CN111939268A - Nano particle compound for responsive deformation of tumor microenvironment - Google Patents
Nano particle compound for responsive deformation of tumor microenvironment Download PDFInfo
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Abstract
The invention discloses a nano particle compound with responsive deformation of a tumor microenvironment, which is formed by combining an amphiphilic oligomeric polypeptide drug conjugate and beta-carboxyamide polylysine through electrostatic interaction. The amphiphilic oligomeric polypeptide drug conjugate is a product obtained by condensing anticancer drug adriamycin and oligomeric polypeptide KIGLFRWR through chemical bonds. The nano particle compound can be passively gathered at a tumor position in a targeted manner, a beta-carboxylic acid amide group connected to polylysine is broken under the acidic pH condition of a tumor area, the charge of the polylysine is changed from negative charge to positive charge and is repelled with an amphiphilic oligomeric polypeptide drug conjugate with positive charge, so that the nano particle compound is dissociated, and then the amphiphilic oligomeric polypeptide drug conjugate can be further assembled into long fibers, so that the long-time retention at the tumor position is realized, the drug adriamycin is slowly released, and the high-efficiency anticancer effect is realized.
Description
Technical Field
The invention relates to the field of medicinal preparations, in particular to a nano particle compound with responsive deformation of tumor microenvironment, which is formed by combining an amphiphilic oligomeric polypeptide drug conjugate and beta-carboxyamide polylysine through electrostatic interaction.
Background
Cancer is the second leading cause of death worldwide, with 429 ten thousands of new tumor cases in our country in 2015, accounting for 20% of 2145 ten thousands of new tumor cases worldwide in this year; the number of the global tumor death cases in the same year reaches 880 ten thousand, wherein the number of the tumor death cases in China reaches 281 ten thousand, and the death rate is the top of the world leaderboard. Although human beings have made good progress in antitumor therapy in the last decades, cytotoxic chemotherapeutic drugs (such as adriamycin and the like) which are commonly used for tumor therapy in clinic still have the problems of poor targeting, difficult accumulation of tumor parts and the like, and serious adverse reactions and toxic and side effects. How to improve the toxic and side effects and improve the in vivo pharmacokinetic behavior of the traditional Chinese medicine is a hot point of research of people.
The polypeptide-drug conjugate composed of the drug, the functional polypeptide sequence and the degradable connecting bond is a novel strategy for improving the physicochemical property of the drug, and has the advantages of high biocompatibility, easy biodegradation, simple preparation and the like. In addition, through the sequence design of the polypeptide constructed by different amino acids, the intermolecular forces such as hydrogen bond action, hydrophilic and hydrophobic action, electrostatic action, pi-pi stacking and the like are utilized to regulate and control the polypeptide to form different shapes such as fibers, tubes, micelles, gels and the like. In earlier researches, a polypeptide-drug conjugate formed by covalent coupling of a functional polypeptide capable of self-assembling to form nano-fibers or gel and a chemical drug with an anti-tumor effect can be used for in-situ injection of solid tumors, and can realize long-term retention at tumor parts to play a long-acting slow-release effect. However, the nanofiber and the nanogel have certain risks in intravenous injection, and are difficult to be clinically used for intravenous injection administration.
The nano particles or the micelles can be used for systemic administration, and due to the EPR effect at the tumor part, the nano particles or the micelles can well penetrate out of capillaries and enter tumor cells, but the retention time at the tumor part is far shorter than that of nano fibers. In order to further develop the application of the polypeptide-drug conjugate in the aspect of anti-tumor intravenous injection treatment, how to make the polypeptide-drug conjugate meet the requirement of intravenous injection administration and have long-term retention capacity is the focus of the current drug development.
