; The purified product that obtains; As follows.

8. a kind of pH and temperature supersensitivity dendritic macromole pharmaceutical carrier three generations half preparation method according to claim 7, is characterized in that, the preparation method that described end becomes the N-propyl group piperazine of carboxyl structure is:

Methylene dichloride is added in container, under agitation in container, slowly add Succinic anhydried, make while stirring Succinic anhydried dissolve;

N-propyl group piperazine, with methylene dichloride dissolving, then packs in constant pressure funnel; At 15 ℃, the aminated compounds that methylene dichloride is dissolved is slowly added drop-wise in there-necked flask, dropwises, and at this temperature, reacts 5h;

After reaction finishes, rotary evaporation steams unnecessary methylene dichloride, and solution becomes reddish-brown viscous liquid, adds a small amount of ether after taking-up, leave standstill after for some time, have pink solid to separate out, filter, washed with dichloromethane twice for filter cake, uses dehydrated alcohol recrystallization, 80 vacuum-drying 24h.

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