Non-cationic polymer siRNA and chemical drug combined delivery system and application

Abstract

This invention discloses a non-cationic polymer siRNA and chemical drug co-delivery system, comprising a non-cationic polymer and a complex of siRNA and drug loaded on the polymer; the complex is composed of a target siRNA and a corresponding drug; the non-cationic polymer contains several branches with aromatic rings, the aromatic rings being selected from one or more of 5- to 15-membered aromatic rings or 5- to 15-membered heteroaromatic rings; the drug is selected from one or more of gemcitabine, mitoxantrone, topotecan, beloteccan, irinotecan, doxorubicin, camptothecin, and derivatives of any of the above drugs. This invention enables the synergistic delivery of siRNA and chemical drugs, effectively overcoming the cytotoxicity problems caused by cationic delivery systems, and demonstrates promising application prospects in the field of modern drug delivery.

Description

Technical Field

[0001] This invention belongs to the field of biomedical polymer materials, specifically relating to a polymeric siRNA and chemical drug co-delivery system based on π-π stacking interaction and its application. Background Technology

[0002] In clinical cancer treatment, single-agent chemotherapy has limitations due to its high toxicity, poor selectivity, and susceptibility to drug resistance. Small interfering RNA (siRNA) forms an RNA-induced silencing complex (RISC) that cleaves target mRNA, thereby achieving gene silencing and effectively inhibiting the overexpression of target mRNA in cancer cells. Numerous studies and clinical trials have demonstrated the great potential and broad clinical application prospects of siRNA-based gene therapy in cancer treatment. Combination therapy strategies that combine chemotherapy with siRNA can compensate for the shortcomings of single-agent therapy and become a more effective alternative.

[0003] Currently, the main challenge in the combined therapy of chemical drugs and siRNA is the development of efficient co-delivery systems to achieve effective joint delivery of chemical drugs and siRNA. Chemical drugs generally suffer from poor water solubility, low bioavailability, and high toxicity due to a lack of targeting ability, severely limiting their clinical application in cancer treatment. On the other hand, siRNA has a large molecular weight, strong hydrophilicity, and a high density of negative charge, making it unable to effectively cross cell membranes to exert its therapeutic effect. Furthermore, unmodified siRNA is easily degraded by nucleases in human blood, losing its therapeutic efficacy. To address these issues, various drug and gene co-delivery systems based on nanocarriers have been successfully developed, mainly including lipid or lipid-like nanoparticles, polymer (natural and synthetic) nanoparticles, and inorganic material nanoparticles. To effectively bind the strongly negatively charged siRNA nucleic acid drugs, the aforementioned effective gene delivery carriers are mostly cationic materials. However, these cationic materials may disrupt biological membranes and cause cytotoxicity, posing significant challenges in clinical translation.

[0004] Therefore, there is an urgent need to develop a non-cationic siRNA and chemical drug co-delivery system. Summary of the Invention

[0005] Existing methods for co-delivering siRNA and chemical drugs typically employ cationic delivery systems. However, these cationic materials often induce significant cytotoxicity in normal tissues, limiting their clinical application. To address this issue, this invention proposes a non-cationic polymer micelle delivery system based on π-π stacking interactions. This system enables the synergistic delivery of siRNA and chemical drugs, effectively overcoming the cytotoxicity problems caused by cationic delivery systems and demonstrating promising application prospects in the field of modern drug delivery.

[0006] The purpose of this invention is to provide a non-cationic siRNA and chemical drug co-delivery system.

[0007] The specific technical solution is as follows:

[0008] A non-cationic polymer siRNA and chemical drug co-delivery system, characterized in that it comprises a non-cationic polymer and a complex of siRNA and drug loaded on the polymer;

[0009] The complex is composed of the target siRNA and the corresponding drug;

[0010] The non-cationic polymer contains several branches with aromatic rings, wherein the aromatic rings are selected from one or more of 5- to 15-membered aromatic rings or 5- to 15-membered heteroaromatic rings.