Disclosure of Invention
In order to overcome the defects of antitumor drugs in the prior art, the invention designs a nanoparticle compound with responsive deformation of tumor microenvironment, which is different from the covalent connection mode in the prior art, and the amphiphilic oligomeric polypeptide drug conjugate of the nanoparticle compound is combined with beta-carboxylic acid amide polylysine through electrostatic interaction. Under the condition of tumor acidic pH, the polylysine of the beta-carboxylic acid amide group of the nanoparticle compound is subjected to charge reversal, the nanoparticle compound is dissociated under the electrostatic action, and the amphiphilic oligomeric polypeptide drug conjugate is released and further assembled into long fibers, so that the effects of long-time retention of tumor parts and tumor metastasis inhibition are achieved.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
an amphiphilic oligomeric polypeptide drug conjugate is characterized in that an amino acid sequence is shown as SEQ ID No: 1 and adriamycin through chemical bond condensation.
The preferable chemical bond of the amphiphilic oligomeric polypeptide drug conjugate is an amido bond or an ester bond.
In a preferred technical scheme, an amido bond or an ester bond is formed between an amino group or a hydroxyl group in the doxorubicin molecular structure and a carboxyl group at the C-terminal of the oligomeric polypeptide, or the carboxyl group at the C-terminal of the oligomeric polypeptide is converted into an amido group or an ester group, and the amino group at the N-terminal reacts with dianhydride to form a carboxyl group, and then forms an amido bond or an ester bond with the amino group or the hydroxyl group in the doxorubicin molecular structure. The dianhydride is selected from malonic anhydride, succinic anhydride, glutaric anhydride or adipic anhydride.
In a preferred technical scheme, the amphiphilic oligomeric polypeptide drug conjugate is Dox-Suc-K (Fmoc) IGLFRWR, and the structural formula is as follows:
the amphiphilic oligomeric polypeptide drug conjugate can be assembled with beta-carboxyamide polylysine to form nanoparticles through electrostatic interaction, and can be self-assembled to form fibers under the condition of tumor weak acid environment.
The invention provides a nano particle compound with responsive deformation of a tumor microenvironment, which is formed by combining an amphiphilic oligomeric polypeptide drug conjugate and beta-carboxyamide polylysine through electrostatic interaction.
Based on the characteristics of the nanoparticle complex, the invention provides an anti-tumor medicament which contains the nanoparticle complex as an active ingredient. Furthermore, the anti-tumor medicine also comprises a pharmaceutically acceptable carrier.
The anti-tumor drug is preferably an intravenous injection.
The invention also aims to provide application of the beta-carboxyamide polylysine in inducing morphological transformation between long fibers and nanoparticles of the amphiphilic oligopolypeptide or the amphiphilic oligopolypeptide drug conjugate.
The invention also aims to provide an oligomeric polypeptide, the amino acid sequence of which is shown as SEQ ID No: 1 is shown.
The invention has the beneficial effects that:
the invention designs an amphiphilic oligomeric polypeptide drug conjugate with the capability of forming fibers by self-assembly by utilizing the characteristic that beta-carboxylic acid amide polylysine has a pH response type charge reversal function, and the beta-carboxylic acid amide polylysine can form a nano particle compound through electrostatic interaction with the beta-carboxylic acid amide polylysine to block the self-assembly behavior of the beta-carboxylic acid amide polylysine so as to form the nano micelle capable of being injected intravenously. After intravenous injection, the nano particle compound is passively targeted to a tumor part, under the environment of low pH value of the tumor part, a beta-carboxylic acid amide group on beta-carboxylic acid amide polylysine on the nano particle compound leaves, so that a polymer is converted from negative charge to positive charge to generate charge inversion, the nano particle compound is dissociated under the action of electrostatic repulsion to release an amphiphilic oligomeric polypeptide drug conjugate, the assembly performance is recovered, the nano particle compound is locally aggregated on the tumor to form a long fiber structure, and the nano particle compound is retained in tumor tissues for a long time to achieve the effect of long-acting treatment.
The nanoparticle compound can realize intravenous injection administration of the antitumor drug, and can be slowly degraded in vivo to release the inner core antitumor drug and exert antitumor effect.
The nanoparticle composite provided by the invention has the advantages of stable structure, high drug loading capacity and small side effect, and provides a new strategy for tumor treatment.
Drawings
FIG. 1 is a mass spectrum of Dox-Suc-KIGLFRWR prepared according to the present invention.
