CN110639025B

CN110639025B - Polyamide-imide drug-loaded nanoparticle and application thereof

Info

Publication number: CN110639025B
Application number: CN201910976388.1A
Authority: CN
Inventors: 李苁; 杨树东
Original assignee: Wuxi Peoples Hospital
Current assignee: Wuxi Peoples Hospital
Priority date: 2019-10-15
Filing date: 2019-10-15
Publication date: 2021-11-09
Anticipated expiration: 2039-10-15
Also published as: CN110639025A

Abstract

The invention discloses a polyamide-imide drug-loaded nanoparticle and application thereof, wherein the structural formula of the polyamide-imide drug-loaded nanoparticle is shown as a formula (I), wherein R is R₁Or R₂Said R is₁Is camptothecin modified by sulfydryl and pyridine disulfide, and R is₂Is PEG₅₀₀Said R is₁And R₂The molar ratio of (10-40): (60-90); x is an integer of 1 to 7, and y is an integer of 1 to 3. The PAI-CPT PNDs can be used as nano-carriers to load other anticancer drugs (such as DOX) for combined chemotherapy and show synergistic therapeutic effect.

Description

Polyamide-imide drug-loaded nanoparticle and application thereof

Technical Field

The invention belongs to the technical field of antitumor drugs, and particularly relates to a polyamide-imide drug-loaded nanoparticle and application thereof.

Background

While small molecule drug chemotherapy is one of the important paradigms for cancer treatment, it often exhibits poor bioavailability and uncontrolled pharmacokinetics, resulting in severe side effects and limited therapeutic efficacy. One effective strategy to address these problems is to utilize nanoformulations by chemically coupling the drug to the nanomaterial or by physically encapsulating the drug in a nanocarrier. Compared with inorganic materials, the polymer nano-carrier has good biocompatibility and adjustable degradability, and is a promising tumor chemotherapy drug delivery system

It is noteworthy that higher drug loading does not guarantee better therapeutic effect, because the physical and chemical properties of the nanocarriers are also linked to cellular uptake and subsequent targeted drug release of the nanocarriers. Yuan et al describe a Conjugated Polyelectrolyte (CPEs) -based polymer prodrug with subsequent DOX attachment, but the cumbersome multi-step synthesis and purification makes its availability challenging.

Disclosure of Invention

This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.

As one aspect of the present invention, the present invention provides a polyamide-imide drug-loaded nanoparticle, wherein: the structural formula of the polyamide-imide medicine-carrying nano particle is shown as the formula (I):

wherein R is R₁Or R₂Said R is₁Is camptothecin modified by sulfydryl and pyridine disulfide, and R is₂Is PEG₅₀₀Said R is₁And R₂The molar ratio of (10-40): (60-90); x is an integer of 1 to 7, and y is an integer of 1 to 3.

As a preferred scheme of the polyamide-imide medicine-carrying nano particle provided by the invention: the R is₁The structural formula is shown as a formula (II):

as a preferred scheme of the polyamide-imide medicine-carrying nano particle provided by the invention: the polyamide-imide drug-loaded nanoparticles are also loaded with adriamycin, and the adriamycin loading efficiency is 50-60%.

As a preferred scheme of the polyamide-imide medicine-carrying nano particle provided by the invention: the polyamide-imide drug-loaded nanoparticle, wherein R is₁The synthetic route of the CPT-DSSP is as follows:

as a preferred scheme of the polyamide-imide medicine-carrying nano particle provided by the invention: the R is₁The synthesis method also includes model reaction, dissolving CPT-DSSP in DMSO and purging with argon, then adding equimolar amount of mercaptoethanol and stirring the reaction mixture in dark to avoid side reaction.

As a preferred scheme of the polyamide-imide medicine-carrying nano particle provided by the invention: the polyamide-imide drug-loaded nanoparticles are reduction-responsive drugs, which are reduced into sulfydryl intermediates in a reduction environment to initiate continuous intramolecular cyclization and cracking of 1, 3-oxathiolane-2-one, so that camptothecin is released.

As another aspect of the invention, the invention provides application of the polyamide-imide drug-loaded nanoparticles as an anti-tumor drug.