[0011] The drug is selected from one or more of gemcitabine, mitoxantrone, topotecan, belotecone, irinotecan, doxorubicin, camptothecin, and derivatives of any of the above drugs.

[0012] The preparation method of the drug complex is as follows: prepare a drug solution and an aqueous solution of siRNA nucleotides, mix the two, incubate at room temperature for 5 to 30 minutes, place the mixed solution in a refrigerated centrifuge, centrifuge at 3 to 5°C at a speed of 10,000 to 15,000 rpm for 10 to 20 minutes, and collect the precipitate and supernatant formed. The precipitate is the drug complex.

[0013] Furthermore, drugs that form complexes with siRNA include:

[0014]

[0015]

[0016] One or more of them.

[0017] Alternatively, the non-cationic polymer may be one or more of the following structures:

[0018]

[0019] R1, R2, R3, and R4 are the branched chains with aromatic rings, and R1, R2, R3, and R4 are selected from one or more of 5- to 15-membered aromatic rings or 5- to 15-membered heteroaromatic rings; n, x, y, a, and b represent the degree of polymerization, where n is 1-5000, x is 1-4000, y is 1-4000, a is 1-4000, and b is 1-4000.

[0020] A non-cationic siRNA-chemical drug co-delivery system, the structure of which is as follows: Figure 22 As shown.

[0021] Furthermore, the branched chain with the aromatic ring, or the substituents R1, R2, R3, and R4, are selected from one or more of the following structures:

[0022]

[0023] Furthermore, the main chain molecular weight of the non-cationic polymer (or the non-cationic polymer based on π-π stacking) is 4000-20000 g / mol.

[0024] Furthermore, the non-cationic polymer is obtained by random polymerization of polyethylene glycol monomethyl ether (as an end group), lactide and / or glycolide, δ-valerolactone and / or aromatic ring-substituted δ-valerolactone.

[0025] Furthermore, the molecular weight of the polyethylene glycol monomethyl ether is 1000-10000 g / mol; more preferably 3000-8000 g / mol.

[0026] Furthermore, the delivery system comprises nanoparticles autonomously packaged from a complex of siRNA and a drug with the non-cationic polymer.

[0027] A drug-loaded nanoparticle comprising a non-cationic polymer as described in any of the above technical solutions and a complex of siRNA and drug loaded on the carrier.

[0028] A method for preparing drug-loaded nanoparticles includes: preparing a 1-20 mg / mL solution I of a non-cationic polymer material based on π-π stacking interaction; preparing a 1-2 mg / mL drug solution II of a complex of siRNA and a drug; mixing the two solutions and incubating for 20-40 minutes (e.g., 30 minutes) to form an oil phase; separately preparing water as an aqueous phase; adding the oil phase dropwise to the continuously stirred aqueous phase to form a milky white nanoemulsion solution; continuing stirring for 15-30 minutes after the addition is complete; then transferring the nanosolution to an activated dialysis bag and dialyzing in ultrapure water for 6-10 hours; finally removing insoluble suspended solids using a filter membrane to obtain the described drug-loaded nanoparticle aqueous solution.

[0029] Furthermore, the mass ratio of the non-cationic polymer material to the complex is 10:1-30:1; the volume ratio of solution I to solution II is 1:20-30:1, preferably 1:20-20:1, and most preferably 1:1; in the complex, the molar ratio of drug to target siRNA is 0.1:1-5:1; the volume ratio of oil phase to water phase is 1:1-1:30, preferably 1:1-1:20, and most preferably 1:10.

[0030] Furthermore, the solvent for the drug-loaded nanoparticle solution is one or both of DMSO and ultrapure water.

[0031] The application of a non-cationic polymer based on π-π stacking interaction in the delivery of siRNA-drug complexes.

