CN110664758A

CN110664758A - PAI-CPT reduction response type dual-drug delivery nanoparticle preparation method

Info

Publication number: CN110664758A
Application number: CN201910976386.2A
Authority: CN
Inventors: 万霞; 蔡英华
Original assignee: Wuxi Peoples Hospital
Current assignee: Wuxi Peoples Hospital
Priority date: 2019-10-15
Filing date: 2019-10-15
Publication date: 2020-01-10
Anticipated expiration: 2039-10-15
Also published as: CN110664758B

Abstract

The invention discloses a preparation method of PAI-CPT reduction response type dual-drug delivery nanoparticles, which comprises the steps of synthesizing DSSP-OH; synthesizing CPT-DSSP; CPT-DSSP model reaction; synthesis of a PAI-CPT prodrug; self-assembly of PAI-CPT prodrugs; dual-drug delivery nanoparticles are prepared, which are capable of rapid cellular internalization within 2 hours and can achieve reduction-reactive drug release due to disulfide bonding between the PAI scaffold and the drug, and the PAI-CPT reduction-responsive dual-drug delivery nanoparticles are used in combination chemotherapy and exhibit synergistic therapeutic effects.

Description

PAI-CPT reduction response type dual-drug delivery nanoparticle preparation method

Technical Field

The invention belongs to the technical field of antitumor drugs, and particularly relates to a preparation method of PAI-CPT reduction response type dual-drug delivery nanoparticles.

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 polymer prodrug with Conjugated Polyelectrolytes (CPEs) as the backbone, side-chain linked to DOX, 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 PAI-CPT reduction responsive dual drug delivery nanoparticle preparation method, comprising,

synthesizing DSSP-OH;

synthesizing CPT-DSSP;

CPT-DSSP model reaction;

synthesis of a PAI-CPT prodrug;

self-assembly of PAI-CPT prodrugs;

preparing dual-drug delivery nanoparticles.

As a preferred scheme of the preparation method of the PAI-CPT reduction response type dual-drug delivery nano-particle, the invention comprises the following steps: the DSSP-OH is synthesized by dissolving 2,2' -dipyridyl disulfide in methanol, adding glacial acetic acid as a catalyst, slowly adding methanol diluent of mercaptoethanol, stirring for 2-4 hours in the dark, and drying in vacuum to obtain yellow oily matter.

As a preferred scheme of the preparation method of the PAI-CPT reduction response type dual-drug delivery nano-particle, the invention comprises the following steps: the addition amount of the 2,2 '-dipyridyl disulfide was 25.5mmol of 2,2' -dipyridyl disulfide per 30mL of methanol, the addition amount of glacial acetic acid was 0.5mL, and the addition amount of mercaptoethanol was 17.1 mmol; the methanol diluent of mercaptoethanol is slowly added, wherein the adding amount of the mercaptoethanol is 17.1mmol, the mercaptoethanol is diluted in 10ml of methanol, and the mercaptoethanol is slowly added within 20 minutes; after drying in vacuo to a yellow oil, the reaction mixture was dried using ethyl acetate: hexane ═ 3: 7 and the crude product is purified by chromatography on silica gel.

As a preferred scheme of the preparation method of the PAI-CPT reduction response type dual-drug delivery nano-particle, the invention comprises the following steps: the CPT-DSSP is synthesized by suspending CPT, 4-dimethylaminopyridine and triphosgene in anhydrous dichloromethane, stirring under argon to obtain a mixture, adding a DSSP-OH solution in anhydrous tetrahydrofuran and dropwise adding the mixture, stirring at room temperature overnight, drying a solvent after reaction, and purifying to obtain white powder CPT-DSSP.

As a preferred scheme of the preparation method of the PAI-CPT reduction response type dual-drug delivery nano-particle, the invention comprises the following steps: the CPT, the 4-dimethylaminopyridine and the triphosgene are suspended in anhydrous dichloromethane, wherein in 50mL of anhydrous dichloromethane, the addition amount of the CPT is 5.74mmol, the addition amount of the 4-dimethylaminopyridine is 17.2mmol, the addition amount of the triphosgene is 1.92mmol, and the addition amount of the DSSP-OH is 6.31 mmol.