As an optimal scheme for the application of the polyamide-imide medicine-carrying nano particles as the antitumor drugs, the polyamide-imide medicine-carrying nano particles comprise the following components in percentage by weight: the polyamide-imide drug-loaded nanoparticle can be used for simultaneously loading camptothecin and adriamycin and can be used for quickly internalizing cells.

As an optimal scheme for the application of the polyamide-imide medicine-carrying nano particles as the antitumor drugs, the polyamide-imide medicine-carrying nano particles comprise the following components in percentage by weight: the tumors comprise cervical cancer, fibrosarcoma and breast cancer.

The invention has the beneficial effects that: the cationic PAI-based polymer prodrug and the self-assembly thereof are used for cancer chemotherapy, sulfhydryl-dithiopyridine exchange reaction is generated in situ from the aminolysis side chain of thiolactone to promote subsequent CPT connection, the synthesis efficiency of the polymer-drug conjugate is obviously improved, the obtained PAI prodrug not only has a hard framework structure, but also has positive charge due to secondary amine groups in the PAI stent, the PAI-CPT PDNPs are rapidly internalized within 2 hours due to the characteristic of EPR effect, and reduction-reactive drug release can be realized due to disulfide bond connection between the PAI stent and drugs, and in addition, the PAI-CPT PNDs can be used as nano carriers to load other anticancer drugs (such as DOX) for combined chemotherapy and show synergistic therapeutic effect.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:

FIG. 1 shows CPT-DSSP¹H and¹³c nuclear magnetic resonance results.

FIG. 2 is a scheme showing the synthesis of a PAI-CPT prodrug.

FIG. 3 shows polyamide-imide drug-loaded nanoparticles¹³C NMR spectrum.

Fig. 4 is a graph of fluorescence concentration of polyamide-imide drug-loaded nanoparticles.

Fig. 5 is a graph of the molecular weight of polyamide-imide drug-loaded nanoparticles.

Fig. 6 is a TEM image, hydrodynamic size, PDI and surface potential of polyamide-imide drug-loaded nanoparticles.

Figure 7 is a reduction response release of polyamide-imide drug loaded nanoparticles.

Fig. 8 is cellular internalization of polyamide-imide drug-loaded nanoparticles.

Figure 9 is the cytotoxicity of polyamide-imide drug-loaded nanoparticles.

Fig. 10 is the hydrodynamic size, PDI, of doxorubicin-loaded polyamide-imide drug-loaded nanoparticles.

Fig. 11 is a schematic diagram of the present invention.

Figure 12 is a synthesis and drug release profile according to the present invention.

FIG. 13 is an HPLC chart of CPT-DSSP.

FIG. 14 is a plot of the fluorescence concentration of the PAI-CPT prodrug during self-assembly.

FIG. 15 shows the critical aggregation concentrations of P1, P2, and P3.

Figure 16 is cellular uptake of polyamide-imide drug-loaded nanoparticles.

Figure 17 is a release profile of doxorubicin-loaded polyamide-imide drug-loaded nanoparticles.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.

Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.

The material and the method of the invention are as follows:

experimental materials: camptothecin (CPT, alatin, 97%), triphosgene (alatin, 99%), mercaptoethanol (TCI, 98%), 2' -bipyridine disulfide (TCI, 98%), 4-dimethylaminopyridine (Acros, 99%), 4, 7, 10-trioxa-1, 13-tridecane diamine (diamine 220, Aldrich, 97%), poly (ethylene glycol) methyl methacrylate (PEG) methyl ester₅₀₀) (Aldrich, average M)_n500), reduced glutathione (GSH, TCI,>97%), doxorubicin hydrochloride (alatin, 98%), triethylamine (Acros, ultrapure, 99.7%), dimethyl sulfoxide (Acros, ultrapure,>99.8%), anhydrous dichloromethane (Innochem, 99.9%), anhydrous pyridine (Innochem, 99%), thiolactone-maleimide monomer. Other reagents were purchased from SCRC and used as received. Cell count kit-8 (cck-8) was purchased from Biyuntian biotech.