[0032] The superior effects of this invention are as follows:

[0033] 1) The present invention provides a non-cationic siRNA and chemical drug co-delivery system, which combines π-π stacking and hydrophobic interaction to have a positive effect on the particle size, distribution and drug loading of nanoparticles, with better effect than single hydrophobic interaction, and the formed nanoparticles have better stability.

[0034] 2) The complex of siRNA and drug can be effectively released from the drug-loaded nanoparticles. The complex nanoparticles all show good pH responsiveness, which is conducive to achieving efficient co-delivery of siRNA and chemical drugs and generating strong chemical gene combination therapy against tumors.

[0035] 3) The siRNA and chemical drug co-delivery system of the present invention has higher safety in terms of delivery materials compared with cationic polymers.

[0036] 4) The siRNA and chemical drug combined delivery system of the present invention has a stronger killing effect on cancer cells due to its combined drug treatment method compared with single drug administration.

[0037] 5) The polymer material prepared by this invention exhibits good effects in terms of cell uptake and drug release, which helps to inhibit tumor cell growth.

[0038] 6) The non-cationic siRNA and chemical drug co-delivery system used in this invention can effectively reduce the side effects of chemotherapy drugs while achieving co-delivery of siRNA and chemical drugs, thus achieving a synergistic therapeutic effect. This delivery system can effectively broaden the development ideas of gene and chemotherapy co-delivery, and provide theoretical and practical basis for new non-cationic co-delivery systems. Attached Figure Description

[0039] Figure 1Synthetic routes for monomers BnVL and NaVL;

[0040] Figure 2 Synthetic routes for PPLB, PPLN copolymers and PPLV copolymers;

[0041] Figure 3 monomer BnVL 1 H NMR spectrum;

[0042] Figure 4 Monomer NaVL 1 H NMR spectrum;

[0043] Figure 5 PPLB6 1 H NMR spectrum;

[0044] Figure 6 PPLN5 1 H NMR spectrum;

[0045] Figure 7 PPLN6 1 H NMR spectrum;

[0046] Figure 8 PPLV4 1 H NMR spectrum;

[0047] Figure 9 The binding of siRNA to IR·HCl;

[0048] Figure 10 The binding status of siRNA to DOX·HCl;

[0049] Figure 11 Changes in the UV-Vis absorption spectrum after IR binds to siRNA;

[0050] Figure 12 Changes in Cy5.5-siRNA fluorescence detection;

[0051] Figure 13 Fluorescence and UV-Vis absorption spectra of DOX after binding with siRNA;

[0052] Figure 14 TEM image of the PPLN5@IR complex;

[0053] Figure 15 Schematic diagram of the stability of IR-loaded composite nanoparticles;

[0054] Figure 16 A schematic diagram illustrating the stability of DOX-loaded composite nanoparticles;

[0055] Figure 17Graph showing drug release results from IR-loaded nanoparticles;

[0056] Figure 18 Graph showing drug release results from DOX-loaded nanoparticles;

[0057] Figure 19 Cell viability graphs of NP cells loaded with siPLK1 & IR and control HeLa-Luc cells;

[0058] Figure 20 Cell viability graphs of NP cells loaded with siPLK1 & DOX and control HeLa-Luc cells;

[0059] Figure 21 Figure 4 shows the results of cell uptake after 4 hours with different doses of PPLB6@siLuc & DOX NP;

[0060] Figure 22 This is a composition diagram of the non-cationic polymer siRNA and chemical drug co-delivery system of the present invention. Detailed Implementation

[0061] The present invention will be further described below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited thereto.