FIG. 2 is a transmission electron microscope inspection of the Dox-Suc-KIGLFRWR self-assembly prepared by the present invention.
FIG. 3 is a spectrum of beta-carboxyamide polylysine (FB) MALDI-TOF MS.
FIG. 4 shows the Zeta potential change of the beta-carboxyamide polylysine (FB) at different pH values.
FIG. 5 shows the particle size of nanoparticles at different ratios of Dox-Suc-KIGLFRWR to β -carboxyamide polylysine.
FIG. 6 Zeta potentials for the nanoparticle complexes FDSPC-NPs of the present invention constructed at a molar ratio of 1: 2.
FIG. 7 particle size of nanoparticle complex FDSPC-NPs according to the present invention constructed at a molar ratio of 1: 2.
FIG. 8 is the creation of a Dox-Suc-KIGLFRWR standard curve in example 5.
FIG. 9 is the creation of DOX standard curve in example 5.
FIG. 10 is a graph showing the cumulative release of FDPC-NPs from example 5 at various pH buffers.
FIG. 11 is a transmission electron microscopy examination of the self-assembly of FDSPC-NPs at different pH values in example 6.
FIG. 12 is an in vitro apoptosis assay of FDSPC-NPs in example 7.
Detailed Description
The following examples illustrate specific steps of the present invention, but are not intended to limit the invention.
Terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified.
The present invention is described in further detail below with reference to specific examples and with reference to the data. It will be understood that these examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.
The present invention is further illustrated by the following specific examples.
Example 1: preparation of amphiphilic oligomeric polypeptide drug conjugate Dox-Suc-KIGLFRWR
1. Material
9-fluorenylmethoxycarbonylacyl (Fmoc) protected glycine (G), Fmoc protected phenylalanine (F), Fmoc and tert-butyloxycarbonyl (Boc) protected tryptophan (W), Fmoc protected leucine (L), Fmoc and 2,2,4,6, 7-pentamethyl-2H-benzofuran-5-sulfonyl (Pbf) protected arginine (R), Fmoc protected isoleucine (I), Fmoc and 2- (4, 4-dimethyl-2, 6-dioxocyclohexylmethylene) ethyl protected lysine (K), Rink Amide-AM Resin (100-200 mesh, substitution coefficient: 0.486mmol/G), O-benzotriazol-tetramethyluronium hexafluorophosphate (HBTU, 99%); n, N-diisopropylethylamine (DIEA, 99%); piperidine (Piperidine, 99%), N-dimethylformamide (DMF, 99%), triethylamine; triisopropylsilane (Tis, 99%); trifluoroacetic acid (TFA, 99%).
2. Preparation method
The oligomeric polypeptide is synthesized by a conventional solid-phase synthesis method, and then the drug is coupled. The method comprises the following specific steps:
synthesis of Suc-K (Fmoc) IGLFRWR:
Fmoc-K (Dde) -IGLFRWR-Resin is synthesized by a Focus XC full-automatic polypeptide synthesizer (AAPPTEC), washed 3 times by DMF and detected by a ninhydrin color development method, and the reaction is completely proved by no blue color. Then, the Dde protection is removed by reaction with 1% -2% hydrazine hydrate solution, the reaction is repeated for 3 times, each time for 3 minutes, and the detection is carried out by a ninhydrin color development method, and the color is blue. The obtained Resin peptide Fmoc-KIGLFRWR-Resin was weighed to calculate the theoretical molar number, and succinic anhydride (Suc) and triethylamine were added to the Resin peptide at a charge ratio (Resin peptide: succinic anhydride: triethylamine: 1:2:3) using DMF as a solvent. N is a radical of2Stirring at room temperature for 1 hr under protection, filtering with sand core funnel, discarding filtrate, washing the obtained resin product with DMF for 5 times to obtain HOOCCH2CH2CO-K (Fmoc) IGLFRWR-Resin (abbreviated as Suc-K (Fmoc) IGLFRWR-Resin), detected with ninhydrin, and no color was used to confirm completion of the reaction. After weighing, the resulting mixture was transferred to a 50mL centrifuge tube, and 1g/10mL of cleavage solution (TFA: Tis: water ═ 95:2.5:2.5) was added per gram of product to obtain HOOCCH2CH2Cutting the CO-K (Fmoc) IGLFRWR from the resin, oscillating and cutting for 90min, filtering by a sand core funnel to collect filtrate, pouring the filtrate into 100mL of glacial ethyl ether (precooled for 2h at (-80 ℃), standing for 30min, 10000rpm, 6 ℃, centrifuging for 5min, discarding the supernatant, washing the precipitate with ethyl ether for 5 times, and carrying out vacuum drying to obtain the Suc-K (Fmoc) IGLFRWR (crude product, purifying the prepared liquid phase, and carrying out freeze drying to obtain a pure product.