The characterization method comprises the following steps: record Nuclear Magnetic Resonance (NMR) spectra on a Bruker Avance II 400 spectrometer ((R))¹The resonant frequency of H is 400MHz,¹³c resonance frequency was 100MHz) and the results were processed using MestReNova-12.0.1 software. The morphology of the PDNPs was examined on a FEI Tecnai G2F 20 field emission transmission electron microscope at an accelerating voltage of 200 kV. The morphology of the PDNPs was observed on a FEI Tecnai G2F 20 field emission transmission electron microscope with an acceleration voltage of 200 kv. Hydrodynamic diameter, zeta potential and Critical Aggregation Concentration (CAC) of PDNPs were measured on a Marlvern Zetasizer nano ZS90 Dynamic Light Scattering (DLS) particle size analyzer using a helium-neon laser. Fluorescence measurements were all measured on an LS55 fluorescence spectrometer (PerkinElmer). UV was performed on a Lambda25 UV-vis spectrophotometer (Perkinelmer). High Performance Liquid Chromatography (HPLC) was performed on Waters 1525(Luna 5. mu. mC18(2)100, LC column 250X 4.6mm) equipped with a 2998 photodiode array detector. Acetonitrile: eluent of water (75/25, v/v) at 25 ℃ for 1.0mL min^-1Is operated at the flow rate of (c). The molecular weight and polydispersity index (PDI) of the PAI-CPT prodrug were measured on a Marlvern Viscotek HT GPC using a Model-1122 syringe pump and Model 350 triple detector array (RI, viscosity and SALLS). The flow rate of DMF as an eluent was set to 1mL min under 30mL using polystyrene (Polymer Laboratories, Inc., MA)^-1For calibration. Cellular uptake of PAI-CPT PDNP was measured on a BD FACSCalibur flow cytometer. Live cell imaging was performed on a Leica TCS SP8 Confocal Laser Scanning Microscope (CLSM).

Example 1:

synthesis of DSSP-OH: 2,2' -dipyridyl disulfide (5.6g, 25.5mmol) was dissolved in 30mL of methanol, 0.5mL of glacial acetic acid was added as a catalyst, and mercaptoethanol (1.34 g, 17.1mmol) was slowly added over 20 minutes in 10mL of methanolAnd stirred at room temperature in the dark for 3 hours, after reaction the mixture was evaporated in vacuo to a yellow oil using ethyl acetate: hexane ═ 3: 7(v/v), purification of the crude product by chromatography on silica gel, yield: 54.8 percent of the total weight of the steel,¹h NMR (400MHz, chloroform-d) Δ 8.52(ddd, 1H), 7.61(m, 1H), 7.40(dt, 1H), 7.15(m, 1H), 5.77(t, 1H), 3.83(m, 2H), 2.95(m, 2H);

synthesis of CPT-DSSP: CPT (2.0g, 5.74mmol), 4-dimethylaminopyridine (DMAP, 2.10g, 17.2mmol) and triphosgene (0.567g, 1.92mmol) were suspended in 50mL of anhydrous dichloromethane and stirred at room temperature under argon for 30 minutes, then a solution of DSSP-OH (1.18g, 6.31mmol) dissolved in anhydrous Tetrahydrofuran (THF) was added dropwise to the above mixture and the reaction mixture was stirred at room temperature overnight, after reaction, the solvent was evaporated and the crude product was purified by silica column chromatography eluting with ethyl acetate to give a white powder in yield: 33.6 percent of the total weight of the mixture,¹h NMR (400MHz, chloroform-d) δ 8.48-8.35 (m,2H),8.22(dd, J-8.5, 1.1Hz,1H), 7.99-7.90 (m,1H),7.84(ddd, J-8.5, 6.9,1.5Hz,1H),7.68(ddd, J-8.2, 6.9,1.2Hz,1H), 7.64-7.60 (m,2H), 7.34(s,1H),7.04(td, J-5.0, 3.2Hz,1H),5.70(d, J-17.2 Hz,1H),5.39(d, J-17.2 Hz,1H),5.29(dd, J-3.2, 1.3, 2H),4.44, 4.7.7H, 7.7H, 7.06 (t, 7.7, 14H), 7.7.06 (J-7.7H), 7.7.7.7.7.7.7H, 14 (t, 3.2Hz,1H, 7.7.7H);