[0062] Special note: The nomenclature of each modified product is as follows: α-benzylvalerol is BnVL, α-naphthylmethylvalerol is NaVL; PPLB is mPEG. n -bP(LA-co-BnVL) x+y PGLB is an abbreviation for mPEG. n -bP(GA-co-BnVL) x+y PLLB is an abbreviation for mPEG. n -bP(LGA-co-BnVL) x+y PCLB is an abbreviation for mPEG. n -bP(CL-co-BnVL) x+y The abbreviation for mPEG; PPLN is for mPEG. n -bP(LA-co-NaVL) x+y The abbreviation for PGLN is mPEG. n -bP(GA-co-NnVL) x+y The abbreviation for PLLN is mPEG. n -bP(LGA-co-NnVL) x+y PCLN is an abbreviation for mPEG. n -bP(CL-co-NnVL) x+y PPLV is an abbreviation for mPEG. n-bP(LA-co-VL) x+y PGLV is an abbreviation for mPEG. n -bP(GA-co-VL) x+y PLLV is an abbreviation for mPEG. n -bP(LGA-co-VL) x+y PCLV is an abbreviation for mPEG. n -bP(CL-co-VL) x+y The abbreviation PPLB@siRNA&IR NP represents nanoparticles formed by the complex of siRNA and drug with polymer materials. PPLB can also be replaced by PGLB, PLLB, PCLB, PPLN, PGLN, PLLN, PCLN, PPLV, PGLV, PLLV, and PCLV. Drug IR can also be replaced by DOX. The number in the lower right corner of the polymer material represents different molecular weights. The actual molecular weights of PPLB6, PPLN5, PPLN6, and PPLV4 are 10600, 11000, 13200, and 11700, respectively. In PPLN6, the ratio of LA to the corresponding monomer NaVL is 3:1, while the ratio of LA to the corresponding monomer in the other mentioned polymer materials is 1:1.

[0063] The structural formula of the non-cationic siRNA-chemical drug co-delivery system is as follows: Figure 22 As shown;

[0064] Figure 22 In the system shown, the substituents R1, R2, R3, and R4 are:

[0065]

[0066] One of them;

[0067] In the formula, the drug that interacts with siRNA to form a complex is:

[0068]

[0069] One of them;

[0070] Example 1: Synthesis of monomer BnVL

[0071] Under nitrogen protection, δ-valerol (VL) (10 g, 99.88 mmol) was added to a 1 L three-necked flask containing 200 mL of redistilled tetrahydrofuran, and the mixture was stirred in an ice-cold ethanol bath at -78 °C for 30 min. Diisopropylaminolithium (LDA) (55 mL, 109.87 mmol) was added dropwise to the reaction mixture at a rate of 2 drops per second using a double-needle method, and the mixture was stirred for 30 min after the addition was complete. Then, a mixture of hexamethylphosphoric triamine (21.48 g, 120 mmol) and benzyl bromide (20.5 g, 120 mmol) was added dropwise to the reaction mixture at a rate of 3 drops per second, and the mixture was stirred for 30 min after the addition was complete. The reaction mixture was then heated to -30 °C and maintained at this temperature for 2 hours. Two hours later, the reaction was confirmed to be complete by TLC (hexane:ethyl acetate = 3:1). The reaction solution was then brought to room temperature, and 300 mL of saturated NH4Cl solution was added to quench the reaction. The mixture was stirred at room temperature for 30 minutes. Tetrahydrofuran was then removed by rotary evaporation. The concentrated reaction solution was extracted three times with 150 mL of ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous Na2SO4, filtered, and finally concentrated by rotary evaporation to remove ethyl acetate, yielding a yellow crude product. The crude product was separated by silica gel column chromatography (hexane:ethyl acetate = 9:1) to obtain a colorless oily liquid (9.85 g, yield 51.84%). Recrystallization from hexane yielded purified BnVL monomer (structure shown in [link to structure]). Figure 1 (6.75g, total yield 35.53%). 1 H NMR (400MHz, CDCl3): δ1.45-1.57(m,1H),1.75-1.95(m,3H),2.69-2.78(m,2H),3.32-3.41(m,1H),4.22-4.33(m,2H),7.19-7.34(m,5H).

