CN111298132B - Tree-shaped molecule gemcitabine self-assembled nano prodrug and preparation method and application thereof - Google Patents
Tree-shaped molecule gemcitabine self-assembled nano prodrug and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a dendrimer gemcitabine self-assembly nano prodrug and a preparation method and application thereof. The microstructure and the enzyme stability of the nanoparticle are researched, and further cell uptake and mouse in-vivo experiments prove that the self-assembled nano prodrug can obviously improve the metabolic stability and the antitumor activity of the prototype drug gemcitabine, can avoid the problems of large toxic and side effects and the like of the parent small-molecule drug gemcitabine, and can provide a basic experiment basis for clinical application.
Description
Technical Field
The invention belongs to the technical field of nano drug delivery systems, and particularly relates to a dendrimer Gemcitabine (Gemcitabine) self-assembled nano prodrug, and a preparation method and application thereof.
Background
Gemcitabine (formula I), approved by FDA in 1996 to enter the market, is a first-line drug for treating advanced non-small cell lung cancer and pancreatic cancer, and is also used for treating malignant tumors such as breast cancer, ovarian cancer and nasopharyngeal carcinoma (biomed. Pharmacother.2017,88,635.). However, gemcitabine is highly susceptible to the removal of the 4-amino group by the presence of a large amount of deoxycytidine deaminase (CDA) in the liver and blood to produce the inactive product 2,2' -difluorodeoxyuridine, resulting in poor metabolic stability and a very short half-life (<17min) in humans (eur.j.pharm.sci.2016,93,147.). Therefore, continuous intravenous administration or combination is usually required in clinic to maintain the cytotoxic effect, which increases the toxic side effects of the drug on normal tissues and organs of the body, such as hepatotoxicity and renal toxicity (eur.j.pharm.sci.2016,93,147). In addition, gemcitabine is a hydrophilic drug that is difficult to penetrate the cell membrane by free diffusion and must be transferred into cells by the corresponding nucleoside transporter (e.g., hENT). However, this transporter appears to be absent or low in expression in most patients (> 65%) (Biomaterials 2014,35,6482.), further limiting the efficacy of gemcitabine, and is one of the major causes of tumor cell resistance to gemcitabine (Cancer treat. res.2002,112, 27.). Therefore, improving the curative effect of gemcitabine and reducing toxic and side effects is a scientific problem to be researched urgently in clinic at present.
The nanometer drug delivery system can accurately convey the drug loaded on the carrier to tumor tissues by relying on an EPR effect, and the administration mode can effectively reduce the toxic and side effects of gemcitabine chemical drugs, thereby improving the chemotherapy effect. However, the drug molecules are passively encapsulated in the carrier like this, and there is usually no strong force between the drug molecules and the carrier to link them except for a few possible hydrophobic or electrostatic interactions. Therefore, most drug carriers have the problems of low drug encapsulation rate and easy leakage, and are likely to occur in the preparation storage stage and in the in vivo circulation, especially for gemcitabine type polar drugs. Furthermore, the carrier material serves only as a drug delivery vehicle without drug effect and takes up a great proportion of the composition of the entire drug delivery system, resulting in a relatively low loading of the drug; the molecular weight and its distribution of the polymeric carrier material are relatively difficult to control; the use of a large amount of auxiliary materials can cause unnecessary metabolic problems and toxic and side effects, and the clinical transformation and wide application of the nano-medicament are seriously limited. Therefore, there is an urgent need to design and develop a novel drug delivery system to improve the drug delivery efficiency.
Prodrug-based self-assembled prodrug nano Delivery Systems (SAPDs) combine the advantages of prodrugs and nanotechnology, and have become a hotspot of chemotherapeutic drug delivery research due to the advantages of high drug loading, good stability, low toxic and side effects and the like (Materials Today Chemistry 2017,4, 26.). The so-called self-assembled prodrug delivery system is mainly characterized in that drug molecules are covalently combined with various types of materials or other drugs to form amphiphilic prodrugs, and then the amphiphilic prodrugs are dispersed into water by a certain method to prepare nanostructures or nano aggregates. Couvreur et al reacted the hydrophilic drug gemcitabine (dFdC) with squalene acid (SQ) to give the amphiphilic conjugated molecule SQdFdC and used chemical precipitation to give SQdFdC self-assembled prodrug nanosystems (SQdFdCNAs) (J.Control Release 2007,124, 20.). The SQdFdCNAs has better in-vivo blood compatibility in tumor-bearing mice than the prototype drug gemcitabine, obviously improves the survival rate of the mice when orally administered, and shows better anti-tumor effect. The Yan research team connects the hydrophilic drug irinotecan (Ir) and the hydrophobic drug chlorambucil (Cb) through esterification reaction to obtain an amphiphilic drug-drug conjugate (ADDC-Ir-Cb). The ADDC-Ir-Cb can self-assemble in water to form a stable nano-drug self-delivery system (J.Am.chem.Soc.2014,136, 11748.). Compared with a single medicine, the nano system can prolong the in vivo circulation time of the loaded medicine and greatly promote the enrichment of the medicine at the tumor part. In addition, the nano-sized particles can effectively overcome the multidrug resistance of tumor cells, and finally show remarkable in vivo and in vitro anti-tumor activity. It can be seen that the drug molecules in the self-assembling prodrug delivery system are not only part of the therapeutic drug but also part of the carrier.