② the synthesis of Dox-Suc-KIGLFRWR:
taking a pure product of Suc-K (Fmoc) IGLFRWR, taking anhydrous DMF as a solvent, adding PyAop and DIEA into the Suc-K (Fmoc) IGLFRWR according to a feeding ratio (Suc-K (Fmoc) IGLFRWR: PyAop: DIEA ═ 1:5:5), and adding N2Under protection, stirring and activating in ice-water bath for 2-3h, adding adriamycin hydrochloride with 3 times of equivalent weight, and adding N2The reaction was stirred under protection from light for 1 day. Then the mixture is put into a 50mL centrifuge tube, 20Ml of 20% piperidine is added into the centrifuge tube to carry out reaction for removing Fmoc protection, after shaking for 5min, the mixture is pumped to dry, the reaction is repeated for 1 time, and methanol is washed for 3 times and pumped to dry. The crude product was purified by preparative liquid phase and identified by ESI-MS.
Mass spectrometry (ESI-MS) measurements are shown in FIG. 1, which gives molecular weights 850.928 and 1700.847 as the molecular ion peaks of double and single charge, respectively, whose calculated molecular weights match the theoretical molecular weight, indicating that the chemical structure of the compound is correct.
Example 2: transmission Electron Microscopy (TEM) examination of Dox-Suc-KIGLFRWR self-Assembly
Preparing Dox-Suc-KIGLFRWR solution with the concentration of 100 mu M, assembling for 0,1,6 and 12 hours, dripping the solution on a copper net coated with a supporting film, staying for 60s, sucking off redundant solution by using filter paper, adding a drop of saturated uranium acetate coloring agent respectively, negatively dyeing for 90s, sucking off redundant coloring agent by using the filter paper, naturally drying, and observing under a transmission electron microscope, wherein the electron microscope images of assembling for 0h, 1h, 3h and 6h are respectively shown from left to right in the figure 2. As can be seen from the figure, the polypeptide-drug conjugate is self-assembled to form 40-50nm spherical micelles at 0h, the micelles are further aggregated to form primary fibers after 1h, and the fibers are intertwined and aggregated after 6h, which shows that the synthesized polypeptide-drug conjugate has good self-assembly capability.
Example 3: preparation of FB-Dox-Suc-KIGLFRWR-NPs
1. Material
2, 3-dimethylmaleic anhydride (DMMA, 99%), -polylysine (-PL, Mw: 3000-5000), triethylamine, N-hydroxysuccinimide (NHS, 99%), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI, 99%); general dialysis bag (molecular weight 2000D), pure water, glacial acetic acid.
2. Preparation method
(1) Synthesis of β -carboxylic acid amide polylysine (FB):
adding 180mg of DMMA into 15mL of a polylysine aqueous solution dissolved with 160mg, adding triethylamine to adjust the pH value to be alkaline, stirring and dissolving, adding 190mg of NHS, dissolving, adding 240mg of EDCI, stirring and adding triethylamine, keeping the pH value of a reaction system to be 8.5-9, and reacting overnight. Transferring the reaction solution into an activated dialysis bag (molecular weight 2000D), dialyzing for 12h by using pure water as a dialysis medium at a volume ratio of 1:1000, and changing the dialysis medium every 3-4h until the reaction solution in the dialysis bag is neutral. And freeze-drying the dialyzed reaction solution to obtain functional beta-carboxylic acid amide polylysine solid (FB) with charge reversal characteristics. The molecular weight of the product FB was analyzed by MALDI-TOF MS.