As a preferred scheme of the preparation method of the PAI-CPT reduction response type dual-drug delivery nano-particle, the invention comprises the following steps: the CPT-DSSP model reaction was performed by dissolving 0.01mmol of CPT-DSSP in 10mL of DMSO and purging with argon, adding an equal amount of mercaptoethanol, and stirring in the dark at room temperature.

As a preferred scheme of the preparation method of the PAI-CPT reduction response type dual-drug delivery nano-particle, the invention comprises the following steps: the synthetic PAI-CPT prodrug is prepared by mixing 0.1mmol of thiolactone maleimide and PEG₅₀₀Dissolved in 1mL DMSO, purged with argon for 10min, then 0.1

mmol

4,7, 10-trioxa-1, 13-tridecane was addedDiamine, initiating polymerization at room temperature for 24 hours, followed by addition of CPT-DSSP and reaction for 1 hour, in a volume ratio of ether: acetone ═ 2: 1, precipitating and purifying in the mixed solvent of 1, and drying in vacuum for 4 h.

As a preferred scheme of the preparation method of the PAI-CPT reduction response type dual-drug delivery nano-particle, the invention comprises the following steps: the PEG₅₀₀And CPT-DSSP in a total molar amount of 0.1mmol, PEG₅₀₀And the molar ratio of CPT-DSSP is (60-90): (10-40); during the polymerization reaction, the concentration of the sulfhydryl group is controlled below 0.25 mM.

As a preferred scheme of the preparation method of the PAI-CPT reduction response type dual-drug delivery nano-particle, the invention comprises the following steps: self-assembly of the PAI-CPT prodrug to dissolve every 3mg of the PAI-CPT prodrug in 0.3mL DMSO, add to 3mL water with vigorous stirring, dialyze in water for 8 hours, and filter by centrifugation at 4000rpm for 5 minutes.

As a preferred scheme of the preparation method of the PAI-CPT reduction response type dual-drug delivery nano-particle, the invention comprises the following steps: the preparation of dual drug delivery nanoparticles was performed by dissolving 5mg of DOX in 0.2mL of DMSO, adding 2mL of triethylamine to obtain a DOX stock solution, dissolving 5mg of the PAI-CPT prodrug and 0.3mL of the DOX stock solution in 5mL of dichloromethane, adding 10mL of deionized water with vigorous stirring, performing pulsed sonication at 500W for 10 minutes, stirring overnight to dry the dichloromethane, and dialyzing to remove DMSO and free DOX.

The invention has the beneficial effects that: the cationic PAI-based polymer prodrug and the self-assembly thereof are used for the corresponding DDS for cancer chemotherapy, the sulfydryl-dithiopyridine exchange reaction is generated in situ by the aminolysis side chain of thiolactone to promote the 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 scaffold, 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 scaffold and the drug, and the PAI-CPT reduction-responsive dual-drug delivery nanoparticle is used for combined chemotherapy and shows 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 SCRS and used as received. Cell count kit-8 (cck-8) was purchased from Biyuntian biotech.

The characterization method comprises the following steps: NMR was recorded on a Bruker Avance II 400 spectrometer (NMR spectrum (C)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 BDFACSCalibur 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, a dilution of mercaptoethanol (1.34 g, 17.1mmol) in 10mL of methanol was added slowly over 20 minutes and stirred at room temperature in the dark for 3 hours, after reaction, the mixture was dried 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,¹HNMR (400MHz, chloroform-d) delta 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: mixing CPT (2.0g, 5.74mmol), 4-bisMethylaminopyridine (DMAP, 2.10g, 17.2mmol) and triphosgene (0.567g, 1.92mmol) were suspended in 50mL of anhydrous dichloromethane and stirred under argon at room temperature for 30 minutes, then a solution of DSSP-OH (1.18g, 6.31mmol) dissolved in anhydrous THF was added dropwise to the above mixture and the reaction mixture was stirred at room temperature overnight, after the reaction, the solvent was dried and the crude product was purified by silica column chromatography eluting with ethyl acetate to give a white powder, 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. Note: 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 the total molar amount of CPT-DSSP was 0.1mmol) and reacted for 1 hour, the reaction mixture was mixed with ethyl ether and acetone (ethyl ether: acetone ═ 2: 1, v/v) mixed solvent, and vacuum drying for 4h 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 the nanoprecipitation method, exemplified by P1, by dissolving P1(3mg) in 0.3mL of DMSO, then slowly adding to 3mL of deionized water with vigorous stirring at 5000rpm, and subsequently, further dialyzing the resulting solution in deionized water for 8 hours, after which the aqueous solution of P1 nanoparticles was concentrated at 4000rpm by an Amicon Ultra ultrafiltration tube (MWCO: 3kDa) for 5 minutes and stored as a stock solution, and the self-assembly process of P2 and P3 was similar;