The key to the construction of SAPDs is the design of the prodrug molecule. The prodrug molecule has amphipathy firstly so as to ensure that the whole system can form a stable self-assembly body; secondly, it is also necessary to be able to release the original drug from the aggregate at the target site with a suitable dissociation rate, so as to obtain a controlled release effect. Therefore, the design and preparation of SAPDs are usually drug-specific, and depend on the chemical structure of the drug, and the preparation of SAPDs of each drug requires selection of a proper assembly strategy and reasonable molecular design, and the proportion of the drug is difficult to regulate and optimize, thus limiting the wide application of such delivery systems to a certain extent. Therefore, there is an urgent need for a straightforward and effective solution to further promote the use of such drug delivery systems.
Disclosure of Invention
In order to overcome the disadvantages and shortcomings of the self-assembled nano prodrug in the prior art, the invention provides a tree-like molecule gemcitabine self-assembled nano prodrug, wherein the hydrophilic chain segment of the self-assembled nano prodrug is polyamide tree-like molecule, and the hydrophobic chain segment of the self-assembled nano prodrug is alkyl long chain or polyethylene glycol; the hydrophilic chain segment and the hydrophobic chain segment are connected through a high-efficiency 'nitrine-alkyne' click chemical reaction; the prodrug molecule of the micromolecule antitumor drug gemcitabine is chemically bonded to the surface of the dendrimer to obtain the dendrimer gemcitabine self-assembled nano prodrug; the amphiphilic nano prodrug is self-assembled in the aqueous solution to form a nano micelle, so that the stability of the original drug can be effectively improved; the self-assembled nano prodrug based on the dendrimer has high drug loading, is convenient to synthesize, and has antitumor activity obviously higher than that of a prototype drug.
The invention also aims to provide a preparation method of the dendrimer gemcitabine self-assembled nano prodrug.
The invention further aims to provide application of the dendrimer gemcitabine self-assembled nano prodrug in preparation of drugs for targeted delivery of antitumor drugs and drugs for inhibiting tumor cell proliferation.
The invention adopts the following technical scheme for realizing the purpose: a dendrimer gemcitabine self-assembled nano prodrug, the self-assembled nano prodrug having a structure represented by formula II:
wherein n is the number of methylene repeating units, and n is any integer between 2 and 500.
The invention also provides a preparation method of the dendrimer gemcitabine self-assembled nano prodrug, which is characterized by comprising the following steps: (1) synthesizing an amphiphilic dendritic molecule with azide-modified tail ends; (2) synthesizing a gemcitabine prodrug molecule containing an alkyne linkage structure; (3) grafting the prodrug molecule synthesized in the step (2) to the surface of the dendrimer synthesized in the step (1) through a Click reaction to finally obtain the dendrimer gemcitabine self-assembled nano prodrug.
The specific process of the step (1) is as follows: the azide-terminated dendrimer analogue is synthesized by utilizing the substitution reaction between the first generation or the second generation of dendrimer containing amido and azide sulfonyl imidazole, the substitution reaction can be realized by conventional reaction, and the azide group in the dendrimer structure is used for connecting drug molecules.
Wherein, the synthesis of the azide-terminated dendrimer with the long alkyl chain as the hydrophobic part comprises two steps of reactions, and the specific reaction formula is shown as the following formula III:
n is the number of methylene repeating units, preferably 5 to 500.