FB was detected by MALDI-TOF MS, see FIG. 3, and the molecular weight of FB obtained by synthesis was mainly distributed in 5500-7200, and the molecular weight was increased by nearly 2000 compared with that of polylysine, which is a raw material, indicating that a large amount of DMMA was covalently modified on polylysine.
FB Zeta potential changes at different pH values:
preparing FB solutions (5mg/mL) with different pH values (pH is 3,4,5,6,7,8 and 9) in parallel, and measuring the Zeta values of the FB in the solutions with different pH values; meanwhile, unmodified bulk drug-polylysine solutions are used as a control group, polylysine solutions (3.5mg/mL) with different pH values (pH values of 3,4,5,6,7,8 and 9) are prepared in parallel, and the Zeta values of the polylysine in the solutions with different pH values are measured. Each sample was assayed 3 times in parallel and the average was taken as the assay result, see fig. 4. The functional material FB has electronegativity under physiological conditions (pH is 7.4), when the pH is lower than 6.95, the FB charge is reversed to be positive, and the Zeta potential is rapidly increased along with the reduction of the pH value, which indicates that the synthesized FB has good weak acid sensitivity.
(2) Synthesis of FB-Dox-Suc-KIGLFRWR-NPs (FDSPC-NPs):
taking 4 test tubes, calculating Dox-Suc-KIGLFRWR (PDC) and a high polymer material (FB) of polylysine connected with beta-carboxylic acid amide according to molar ratios of 1:0, 1:1, 1:2 and 1:3, weighing FB 0.00, 6.50, 13.00 and 19.50mg, respectively adding the FB 0.00, the FB 6.50, the FB 13.00 and the FB 19.50mg into the test tubes, sequentially adding 2mL of ultrapure water into each test tube for dissolving, then respectively adding PDC 1.70mg, adjusting the pH to be neutral by using 0.1M hydrochloric acid and sodium hydroxide solution after dissolving, respectively measuring the particle sizes of the test tubes in 0,1, 3, 6 and 12 hours, and inspecting the change of the particle sizes of a self-assembly system along with the two ratios. The result is shown in fig. 5, the self-assembly particle size of the nanoparticles gradually increases in speed and degree with the increase of the addition ratio of the polymer material, which indicates that the addition of the polymer material can limit the growth of the polypeptide-drug conjugate nanofibers. When the proportion of the high polymer material is more than or equal to 2, the particle size of the nano particles is not increased along with the prolonging of the time, but the proportion of the added high polymer material is increased, and the particle size of the system is slightly increased. Therefore, PDC: FB 1:2 is the optimal ratio for the construction of FDSPC-NPs.
The particle size and Zeta potential were determined by constructing FDSPC-NPs at a concentration of 100. mu.M in a molar ratio of 1: 2. As a result, as shown in fig. 6 and 7, the particle size distribution of FDSPC-NPs was about 80nm, the particle size distribution was uniform (PDI 0.165), and the band average potential of FDSPC-NPs was-9.03 mv.
Under the neutral condition, Dox-Suc-KIGLFRWR takes positive charge as a core molecule of the inner layer, FB takes negative charge as a shell molecule, and FDSPC-NPs can be formed between the Dox-Suc-KIGLFRWR and the FB through electrostatic force.
Example 5: in vitro Release study of FDSPC-NPs
(1) Establishment of Dox-Suc-KIGLFRWR Standard Curve
Precisely weighing 16.99mg of Dox-Suc-KIGLFRWR pure product, placing the product in a 10mL volumetric flask, adding a proper amount of methanol to dissolve DSPC, fixing the volume of the methanol to scale, shaking up to obtain a stock solution with the molar concentration of 1mM, and storing the stock solution at 4.0 ℃ for later use. Precisely measuring appropriate amount of stock solutions, placing in 65 mL volumetric flasks, adding appropriate amount of methanol to dilute to scale, shaking to obtain solutions with molar concentrations of 2.5,5,10,50,100,250 μ M, respectively, recording peak area by High Performance Liquid Chromatography (HPLC), and drawing standard curve, see FIG. 8.