preparation of double drug-loaded PAI PDNPs: DOX (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 as follows, P1(5mg) and DOX stock solution (0.3mL DMSO) were dissolved in 5mL dichloromethane, then 10mL deionized water was added with vigorous stirring, then the mixture was subjected to pulsed sonication (500W, pulse 1 second, pulse 2 seconds) for 10 minutes, the resulting emulsion was stirred overnight to dry the dichloromethane and further dialyzed against water (MWCO: 3500Da) to remove DMSO and free DOX, the Drug Loading Efficiency (DLE) and drug loading equation (DLC) for P2/DOX and P3/DOX PDNPs, DOX were performed using similar procedures as follows:

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 a dialysis tube (MWCO: 3500Da) and dialyzed against PBS (10mM) at 37 ℃. 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 Memori 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: the biocompatibility of PAI, as well as the cytotoxicity of PAI-CPTPDNPs or dual drug-loaded PAI-CPT PDNPs against different cells, was assessed by a standard CCK-8 assay. 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 not applied to the preparation of polymer drug conjugates, and the invention respectively regulates the amphipathy of the PAI-CPT prodrugWith 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. Absorbance and fluorescence spectra were recorded to track PAI-CPT coupling due to intrinsic fluorescence of PAI and CPT. CPT has binary absorption peaks at 367nm and 385nm, with a typical fluorescence wavelength of 435nm (see FIG. 4), and on the other hand PAI has weak absorption around 365nm and exhibits intrinsic blue/green fluorescence (E in DMSO) due to the formation of an amine-succinimide chromophore_m473 nm). 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 of CPT at 4.15 ppm (n) and 5.42ppm (i), the degree of CPT loading, es, in P1, P2 and P3 was calculated as 7.5 mol%, 13.4 mol% and 22.7 mol%. 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 hydrophilic diamine 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 showed that all three PAI-CPT PDNPs self-assembled into spherical nanoparticles, and the average size of P1 nanoparticles was23.8 +/-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-CPTPDNPs 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 cell nuclei after 8 hours of incubation with all three PDNPs, and flow cytometry analysis was further used to confirm cellular uptake of HeLa cells by P1-P3 PDNPs, as shown in FIGS. 8b-d, the mean fluorescence intensity of P1-P3 PDNPs internalized in 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 due to their excellent cellular internalization behavior and time-dependent CPT (camptothecin) release within tumor cells, 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 wrapped in PAI-CPT PDNPs to construct a combinatorial nanomedicine, P1PDNP was used as a nanocarrier to load DOX by continuous sonication/dialysis method, and equal mass feed ratio (DOX/P1PDNP) 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 P1PDNP (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 into the corresponding DDS are used for cancer chemotherapy, the thiol-dithiopyridine exchange reaction generated in situ by the aminolysis side chain of the thiolactone facilitates the subsequent connection of CPT, 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 scaffold, the PAI-CPT PDNPs are quickly internalized in cells within 2 hours due to the characteristic of EPR effect, and reduction-reactive drug release can be realized due to the disulfide bond connection between the PAI scaffold and the drug, and the PAI-CPT reduction-responsive dual-drug delivery nanoparticle is used for combined chemotherapy and shows synergistic treatment 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

A method for preparing PAI-CPT reduction responsive dual drug delivery nanoparticles, characterized by: comprises the steps of (a) preparing a mixture of a plurality of raw materials,

synthesizing DSSP-OH;

synthesizing CPT-DSSP;