Firstly, synthesizing an amphiphilic dendrimer modified by a terminal amino group, wherein the amphiphilic dendrimer is obtained by utilizing a reducing amine reaction between ethylenediamine and a dendrimer ester group, the dendrimer is one or two generations, the molar ratio of the dendrimer to the ethylenediamine is 1: 10-1: 80, the reaction time is 24-168 h, the reaction process is preferably carried out at normal temperature, the solvent system is an organic solvent, specifically methanol, the product is obtained by silica gel column chromatography or dialysis, and the product is freeze-dried; and secondly, synthesizing the terminal azide-modified amphiphilic dendritic molecule, wherein the terminal azide-modified amphiphilic dendritic molecule, imidazole azide sulfonate and potassium carbonate react under the catalysis of copper sulfate pentahydrate, the molar ratio of the terminal amine-modified amphiphilic dendritic molecule to imidazole azide sulfonate to potassium carbonate to copper sulfate pentahydrate is 1:1.1:1.5: 0.1-1: 2.2:2:0.5, the specific operation process is preferably that imidazole azide sulfonate is dissolved in a mixed solvent of ethanol and methanol, the amphiphilic dendritic molecule modified by terminal amine, potassium carbonate and copper sulfate pentahydrate are sequentially added and then continuously react for 12-36 hours to obtain a product, and the system after the reaction is extracted by an ethylenediamine tetraacetic acid disodium salt (EDTA) aqueous solution/ethyl acetate system, and is subjected to suction filtration and column chromatography to obtain a purified product.
The synthesis of the second-generation amphiphilic polyamide azide-terminated dendritic molecule comprises two-step reaction, and the specific reaction formula is shown as the following formula IV:
n is the number of methylene repeating units, and n is any integer between 2 and 500.
Firstly, synthesizing an amphiphilic dendrimer modified by a terminal amino group, wherein the amphiphilic dendrimer is obtained by utilizing a reducing amine reaction between ethylenediamine and a dendrimer ester group, the molar ratio of the dendrimer to the ethylenediamine is 1: 10-1: 80, the reaction time is 24-168 h, the reaction process is preferably carried out at normal temperature, the solvent system is an organic solvent, specifically methanol, and the product is obtained by settling; and secondly, synthesizing the terminal azide-modified amphiphilic dendritic molecule, wherein the terminal azide-modified amphiphilic dendritic molecule, imidazole azide sulfonate and potassium carbonate react under the catalysis of copper sulfate pentahydrate, the molar ratio of the terminal amine-modified amphiphilic dendritic molecule to imidazole azide sulfonate to potassium carbonate to copper sulfate pentahydrate is 1:1.1:1.5: 0.1-1: 2.2:2:0.5, the specific operation process is preferably that imidazole azide sulfonate is dissolved in a mixed solvent of ethanol and methanol, the terminal amine-modified dendritic molecule, potassium carbonate and copper sulfate pentahydrate are sequentially added and then continue to react for 12-36 hours to obtain a product, and the system after the reaction is extracted by an ethylenediamine tetraacetic acid disodium salt (EDTA) aqueous solution/ethyl acetate system, is subjected to suction filtration and column chromatography to obtain a purified product.
The specific process of the step (2) is as follows: the method comprises the following steps of synthesizing gemcitabine prodrug molecules with an acetylene bond structure by utilizing acylation reaction between carboxylic acid containing acetylene bonds and gemcitabine, wherein the acylation reaction can be realized through conventional reaction, amide bonds in the prodrug molecular structure can be broken under in vivo enzyme catalysis, the specific reaction comprises three steps of reactions, and the reaction formula is shown as a formula V:
step one, gemcitabine and TBSCl are used as raw materials, substitution reaction is carried out in an organic solvent under the condition of low temperature, and silica gel column chromatography is carried out for separation and purification to obtain gemcitabine intermediate products protected by 4 'and 5' silicon methyl; the organic solvent is dichloromethane or DMF, the molar ratio of gemcitabine to TBSCl is 1: 2.5-1: 5, the reaction temperature is 15-30 ℃, and the reaction time is 12-48 h; the specific process is as follows: dissolving gemcitabine in an organic solvent, adding TBSCl and imidazole, stirring at room temperature for reaction, quenching with 3-10 times of distilled water for reaction, extracting with ethyl acetate, collecting an organic phase, drying, filtering, concentrating, and separating by silica gel column chromatography, wherein the eluent is dichloromethane/methanol to obtain the gemcitabine protected by 4 'and 5' silicon methyl.