(2) Establishment of DOX Standard Curve
Precisely weighing 5.80mg of DOX standard substance, placing the DOX standard substance in a 10mL volumetric flask, adding a proper amount of methanol to dissolve the DOX standard substance, fixing the volume to a scale, shaking up to obtain a stock solution with the molar concentration of 500 mu M, and storing the stock solution at 4.0 ℃ for later use. Precisely measuring appropriate amount of stock solutions, placing in 65 mL volumetric flasks, adding appropriate amount of methanol to dilute to scale, shaking up to obtain standard solutions with molar concentrations of 2.5,5,10,50,100,250 μ M, respectively, recording peak areas by High Performance Liquid Chromatography (HPLC), and drawing standard curves, see FIG. 9.
(3) In vitro release behavior of FDPC-NPs (fully drawn Poly carbonate-NPs) in buffer solutions with different pH values
1mL of each of FDPC-NPs and DOX solutions with a concentration of 1mM was prepared, and placed in an activated dialysis bag (1000D), 10mL of neutral PBS (pH 7.4) and acidic PBS (pH 6.5) were used as dialysis media, and the tubes of each group were placed in a constant temperature oscillator, and subjected to in vitro release investigation at 37 ℃ for 100r min-1. Sampling for 0.25, 0.5, 1, 3, 6,12, 24, 48, 72, 96, 120 and 144 hours respectively, wherein the sampling volume is 0.3mL, supplementing an equal volume of isothermal fresh release medium after sampling, centrifuging the sample for 5min at 5000r min-1, analyzing by high performance liquid chromatography, and substituting into standard curve concentrations of DPCs and DOX under item 2.5.1.4 respectively. The cumulative release rates of DPCs and DOX were calculated according to the formula, 3 replicates of each sample were prepared, run in parallel, averaged, and the in vitro release curves of FDPC-NPs were plotted, see FIG. 10. The result shows that the release rate of the prepared FDPC-NPs is high under a weak acid condition (pH 6.5), the cumulative release rate of 12h reaches 50%, the release rate is slow after 12h, and the cumulative release rate of 196h reaches 85%; under the neutral condition (pH 7.4), the release is slowly released, the cumulative release rate after 12h only reaches 5 percent, and the cumulative release rate after 196h is less than 50 percent. The result proves that the constructed FDPC-NPs have good acid-responsive slow-release function.
Example 6: transmission Electron Microscopy (TEM) examination of self-Assembly of FDSPC-NPs at different pH values
FDSPC-NPs solution was prepared at a concentration of 100. mu.M, pH-adjusted to neutral (7.0) with 0.1M hydrochloric acid and sodium hydroxide solution, and TEM samples were taken at 0.5 h. After 4h, the solution was divided into 2 portions, one portion was kept at the original neutral pH and sampled for 12h to prepare TEM samples, and the other portion was adjusted to pH6.5 with 0.1M hydrochloric acid and sodium hydroxide solution and sampled for 0.5h and 12h to prepare TEM samples. Observed by a transmission electron microscope, see FIG. 11, from left to right are electron micrographs of pH7.0-0.5h, pH7.0-12h, pH6.5-0.5h and pH6.5-12h, respectively. It can be seen that under neutral conditions, FDSPC-NPs can aggregate to form injectable nanospherical structures (40-50nm) and can responsively self-assemble to form fibrous structures under mildly acidic conditions.