CPT-DSSP model reaction;

synthesis of a PAI-CPT prodrug;

self-assembly of PAI-CPT prodrugs;

preparing dual-drug delivery nanoparticles.
2. The method of making a PAI-CPT reduction-responsive dual drug delivery nanoparticle of claim 1, wherein: the DSSP-OH is synthesized by dissolving 2,2' -dipyridyl disulfide in methanol, adding glacial acetic acid as a catalyst, slowly adding methanol diluent of mercaptoethanol, stirring for 2-4 hours in the dark, and drying in vacuum to obtain yellow oily matter.
3. The method of making a PAI-CPT reduction-responsive dual drug delivery nanoparticle of claim 2, wherein: the addition amount of the 2,2 '-dipyridyl disulfide was 25.5mmol of 2,2' -dipyridyl disulfide per 30mL of methanol, the addition amount of glacial acetic acid was 0.5mL, and the addition amount of mercaptoethanol was 17.1 mmol; the methanol diluent of mercaptoethanol is slowly added, wherein the adding amount of the mercaptoethanol is 17.1mmol, the mercaptoethanol is diluted in 10ml of methanol, and the mercaptoethanol is slowly added within 20 minutes; after drying in vacuo to a yellow oil, the reaction mixture was dried using ethyl acetate: hexane ═ 3: 7 and the crude product is purified by chromatography on silica gel.
4. The PAI-CPT reduction responsive dual drug delivery nanoparticle of any of claims 1 to 3, wherein: the CPT-DSSP is synthesized by suspending CPT, 4-dimethylaminopyridine and triphosgene in anhydrous dichloromethane, stirring under argon to obtain a mixture, adding a DSSP-OH solution in anhydrous tetrahydrofuran and dropwise adding the mixture, stirring at room temperature overnight, drying a solvent after reaction, and purifying to obtain white powder CPT-DSSP.
5. The method of making a PAI-CPT reduction-responsive dual drug delivery nanoparticle of claim 4, wherein: the CPT, the 4-dimethylaminopyridine and the triphosgene are suspended in anhydrous dichloromethane, wherein in 50mL of anhydrous dichloromethane, the addition amount of the CPT is 5.74mmol, the addition amount of the 4-dimethylaminopyridine is 17.2mmol, the addition amount of the triphosgene is 1.92mmol, and the addition amount of the DSSP-OH is 6.31 mmol.
6. The method for preparing PAI-CPT reduction-responsive dual drug delivery nanoparticles as claimed in any one of claims 1-3 and 5, wherein: the CPT-DSSP model reaction was performed by dissolving 0.01mmol of CPT-DSSP in 10mL of DMSO and purging with argon, adding an equal amount of mercaptoethanol, and stirring in the dark at room temperature.
7. The PAI-CPT reduction-responsive dual drug delivery nanoparticle as claimed in any one of claims 1 to 3 and 5, wherein the nanoparticle is prepared by the methodThe method comprises the following steps: the synthetic PAI-CPT prodrug is prepared by mixing 0.1mmol of thiolactone maleimide and PEG₅₀₀Dissolved in 1ml of DMSO, purged with argon for 10min, then added with 0.1mmol of 4,7, 10-trioxa-1, 13-tridecanediamine, and the polymerization initiated at room temperature for 24 hours, followed by addition of CPT-DSSP and reaction for 1 hour, in the volume ratios of ether: acetone ═ 2: 1, and drying in vacuum for 4 hours.
8. The method of making a PAI-CPT reduction-responsive dual drug delivery nanoparticle of claim 7, wherein: the PEG₅₀₀And CPT-DSSP in a total molar amount of 0.1mmol, PEG₅₀₀And the molar ratio of CPT-DSSP is (60-90): (10-40); during the polymerization reaction, the concentration of the sulfhydryl group is controlled below 0.25 mM.
9. The method for preparing PAI-CPT reduction-responsive dual drug delivery nanoparticles as claimed in any one of claims 1-3, 5, 8, wherein: self-assembly of the PAI-CPT prodrug to dissolve every 3mg of the PAI-CPT prodrug in 0.3mL of DMMSO, add to 3mL of water with vigorous stirring, dialyze against water for 8 hours, and centrifuge at 4000rpm for 5 minutes.
10. The method for preparing PAI-CPT reduction-responsive dual drug delivery nanoparticles as claimed in any one of claims 1-3, 5, 8, wherein: the preparation of dual drug delivery nanoparticles was performed by dissolving 5mg of DOX hydrochloride in 0.2mL of LDMSO, adding 2mL of triethylamine to obtain a DOX stock solution, dissolving 5mg of the PAI-CPT prodrug and 0.3mL of the DOX stock solution in 5mL of dichloromethane, adding 10mL of deionized water with vigorous stirring, sonicating at 500W pulses for 10 minutes, stirring overnight to dry dichloromethane, and dialyzing to remove DMSO and free DOX.