Secondly, reacting the product of the first step, namely 4 'and 5' position silicon methyl protected gemcitabine, with a terminal alkynyl carboxylic acid compound in an organic solvent under the catalysis of 1-ethyl- (3-dimethylaminopropyl) carbonyl diimine hydrochloride (EDCl) and 1-hydroxy benzotriazole (HOBt) at low temperature, and separating and purifying by silica gel column chromatography to obtain N4 position and 4 'and 5' position protected gemcitabine derivatives; the organic solvent is dichloromethane, tetrahydrofuran or dioxane, the molar ratio of the gemcitabine protected by 4 '-and 5' -silicon methyl groups to the terminal alkynyl carboxylic acid compound is 1: 1-1: 1.2, the low-temperature condition refers to that the reaction temperature is-10-0 ℃, and the reaction time is 12-48 h; the specific process is as follows: dissolving the terminal alkynyl carboxylic acid compounds and the coupling reagent in an organic solvent at low temperature, continuously stirring for 30-60 min, slowly adding the product obtained in the first step and gemcitabine protected by 5 ' -site silicon methyl into a reaction system, stirring for reaction at room temperature, quenching with distilled water, extracting, collecting an organic phase, drying, filtering, separating by silica gel column chromatography, and obtaining the product N4-site 4 ' -and 5 ' -site protected gemcitabine derivatives containing alkyne bonds, wherein the eluent is petroleum ether ethyl acetate.
And thirdly, carrying out substitution reaction on the 4 'and 5' alkyne-containing gemcitabine derivatives at the N4 position obtained in the second step and tetra-N-butylammonium fluoride (TBAF) to obtain gemcitabine prodrug molecules containing alkyne structures, wherein the molar ratio of the N4 4 'and 5' alkyne-containing gemcitabine derivatives to the TBAF is 1: 2-1: 4, the reaction temperature is 15-40 ℃, and the reaction time is 15-60 min.
The specific process and product structure of step (3) are shown below. Wherein, formula VI is a reaction formula for preparing a generation of dendrimer gemcitabine nano prodrug; formula VII is a reaction formula for preparing the second generation dendrimer gemcitabine nano prodrug.
Wherein n is the number of repeating units of methylene, and n is any integer between 2 and 500.
The specific process of the step (3) is as follows: grafting gemcitabine prodrug molecules containing alkyne bond structures obtained in the step (2) to the amphiphilic dendritic molecules subjected to end azide modification obtained in the step (1) by utilizing an azide-alkyne click chemical reaction to obtain amphiphilic dendrimer gemcitabine self-assembled nano prodrugs, wherein the molar ratio of the amphiphilic dendritic molecules subjected to end azide modification to the gemcitabine prodrug molecules containing alkyne bond structures is 1: 1.0-1: 2.0, and preferably 1: 1.0-1: 1.5; the reaction temperature is 40-80 ℃, and preferably 40-60 ℃; the solvent is preferably a mixed solvent of tetrahydrofuran and water; the reaction time is 1-8 h, preferably 3-5 h; and carrying out silica gel column chromatography to obtain a purified product.
The amphiphilic dendrimer gemcitabine self-assembled nano prodrug disclosed by the invention can be self-assembled in an aqueous solution to form nanoparticles, and the specific process is as follows: dissolving the amphiphilic dendrimer gemcitabine self-assembled nano prodrug into a DMSO solution, wherein the concentration is 1.0-25.0 mg/mL, and preferably 5.0-10.0 mg/mL; then, a dialysis bag is filled, and the amphiphilic dendrimer gemcitabine self-assembled nano-particles are obtained through dialysis in deionized water at room temperature; the molecular weight of the dialysis bag is 500-3000 Da; the dialysis time is 5-48 h; the average particle size of the amphiphilic dendrimer gemcitabine self-assembled nanoparticles is 7-200 nm, and preferably 7-100 nm.
The amphiphilic dendrimer gemcitabine nano prodrug is prepared by coupling amphiphilic dendrimer which is rich in surface groups, easy to modify and controllable in structure with anticancer drug gemcitabine through 'azide-alkyne' click chemistry, has amphipathy, and can be self-assembled to form nanoparticles under certain conditions. The obtained gemcitabine serves as a carrier and a chemotherapeutic drug in the obtained nano prodrug, so that the drug loading capacity is greatly improved, and the stability, the delivery efficiency and the antitumor activity of the nano prodrug are enhanced. The nano prodrug has wide application prospect in the anti-tumor field.
Compared with the prior art, the invention has the following beneficial effects:
(1) the surface groups of the dendrimer serving as the carrier are rich, the modification is easy, the synthesis is simple, and the amphiphilic universal nano prodrug delivery system is favorably constructed.
(2) The dendrimer gemcitabine nano prodrug disclosed by the invention can be self-assembled in an aqueous solution to form nanoparticles, and the loading efficiency of chemotherapeutic drugs is high.
(3) The prodrug molecule has amphipathy, and nanoparticles formed by self-assembly can improve the metabolic stability and delivery efficiency of gemcitabine, and show lower toxicity but higher antitumor activity than that of a parent drug.
(4) The invention provides a preparation method of a dendrimer gemcitabine self-assembled nano prodrug, which can obtain an intelligent nano drug with high drug loading rate and has wide application prospect in the aspect of tumor chemotherapy.