CPT-DSSP model reactions: CPT-DSSP (0.01mmol) was dissolved in 10mL DMSO and purged with argon for 10 minutes, then an equal amount of mercaptoethanol was added and the reaction mixture was stirred at room temperature in the dark and the composition of the reaction mixture was monitored dynamically by HPLC;

synthesis of PAI-CPT prodrug: thiolactone maleimide (36.3mg, 0.1mmol) and PEG₅₀₀(feed varied with the addition of CPT-DSSP) was dissolved in 1mL DMSO, purged with argon for 10min, and then diamine 220(22.1mg, 0.1mmol) was added to initiate polymerization at room temperature, noting: the sulfhydryl concentration of the mixture is strictly controlled below 0.25mM, after 24 hours CPT-DSSP (starting material based on the original PEG) is added₅₀₀Varying the amount of PEG added₅₀₀And total moles of CPT-DSSPMolar amount 0.1mmol) and reacted for 1 hour, the reaction mixture was quenched in diethyl ether and acetone (diethyl ether: acetone ═ 2: 1, v/v) mixed solvent, and drying for 4h in vacuum. For different PEGs₅₀₀In the case of the/CPT-DSSP dosage ratio, the mixture in each case should be completely dissolved without precipitation, and PEG was used in this study₅₀₀Initial charge ratios of/CPT-DSSP of 9/1, 8/2, and 7/3 three PAI-CPT prodrugs were prepared, designated P1, P2, and P3, respectively;

self-assembly of PAI-CPT prodrugs: the self-assembly process for the preparation of PAI-CPT prodrugs (P1-P3) by nanoprecipitation, exemplified by P1, by dissolving P1(3mg) in 0.3mL of DMSO, then adding to 3mL of deionized water with vigorous stirring, then dialyzing the resulting solution further in deionized water for 8 hours, after which the aqueous solution of P1 nanoparticles was concentrated at 4000rpm through an Amicon Ultra ultrafiltration tube (MWCO: 3kDa) for 5 minutes and stored as a stock solution, similar to that of P2 and P3;

preparation of double drug-loaded PAI PDNPs: DOX hydrochloride (5mg) was dissolved in 0.2mL DMSO, then 2mL triethylamine was added and the neutralized DOX was distributed as a stock solution in DMSO, typically DOX-loaded P1(P1/DOX) PDNPs were prepared by dissolving P1(5mg) and DOX stock solution (0.3mL DMSO) in 5mL dichloromethane, then adding 10mL deionized water under vigorous stirring, then subjecting the mixture to pulsed sonication (500W, pulse 1 second, pulse 2 seconds) for 10 minutes, stirring the resulting emulsion overnight to evaporate dichloromethane, and further dialyzing against water (MWCO: 3500Da) to remove DMSO and free DOX, using a similar procedure for P2/DOX and P3/DOX PDNPs, the Drug Loading Efficiency (DLE) and drug loading equation (DLC) for DOX were calculated according to:

DLE (%) - (weight of DOX in PDNP)/(weight of PDNP) × 100%

DLC (%) - (weight of DOX in PDNP)/(weight of DOX fed) × 100%.

Example 2:

in vitro drug release: the kinetics of the reduction-responsive drug release of P1-P3 PDNPs were carried out by dialysis. Typically, 1mL of P1-P3 PDNPs stock solution was added to dialysis tubing (MWCO: 3500Da) at 37 deg.CNext, the mixture was dialyzed against PBS (10 mM). For each PDNPs, different concentrations of GSH (2. mu.M, 5mM and 10mM) were used in dialysis. At different predetermined time points, aliquots of the solution were collected and replaced with the same volume of PBS. According to the principle of passing through a fluorescence spectrophotometer (E)_x＝ 370nm，E_m435nm) was plotted to calculate the amount of CPT released. In addition to the DOX fluorescence detection wavelength, E_x＝488nm，E_mIn vitro drug release of double drug-loaded P1-P3 PDNPs was determined by the same method except 591 nm.