Example 7: FDSPC-NPs in vitro apoptosis assay
The Annexin-V FITC/PI apoptosis detection kit is adopted to examine the apoptosis effect of DOX with different concentrations and FDSPC-NPs prepared in example 3 on SMMC7721 cells. After counting SMMC7721 in logarithmic growth phase, cells were counted at 2X 105Inoculating each cell in 6-well plate, adding 2mL of whole culture medium into each well, culturing, and placing the inoculated cells at 37 deg.C with CO2Saturated with 5% concentrationAnd culturing under humidity for 24h, removing culture solution by suction, and washing with PBS 3 times. Adding 2mL of single culture diluent of DOX and FDSPC-NPs with different concentrations into each well, incubating for 24h, carefully sucking the upper layer of drug-containing culture solution into a 4mL centrifuge tube, digesting each well with 0.5mL of pancreatin without EDTA for 4min, adding 0.5mL of total culture medium to terminate digestion, respectively collecting cells from each well, mixing with the corresponding drug-containing culture solution, and culturing at 2000rmin-1And centrifuging for 5 min. The supernatant was carefully aspirated and the cells were washed 1 time with ice PBS. Adding 195 mu L of Annexin-V working solution into each tube of cells to resuspend the cells, adding 5 mu L of Annexin-V FITC, uniformly mixing, adding 10 mu L of PI, uniformly mixing, reacting at room temperature in a dark place for 10min, and detecting the sample by using a flow cytometer. Cells were incubated with single medium cultured cells as control. Results of the assay referring to fig. 12, the apoptosis rate was significantly increased in the FDPC-NPs and DOX groups compared to the control group (p < 0.01), and the apoptosis rate was gradually increased in each group with increasing dosing concentration (calculated as DOX concentration). The FDPC-NPs and DOX have obvious apoptosis promoting effect on SMMC7721 liver cancer cells; compared with the DOX group, the FDPC-NPs have no statistical difference in apoptosis rate under the same concentration (calculated by DOX concentration), which indicates that the FDPC-NPs have tumor killing effect equivalent to that of the DOX.
Example 8
The injection is prepared by a conventional pharmaceutical method and is used for the anti-tumor treatment of solid tumors. The experimental result shows that the preparation has good inhibition effect on liver cancer cells.
Sequence listing
<110> Nanjing university of traditional Chinese medicine
Nanjing haikerui Pharmaceutical Technology Co.,Ltd.
<120> nanoparticle complex for responsive deformation of tumor microenvironment
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Lys Ile Gly Leu Phe Arg Trp Arg
1 5
Claims (10)
1. An amphiphilic oligomeric polypeptide drug conjugate is characterized in that an amino acid sequence is shown as SEQ ID No: 1 and adriamycin through chemical bond condensation.
2. The amphiphilic oligomeric polypeptide drag conjugate according to claim 1, characterized in that said chemical bond is an amide or ester bond.
3. The amphiphilic oligomeric polypeptide drug conjugate of claim 2, wherein amino or hydroxyl groups in the doxorubicin molecular structure form amide bonds or ester bonds with carboxyl groups at the C-terminus of the oligomeric polypeptide, or the carboxyl groups at the C-terminus of the oligomeric polypeptide are converted into amide or ester groups, and the amino groups at the N-terminus are reacted with dianhydride to form carboxyl groups, which are then reacted with amino or hydroxyl groups in the doxorubicin molecular structure to form amide bonds or ester bonds.
4. The amphiphilic oligomeric polypeptide drug conjugate according to claim 3, characterized in that said dianhydride is selected from the group consisting of malonic anhydride, succinic anhydride, glutaric anhydride or adipic anhydride.
5. The amphiphilic oligomeric polypeptide drag conjugate of claim 4, characterized by the structural formula:
6. a tumor microenvironment responsive deformable nanoparticle complex, characterized in that the amphiphilic oligomeric polypeptide drug conjugate of any one of claims 1 to 5 and beta-carboxyamide polylysine are combined through electrostatic interaction.
7. An antitumor agent characterized by containing the tumor microenvironment-responsively deformed nanoparticle complex of claim 6 as an active ingredient.
8. Antitumor drug according to claim 7, characterized in that it is an intravenous injection.
9. The application of beta-carboxyamide polylysine in inducing morphological transformation between long fibers and nanoparticles of amphiphilic oligomeric polypeptide or amphiphilic oligomeric polypeptide drug conjugates.
10. An oligomeric polypeptide characterized by an amino acid sequence as set forth in SEQ ID No: 1 is shown.
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