Drawings
FIG. 1 shows the starting and end products of example 11H NMR；
FIG. 2 shows the intermediate and final products of example 21H NMR；
FIG. 3 shows the end product of example 31H NMR；
FIG. 4 shows the particle size distribution and morphology of dendrimer-gemcitabine self-assembled nano-drug C1;
figure 5 is the stability of gemcitabine and dendrimer-gemcitabine self-assembling nano-prodrug C1 to CDA;
FIG. 6 is a graph of the effect of nucleic acid transporters on gemcitabine and dendrimer-gemcitabine self-assembling nano-prodrug C1 delivery;
figure 7 is the in vivo antitumor activity of gemcitabine and dendrimer-gemcitabine self-assembling nano-prodrug C1.
Detailed Description
The examples set out below are intended to facilitate a better understanding of the invention by a person skilled in the art and are not intended to limit the invention in any way.
The following examples describe the preparation process, all chemical reagents used were analytically pure, unless otherwise noted.
Example 1: preparation of second generation azide-terminated dendritic molecule with C18 long chain as hydrophobic part
First, 99.8mg (0.11mmol) of second-generation ester-group-end amphiphilic dendrimer is weighed, 5.0mL of methanol is added to dissolve the second-generation ester-group-end amphiphilic dendrimer, then 1.0mL (15mmol) of ethylenediamine is added under stirring, stirring is carried out at 30 ℃ for reaction for 72h, a proper amount of methanol is added after the reaction, decompression and spin-drying are carried out to obtain oily liquid, and methanol/ether is used for settling for three times to obtain an amino-end-modified dendrimer product (101.8mg, 91%).
In the second step, 105mg (0.6mmol) of azidosulfonylimidazole was weighed, dissolved in 4.0mL of a mixed solvent of methanol and acetonitrile, and then 78mg (0.075mmol) of amino-terminated dendrimer and CuSO were added thereto under stirring4·5H2O (3.0mg,5.0 mol%) and K2CO3(83mg,0.60mmol), stirred at room temperature for 24h, reacted, spin-dried under reduced pressure, extracted three times with EDTA/ethyl acetate, and then purified on a silica gel column with dichloromethane/methanol 1/1 as eluent to give the product azide-terminated dendrimer (33.mg, 40%).
The synthesis of azide-terminated dendrimers with the C18 long chain as the hydrophobic moiety or the polyethylene glycol as the hydrophobic moiety at the lower generation is the same as that described above.
Example 2: synthesis of acetylenic gemcitabine prodrugs
In the first step, 263mg (1.0mmol) of gemcitabine is weighed and dissolved in 5mL of DMF, then 603mg (4mmol) of TBSCl and 272mg of imidazole are added into the reaction system, the reaction system is kept overnight, 30mL of distilled water is added for quenching reaction, extraction is carried out for 3 times by ethyl acetate, 15mL of the extraction is carried out for each time, the extract liquor is combined, dried by anhydrous sodium sulfate, reduced pressure concentration is carried out, silica gel column chromatography separation is carried out, and dichloromethane/methanol gradient elution is carried out to obtain 310mg of white solid with the yield of 63.3%.
And secondly, dissolving 59.6mg (0.6mmol) of 4-pentynoic acid, 191.3mg (1.0mmol) of HOBt and 135.8mg (1.0mmol) of HOBt in 4.5mL of THF, stirring for 30min in ice bath, adding 250mg (0.5mmol) of gemcitabine protected at the 4 'and 5' positions obtained in the first step, stirring at room temperature, monitoring the reaction by TLC (thin layer chromatography), stopping the reaction, evaporating the solvent under reduced pressure, adding 15mL of distilled water, extracting for 3 times with ethyl acetate, 15mL of each time, combining organic phases, drying with anhydrous sodium sulfate, filtering, evaporating the solvent under reduced pressure, and separating and purifying by silica gel column chromatography to obtain 94.2mg of white solid with the yield of 33%.
And a third step of weighing the product (113mg,0.20mmol) obtained in the second step, adding THF (3.0mL) and TBAF (0.50mL,0.50mmol), stirring at room temperature for 40min, then concentrating under reduced pressure, and purifying by column chromatography to obtain the target product as a white solid (40 mg, 58.1%).