Cell culture: mouse breast cancer cells (4T1 cells), cervical cancer cells (HeLa cells) and human fibrosarcoma cells (HT-1080 cells) were obtained from cell banks of Shanghai institute of cell biology, China. Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) or Roswell Park Medical Institute (RPMI)1640 containing 10% Fetal Bovine Serum (FBS) and 0.1% penicillin-streptomycin. All cells were incubated with 5% CO at 37 deg.C₂And (4) incubation.

Cellular uptake of PAI-CPT PDNPs: the cellular uptake process of PAI-CPT PDNPs was measured by CLSM and flow cytometry. In the samples used for the CLSM test, different cells were tested at 8 × 10 per well³The density of individual cells was seeded in Lab-Tek chamber lid slide wells. After 24 hours, the cells were treated with small amounts of P1, P2 and P3 PDNPs (equivalent CPT concentration: 10. mu.g/mL) and incubated at 37 ℃. For predetermined time intervals, target wells were washed twice with PBS for CLSM analysis.

Among samples for flow cytometry testing, 6-well tissue culture plates (2X 10 per well)⁵Individual cells) were cultured. After 24 hours, P1, P2 and P3 PDNPs (equivalent CPT concentration: 10. mu.g/mL) were added to each well, and the cells were incubated at 37 ℃. For a predetermined time interval, the target wells were removed by successive media removal, PBS washing and trypsinization. Finally, cells were dispersed in 0.5mL PBS for further flow cytometry analysis. All data were processed and analyzed by FlowJo 10 and Origin 9.0 software.

In vitro cytotoxicity studies: assessment of the biocompatibility of PAI by the Standard CCK-8 assay, and PAI-CPT PDNPs or Dual drug LoadingCytotoxicity of PAI-CPT PDNPs on different cells. A typical procedure is as follows, 8X 10 cultures in 96-well plates³Density of individual cells/well and incubation for 24 hours. Then, the cells were treated with different concentrations of PAI, P1, P2 or P3 PDNPs for another 24 hours. After washing the cells twice with medium (DMEM or RPMI1640), 100. mu.L of medium plus 10. mu.L of CCK-8 solution was added to each well and incubated for 1 hour. The absorbance of the sample was measured on a Bio-Rad Model 680 microplate reader (OD 450 nm).

Example 3:

experimental results for example 1 and example 2:

synthesis and characterization of PAI-CPT prodrugs: in the research, a previous research method is adopted, and a sultone maleimide monomer and 4, 7, 10-trioxa-1, 13-tridecane diamine (diamine 220) are subjected to condensation polymerization in an equimolar ratio to form aliphatic PAI (polyamide-imide), and a synthetic scheme is shown in figure 2. The quantitative nature of the aminolysis of thiolactones and amine-maleimide michael additions allows for very efficient synthesis of PAIs and facilitates in-situ one-pot modification of PAIs. Synthesis of pyridine disulfide-modified CPT (i.e., CPT-DSSP) prodrug by a two-step process prior to PAI-CPT conjugation, with an overall yield of about 33%,¹h and¹³the chemical structure was confirmed by the C NMR results (see FIG. 1).

In the present invention, due to the presence of the pyridine disulfide moiety, the CPT-DSSP prodrug not only releases CPT in a reducing environment, but also competitively undergoes a thiol-dithiopyridine exchange reaction, it has been found by the present inventors that the model reaction of CPT-DSSP with thiol-containing molecules can avoid side reactions in the subsequent PAI-CPT coupling, and it has been found that the selection of mercaptoethanol as a representative thiol compound is more reactive than PAI (with equivalent side chain thiol), which has high reducibility and small steric hindrance. As shown in fig. 2, the HPLC results indicated that CPT and CPT-DSSP had different retention times at 2.9 min and 4.9 min, respectively, and that CPT-DSSP was gradually consumed after 1 hour after the addition of mercaptoethanol (equivalent to CPT-DSSP), with a composition that varied with the change in thiol concentration, e.g., 87.5% of CPT-DSSP was converted to CPT-OH in a highly diluted solution (mSH ═ 0.05mM), which eluted at 3.4 min (see fig. 13), and that CPT-DSSP was completely converted to CPT-OH without any formation of byproducts when the thiol concentration increased to 0.20mM, and CPT-OH signals were observed in the HPLC curves when the thiol concentration was further increased to higher concentrations (1mM and 5mM), and therefore, the thiol concentration in the below PAI-CPT prodrug formulation was strictly controlled below 0.25 mM.