Example 3: preparation of second generation dendrimer-gemcitabine amphiphilic prodrug with C18 long chain as hydrophobic part
Weighing gemcitabine prodrug (41.5mg,0.12mmol) containing acetylene bond and CuSO4·5H2O (2.4mg,10 mol%) and sodium ascorbate (2.6mg,15 mol%), followed by addition of terminal azide-modified dendrimers under nitrogen, tetrahydrofuran and water in sequence, stirring at 60 deg.C for 5h, and then concentration with CH2Cl2/Et2The O is settled overnight and then is further purified by silica gel column chromatography, and gradient elution is carried out on ethyl acetate/methanol (volume ratio is 2:1-1:3) to obtain 19.7mg of white solid with the yield of 39.4%.
Example 4: preparation of amphiphilic dendrimer-gemcitabine prodrug nanoparticles
The product of example 3 was dissolved in DMSO (a generation of dendrimer-gemcitabine amphiphile prodrugs with the long C18 chain as the hydrophobic moiety is exemplified) and loaded into dialysis bags with molecular weight of 500-1000; the deionized water is changed every 0.5h in the first two hours, then the water is changed every 1h, and the liquid in the dialysis bag is collected after 8h, so that the micellar solution C1 of the nano prodrug is obtained.
Example 5: characterization of novel amphiphilic dendrimer-gemcitabine nanoproberide (C1)
The particle size distribution of the nanoparticles is determined by a dynamic light scattering method by using a Malvern particle size analyzer, and a sample is diluted to a proper concentration by deionized water before determination. Taking a proper amount of nanoparticle solution, dropwise adding the nanoparticle solution on a copper net, dyeing phosphotungstic acid for 1min, sucking dry filter paper, drying, then placing the dried nanoparticle solution under a transmission electron microscope to observe the appearance form of the nanoparticles and taking a picture, as shown in figure 4, C1 can spontaneously self-assemble in aqueous solution to form stable nano micelles, and the C1 nanoparticles are all in a regular spherical shape, are uniformly dispersed, and are suitable for targeted drug delivery.
Example 6: gemcitabine and dendrimer gemcitabine self-assembled nano prodrug stability
mu.L of Tris-HCl buffer (pH7.5, 50mM) containing gemcitabine (100. mu.M) or AmDM-Gem nanomicelles (100. mu.M) was added to a 96-well plate using a pipette, followed by 50. mu.L of CDA (0.25. mu.g/50. mu.L) enzyme per well and shaking of the plate for 2 min. Setting the absorption wavelength of the microplate reader at 280nm, and measuring the absorbance of each blank at 1min, 2min, 3min, 4min, 5min, 20min and 45 min. As shown in FIG. 5, the absorbance of the dendrimer gemcitabine self-assembled nano-prodrug in the presence and absence of CDA enzyme is not significantly different, while the absorbance of gemcitabine in the presence of CDA is significantly lower than that in the absence of enzyme, which proves that gemcitabine is easily reduced by CDA and is unstable, while the dendrimer gemcitabine self-assembled nano-prodrug shows better stability without being affected by CDA.
Example 7: effect of nucleic acid vector (hENT1) on the delivery efficiency of gemcitabine and the dendrimer gemcitabine self-assembled nano-prodrug
Cancer cell mimic drug resistant strains were constructed using the hENT1 inhibitor dipyridamole. Cells in the logarithmic growth phase are taken, digested and counted, 96-well plates are inoculated according to the cell density of about 6000/well, and the cells are cultured in an incubator for 24 hours. The cells were then pre-loaded with the hENT1 inhibitor dipyridamole (4 μ g/mL) or a blank solvent, placed in a cell incubator and incubated for 30min, followed by addition of a range of concentrations of dendrimer-gemcitabine nanomicelles and functionalized gemcitabine along with free gemcitabine, and the change in IC50 values was determined 72h later using the CCK8 method. As shown in FIG. 6, the IC50 values of C1 for pancreatic cancer cells PANC-1 were 9.4. mu.M and 10.7. mu.M, respectively, with no significant change in the absence of the inhibitory nucleic acid vector hENT inhibitor dipyridamole (dipyridamole). Whereas the antitumor activity of the corresponding gemcitabine (Gem) and functionalized gemcitabine (V) was significantly reduced after the use of the nucleic acid transporter carrier inhibitor (fig. 6). Therefore, the preliminary result shows that the amphiphilic dendrimer-gemcitabine nano-micelle can enter cells to play an anti-tumor effect in a nucleic acid transporter independent mode, and the delivery efficiency of loading gemcitabine is expected to be improved.