The sulfhydryl-dipyridyl disulfide exchange reaction is never applied to the preparation of polymer drug conjugates, and the invention respectively regulates the amphipathy of the PAI-CPT prodrug by PEG₅₀₀Initial charge ratios of/CPT-DSSP were 9/1, 8/2, and 7/3, and 3 PAI-CPT prodrugs were prepared, designated P1, P2, and P3, respectively. The proportion of the CPT-DSSP is further improved, a dissolved reaction mixture cannot be obtained, and the reaction of the CPT-DSSP and the mercapto-disulfide bond of the mercapto group is completed within 1 hour. Subsequently, PEG was additionally added₅₀₀(initial PEG ₅₀₀30% of the starting material) to consume residual thiol groups in the PAI scaffold. As shown in FIG. 3, the signals for thiolactone and maleimide units at 4.65ppm and 7.10ppm both disappeared, and PEG appeared₅₀₀And the typical proton signal of CPT. In that¹³The C NMR spectrum (FIG. 3) shows that the carbon signal of the methylene group at 21ppm for the thiol group also disappeared, indicating complete conversion of the thiolactone maleimide monomer and the thiol intermediate formed in situ. Due to intrinsic fluorescence of PAI and CPT, absorbance and fluorescence spectra were recorded to track PAI-CPT coupling. CPT has binary absorption peaks at 367nm and 385nm, with a typical fluorescence wavelength of 435nm (see fig. 4), on the other hand PAI has weak absorption around 365nm and exhibits intrinsic blue/green fluorescence (Em ═ 473nm in DMSO) due to the formation of amine-succinimide chromophore. After coupling, PAI-CPT has single absorbance at 367nm, half-peak width and stronger fluorescence above 477 nm. All these results indicate successful thiol pegylation and CPT coupling. Furthermore, according to PEG₅₀₀And integration of typical proton signals at 4.15ppm (n) and 5.42ppm (i) for CPT, the CPT grafting in P1, P2 and P3 was 7.5 mol%, 13.4 mol% and 22.7 mol%, respectively. GPC analysis showed that the molecular weights of P1, P2 and P3 were 8kDa, 7kDa and 9.5kDa, respectively (see FIG. 5).

Self-assembly of PAI-CPT prodrugs: the invention uses hydrophilicityDiamine 220, PEG₅₀₀The fragment is longer than the hydrophobic CPT fragment, therefore, spherical nanoparticles should be formed during the self-assembly of PAI-CPT prodrug, the fluorescence concentration curve is shown in FIG. 14, the invention adopts DLS method to determine the Critical Aggregation Concentration (CAC) of PAI-CPT PDNP, and the CAC of P1, P2 and P3 is respectively reduced by 34.6mg/L, 21.3mg/L and 17.4mg/L with the increase of CPT load (see FIG. 15). In fig. 6, TEM images show that all three PAI-CPT PDNPs self-assemble into spherical nanoparticles, and the average size of the P1 nanoparticles is 23.8 ± 5.3 nm. The higher CPT loading increased the size of the P2 and P3 nanomicelles to 25.2 ± 6.9nm and 27.4 ± 7.5nm, respectively. In aqueous solution (0.50mg/mL), PAI-CPT nanoparticles swelled to a larger hydrodynamic size due to extension of the hydrophilic segment (FIGS. 6 d-f), and all PAI-CPT PDNPs exhibited positive surface potentials due to the presence of secondary amine groups in the PAI backbone and nitrogen atoms in CPT. The size, PDI and zeta potential data of P1-P3 nanoparticles as measured by DLS are summarized in Table 1. As a potential polymeric drug carrier, the P1-P3 nanoparticles showed excellent stability within 7 days, and no significant changes in size, PDI and surface potential were observed (FIGS. 6 g-h).