Example 8: in vivo antitumor Activity of Gemcitabine and dendrimer Gemcitabine self-assembled Nanopropions
Tumor cells are inoculated to female mice of 6-8 weeks of age by subcutaneous injection, gemcitabine or amphiphilic dendrimer-gemcitabine nano prodrug is injected into tail vein when tumors grow to a certain volume, the administration frequency is 2 times per week, and 7 mice are in each group. The PBS buffer solution treatment group, the dendrimer nano-micelle treatment group and the gemcitabine treatment group were used as control groups. The body weight changes of the mice were recorded after administration to examine the toxicity of both drugs, and the tumor volume was measured to evaluate the proliferation of the tumor. As shown in fig. 7, it is shown that dendrimer gemcitabine self-assembled nano prodrug C1 can significantly inhibit tumor growth, and the activity of inhibiting tumor growth is significantly stronger than that of bulk gemcitabine.
The foregoing is illustrative of specific embodiments of the present invention and reference to reagents, equipment, procedures and the like not specifically described herein is to be understood as being modified in light of the common and routine experimentation in the art.
The foregoing embodiments illustrate the principles, principal features and advantages of the invention, and it will be understood by those skilled in the art that the invention is not limited to the foregoing embodiments, which are merely illustrative of the principles of the invention, and that various changes and modifications may be made therein without departing from the scope of the principles of the invention.
Claims (7)
1. A dendrimer gemcitabine self-assembled nano prodrug, wherein the self-assembled nano prodrug has a structure represented by formula II (1) or formula II (2):
wherein n in the formula II (1) and the formula II (2) is the number of methylene repeating units, and n is any integer between 2 and 500.
2. The dendrimer gemcitabine self-assembling nano-prodrug of claim 1, wherein the dendrimer gemcitabine self-assembling nano-prodrug is capable of self-assembling in an aqueous solution to form nanoparticles by: dissolving the amphiphilic dendrimer gemcitabine self-assembled nano prodrug in a DMSO solution, wherein the concentration of the amphiphilic dendrimer gemcitabine self-assembled nano prodrug is 1.0-25.0 mg/mL, then filling the amphiphilic dendrimer gemcitabine self-assembled nano prodrug into a dialysis bag, and dialyzing the amphiphilic dendrimer gemcitabine self-assembled nano prodrug in deionized water at room temperature to obtain amphiphilic dendrimer gemcitabine self-assembled nano particles, wherein the molecular weight of the dialysis bag is 500-3000 Da, the dialysis time is 5-48 h, and the particle size of the obtained amphiphilic dendrimer gemcitabine self-assembled nano particles is 7-200 nm.
3. A method of preparing the dendrimer gemcitabine self-assembled nano-prodrug of claim 1, comprising the steps of: (1) synthesizing an amphiphilic dendritic molecule with azide-modified tail ends; (2) synthesizing a gemcitabine prodrug molecule containing an alkyne linkage structure; (3) grafting gemcitabine prodrug molecule prodrug molecules containing acetylene bond structures synthesized in the step (2) to the surfaces of the amphiphilic dendritic molecules subjected to end azide modification synthesized in the step (1) through a Click reaction to finally obtain the dendrimer gemcitabine self-assembled nano prodrug.
4. The method of preparing the dendrimer gemcitabine self-assembling nano-prodrug of claim 3, wherein: for the synthesis of formula II (1), step (1) comprises a two-step reaction, the specific reaction formula is shown in formula III below:
n is the number of methylene repeating units, and n is any integer between 2 and 500;
the specific process is as follows: firstly, synthesizing an amphiphilic dendrimer modified by a terminal amino group, specifically, obtaining the amphiphilic dendrimer by utilizing a reducing amine reaction between ethylenediamine and a dendrimer ester group, wherein the molar ratio of the dendrimer to the ethylenediamine is 1: 10-1: 80, the reaction time is 24-168 h, the reaction process is carried out at normal temperature, the solvent system is an organic solvent, and the product is obtained by silica gel column chromatography or dialysis and freeze-drying; secondly, synthesizing a terminal azide-modified amphiphilic dendritic molecule, specifically, reacting the terminal amino-modified amphiphilic dendritic molecule, imidazole azide sulfonate and potassium carbonate under the catalysis of copper sulfate pentahydrate, wherein the molar ratio of the terminal amino-modified amphiphilic dendritic molecule to the imidazole azide sulfonate to the potassium carbonate to the copper sulfate pentahydrate is 1:1.1:1.5: 0.1-1: 2.2:2:0.5, extracting the reacted system with an oxalic acid diethylamine disodium salt aqueous solution/ethyl acetate system, performing suction filtration, and performing column chromatography to obtain a purified product;
for the synthesis of formula II (2), step (1) comprises a two-step reaction, the specific reaction formula is shown in formula IV below:
n is the number of methylene repeating units, and n is any integer between 2 and 500;
the specific process is as follows: firstly, synthesizing an amphiphilic dendrimer modified by terminal amino, specifically, obtaining the amphiphilic dendrimer by using a reducing amine reaction between ethylenediamine and a dendrimer ester group, wherein the molar ratio of the dendrimer to the ethylenediamine is 1: 10-1: 80, the reaction time is 24-168 h, the reaction process is carried out at normal temperature, the solvent system is an organic solvent, and the product is obtained by settling; and secondly, synthesizing a terminal azide-modified amphiphilic dendritic molecule, specifically, reacting the terminal amino-modified amphiphilic dendritic molecule, imidazole azide sulfonate and potassium carbonate under the catalysis of copper sulfate pentahydrate, wherein the molar ratio of the terminal amino-modified amphiphilic dendritic molecule to the imidazole azide sulfonate to the potassium carbonate to the copper sulfate pentahydrate is 1:1.1:1.5: 0.1-1: 2.2:2:0.5, extracting the reacted system by using an oxalic acid diethylamine disodium salt aqueous solution/ethyl acetate system, performing suction filtration, and performing column chromatography to obtain a purified product.
5. The method of preparing the dendrimer gemcitabine self-assembling nano-prodrug of claim 3, wherein: in the step (2), an acylation reaction between carboxylic acid containing an acetylene bond and gemcitabine is utilized to synthesize gemcitabine prodrug molecules with acetylene bond structures, the acylation reaction is realized through conventional reactions, amide bonds in the prodrug molecular structures can be broken under the catalysis of in vivo enzymes, the specific reaction comprises three steps of reactions, and the reaction formula is shown as formula V:
the first step, gemcitabine and TBSCl are used as raw materials, substitution reaction is carried out in an organic solvent under the low temperature condition, silica gel column chromatography separation and purification are carried out to obtain a gemcitabine intermediate product protected by 4 'and 5' silicon methyl, the organic solvent is dichloromethane or DMF, the molar ratio of gemcitabine to TBSCl is 1: 2.5-1: 5, the reaction temperature is 15-30 ℃, and the reaction time is 12-48 hours;
secondly, reacting the 4 'and 5' silicon methyl protected gemcitabine obtained in the first step with a terminal alkynyl carboxylic acid compound in an organic solvent under the catalysis of 1-ethyl- (3-dimethylaminopropyl) carbonyl diimine hydrochloride (EDCI) and 1-hydroxy benzotriazole (HOBt) at a low temperature, and performing silica gel column chromatography separation and purification to obtain N4 bit and 4 'and 5' protected gemcitabine derivatives, wherein the organic solvent is dichloromethane, tetrahydrofuran or dioxane, the molar ratio of the 4 'and 5' silicon methyl protected gemcitabine to the terminal alkynyl carboxylic acid compound is 1: 1-1: 1.2, and the low temperature refers to a reaction temperature of-10-0 ℃ and a reaction time of 12-48 hours;
and thirdly, carrying out substitution reaction on the 4 'and 5' protected alkyne-containing gemcitabine derivatives at the N4 site obtained in the second step and tetra-N-butylammonium fluoride (TBAF) to obtain gemcitabine prodrug molecules containing alkyne structures, wherein the molar ratio of the 4 'and 5' protected alkyne-containing gemcitabine derivatives at the N4 site to the TBAF is 1: 2-1: 4, the reaction temperature is 15-40 ℃, and the reaction time is 15-60 min.
6. The method of preparing the dendrimer gemcitabine self-assembling nano-prodrug of claim 3, wherein: for the synthesis of formula II (1), the reaction formula in step (3) is shown as formula VI; for the synthesis of formula II (2), the reaction in step (3) is as shown in formula VII:
wherein n in the formula II (1) and the formula II (2) is the number of methylene repeating units, and n is any integer between 2 and 500;
the specific process of the step (3) is as follows: and (2) grafting gemcitabine prodrug molecules containing alkyne bond structures obtained in the step (2) to the terminal azide-modified amphiphilic dendritic molecules obtained in the step (1) by using an azide-alkyne click chemical reaction to obtain amphiphilic dendrimer gemcitabine self-assembled nano prodrug, wherein the molar ratio of the terminal azide-modified amphiphilic dendritic molecules to the gemcitabine prodrug molecules containing alkyne bond structures is 1: 1.0-1: 2.0, the reaction temperature is 40-80 ℃, the solvent system is a mixed solvent of tetrahydrofuran and water, and the reaction time is 1-8 hours.
7. Use of the dendrimer gemcitabine self-assembled nano prodrug as claimed in any one of claims 1 to 2 in the preparation of drugs for targeted delivery of antitumor drugs or drugs for inhibiting tumor cell proliferation.
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