Reduction-responsive drug release: as shown in FIG. 12, disulfide bonds were reduced to sulfhydryl intermediates in a reducing environment to initiate successive intramolecular cyclization and cleavage of 1, 3-oxathiolan-2-one and release of parent CPT from PAI-CPT PDNPs, and to evaluate the release profile of the PAI-CPT PDNPs from the reduction reaction, in vitro cumulative CPT release was studied by dialysis methods and characterized by fluorescence spectroscopy, and in addition, different concentrations of GSH were used to stimulate different levels of GSH in the extracellular and intracellular environments, 2-20. mu.M and 2-10 mM, respectively, incubation in 2. mu.M GSH with cumulative CPT release after 48 hours of less than 6%, indicating no significant premature release of drug during the blood circulation, in contrast, increasing the GSH concentration up to 10mM 48 hours significantly increased CPT cumulative release, 83.3% of P1, P2 and P3 PDNPs, respectively, 88.6% and 93.3%, as shown in fig. 7, the difference in cumulative CPT release between the three nanoparticles may be due to their different degrees of CPT-DSSP coupling, which upon GSH triggering, results in different concentrations of sulfhydryl intermediate.

Cellular uptake: small drug molecules can enter tumor cells through an active diffusion process, but escape from the cells is rare, and as a hard crystalline polymer, the PAI scaffold contributes to the effective cellular uptake of PAI-CPT PDNPs. Furthermore, many polymeric scaffolds for conjugation to CPT are neutral, in contrast, the PAIs in our design exhibit positive charge due to the large number of secondary amine groups in the PAI backbone and thus have better affinity for negatively charged cell membranes, the living cell imaging system was used to assess the cellular uptake behavior of HeLa cells for P1-P3 PDNPs, the more cellular internalization of PDNPs, the stronger the fluorescence intensity of the cells, figure 8 shows that all three PDNPs exhibit similar cellular internalization at different incubation periods (2, 4 and 8 hours), probably due to their size and surface potential equivalence. As expected, strong fluorescence was observed in the cells after 2 hours and increased significantly after 4 and 8 hours of incubation, much faster than reported in the prior art. FIG. 16 clearly demonstrates that most of the blue fluorescence is located in HeLa nuclei after 8 hours of incubation with all three PDNPs, and flow cytometry analysis was further used to confirm cellular uptake of P1-P3 PDNPs into Hela cells, as shown in FIGS. 8b-d, the mean fluorescence intensity of P1-P3 PDNPs internalized by Hela cells was significantly increased after 2 hours of incubation, and the longer incubation times of 4 hours and 8 hours further increased the cellular uptake of P1-P3 PDNPs.

In vitro cytotoxicity: evaluation of cytotoxicity of P1-P3 PDNPs against various tumor cell lines (HeLa cells, 4T1 cells and HT-1080 cells) in vitro by CCK-8 assay, PAIs have no significant cytotoxicity against all three cell lines at concentrations up to 800 μ g/mL, although having positive surface charge, and the above-mentioned results of cellular internalization show rapid uptake and sufficient release and diffusion of CPT into the nucleus for P1-P3 PDNPs, and thus their anticancer effects against tumor cells can be expected, as shown in FIG. 9, P1-P3 PDNPs show cell viability curves similar to the increased CPT concentration. Especially at higher CPT concentrations (>10 mug/mL), P1-P3 PDNPs have better anticancer effect than free CPT. This may be attributed to their excellent internalization behavior within tumor cellsFor time-dependent CPT (camptothecin) release, whereas free CPT may precipitate at higher CPT concentrations and fail to enter tumor cells. IC of P1-P3 PDNPs₅₀The (half maximal inhibitory concentration) values are summarized in table 2.

Double drug loading PDNPs: DOX (doxorubicin) was further encapsulated in PAI-CPT PDNPs to construct a combinatorial nanomedicine, P1 PDNP was used as a nanocarrier to load DOX by continuous sonication/dialysis method, and equal mass feed ratio (DOX/P1 PDNP) gave 54.8% DLE and 2.3% DLC, respectively; after loading with DOX, P1 PDNPs self-assemble into 170.3 + -47.8 nm larger nanoparticles by TEM and DLS analysis showed a hydrodynamic size of 222.7nm, PDI of 0.26 (FIG. 10) and a slight increase in surface potential to 22.6mV for DOX-loaded P1 PDNP (P1/DOX PDNPs). CLSM results indicated that P1/DOX PDNP was completely internalized into HeLa cells after 1 hour of incubation and fluorescence intensity increased significantly with increasing incubation time (2 and 4 hours).

CPT and DOX both exhibited time-dependent release profiles and were localized in the nucleus (FIG. 17), P1/DOX PDNP had synergistic anti-cancer efficacy, and CCK-8 assays indicated IC of P1/DOX PDNP on 4T1, HT-1080 and HeLa cells₅₀Values of 0.26. mu.g/mL, 0.39. mu.g/mL and 0.30. mu.g/mL (Table 3), respectively, are well below free DOX (4T1 cells: 0.71. mu.g/mL; HT-1080 cells: 0.50. mu.g/mL; HeLa cells: 0.49. mu.g/mL) or free CPT (4T1 cells: 3.0. mu.g/mL; HT-1080 cells: 2.4. mu.g/mL; HeLa cells: 2.8. mu.g/mL) (Table 2), P1/DOX PDNPs can be used as carriers for loading additional hydrophobic drugs and show synergistic effects in combination chemotherapy. The schematic diagram of the invention is shown in figure 11.

TABLE 1

TABLE 2

TABLE 3

In conclusion, the cationic PAI-based polymer prodrug and the self-assembly thereof are used for cancer chemotherapy, the thiol-dithiopyridine exchange reaction generated in situ by the aminolysis side chain of thiolactone facilitates the subsequent CPT connection, the synthesis efficiency of the polymer-drug conjugate is remarkably improved, the obtained PAI prodrug not only has a hard framework structure, but also has positive charge due to the secondary amine group in the PAI scaffold, the PAI-CPT PDNPs are quickly internalized in cells within 2 hours due to the characteristic of EPR effect, and the reduction-reactive drug release can be realized due to the disulfide bond connection between the PAI scaffold and the drugs, and in addition, the PAI-CPT PNDs can be used as nano-carriers for loading other anticancer drugs (such as DOX) for combined chemotherapy and show synergistic therapeutic effect.

It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

Claims

1. A polyamide-imide medicine-carrying nanoparticle is characterized in that: the structural formula of the polyamide-imide medicine-carrying nano particle is shown as the formula (I):

（I）

2. The polyamide-imide drug-loaded nanoparticle of claim 1 wherein: the R is₁The structural formula is shown as a formula (II):

（Ⅱ）。

3. the polyamide-imide drug-loaded nanoparticle of claim 1 or 2 wherein: the polyamide-imide drug-loaded nanoparticles are also loaded with adriamycin, and the adriamycin loading efficiency is 50-60%.

4. The polyamide-imide drug-loaded nanoparticle of claim 1 or 2 wherein: the polyamide-imide drug-loaded nanoparticle is characterized in that R is₁The synthetic route of the CPT-DSSP is as follows:

。

5. the polyamide-imide drug-loaded nanoparticle of claim 4 wherein: the R is₁The synthesis method also includes model reaction, dissolving CPT-DSSP in DMSO and purging with argon, then adding equimolar amount of mercaptoethanol and stirring the reaction mixture in dark to avoid side reaction.

6. The polyamide-imide drug-loaded nanoparticle of any one of claims 1, 2, 5 wherein: the polyamide-imide medicine carrying nano particles are reduction response type medicines, and are reduced into sulfydryl intermediates in a reduction environment to initiate continuous intramolecular cyclization and cracking of 1, 3-oxathiolane-2-ketone so as to release camptothecin.

7. The use of the polyamide-imide drug-loaded nanoparticle of any one of claims 1 to 6 in the preparation of an anti-tumor drug.

8. The use of claim 7, wherein: the polyamide-imide drug-loaded nanoparticle can be used for simultaneously loading camptothecin and adriamycin and can be used for quickly internalizing cells.

9. The use of claim 8, wherein: the tumors comprise cervical cancer, fibrosarcoma and breast cancer.