[0072] Example 2 Synthesis of monomer NaVL

[0073] Under nitrogen protection, δ-valerol (10 g, 99.88 mmol) was added to a 1 L three-necked flask containing 200 mL of redistilled tetrahydrofuran, and the mixture was stirred in an ice-cold ethanol bath at -78 °C for 30 min. Diisopropylaminolithium (LDA) (55 mL, 109.87 mmol) was added dropwise to the reaction mixture at a rate of 2 drops per second using a double-needle method, and the mixture was stirred for 30 min after the addition was complete. Then, a mixture of hexamethylphosphoric triamine (21.48 g, 120 mmol) and 2-(bromomethyl)naphthalene (26.5 g, 120 mmol) dissolved in 200 mL of tetrahydrofuran was added dropwise to the reaction mixture at a rate of 3 drops per second, and the mixture was stirred for 30 min after the addition was complete. The reaction mixture was then heated to -30 °C and maintained at this temperature for 2 hours. Two hours later, the reaction was confirmed to be complete by TLC (hexane:ethyl acetate = 5:1). The reaction solution was then brought to room temperature, and 300 mL of saturated NH4Cl solution was added to quench the reaction. The mixture was stirred at room temperature for 30 minutes. Tetrahydrofuran was removed by rotary evaporation. The concentrated reaction solution was extracted three times with 150 mL of ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous Na2SO4, filtered, and finally concentrated by rotary evaporation to remove ethyl acetate, yielding a yellow crude product. The crude product was separated by silica gel column chromatography (hexane:ethyl acetate = 12:1) to give a pale yellow oily liquid (7.15 g, yield 29.79%). Recrystallization from hexane yielded purified NaVL monomer (structure shown in [link to structure]). Figure 1 (6.25g, yield 26.04%). 1 HNMR (400MHz, CDCl3): δ1.48-1.60(m,1H),1.71-1.96(m,3H),2.77-2.93(m,2H),3.47-3.57(t,1H),4.21-4.34(m,2H),7.24-7.85(m,7H).

[0074] Example 3 Synthesis of aromatized modified PPLB and PPLN copolymers and PPLV copolymers using mPEG 4k -bP(LA-co-BnVL) 4k(LA:BnVL = 1:1, referring to the monomer molar ratio) (PPLB4) is used as an example. All glassware and stir bar used in the reaction must be dried beforehand. Weigh out purified BnVL (0.685g, 3.60mmol), lactide (LA) (0.519g, 3.60mmol), and mPEG (polyethylene glycol monomethyl ether, 1.0g, 0.25mmol) with a molecular weight of 4k, and add them to a 50mL three-necked flask. Then add 25mL of anhydrous toluene, set up the experimental setup, and transfer it to an oil bath. The entire reaction is carried out under nitrogen protection. Under nitrogen protection, stir magnetically to completely dissolve the toluene. Then, heat to 150℃, separate 20mL of the toluene-water mixture, remove the small amount of water present in the reaction solution, and quickly add 6-7 drops of stannous octoate (Sn(Oct)2) catalyst. React for 48 hours. After the reaction was complete, the reaction solution was cooled to room temperature, and 200 mL of dichloromethane (DCM) and 4 drops of 0.1 M dilute hydrochloric acid solution were added to quench the reaction. The quenched reaction solution was washed with water several times until the upper aqueous phase changed from acidic to neutral. Then the organic phase was separated, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to remove DCM, yielding a pale yellow, oily, viscous crude product. The crude product was dissolved in a small amount of DCM, and then n-hexane (dichloromethane:n-hexane = 1:10) was added dropwise while stirring, accompanied by the appearance of a white precipitate. After the addition was complete, the mixture was allowed to stand for a period of time, and the supernatant was discarded. The above process was repeated three times. The precipitate was dried in a vacuum drying oven to obtain the final product PPLB4 (1.72 g, yield 86.0%).

[0075] Using the same ring-opening polymerization method, by controlling the amount of initiator (mPEG) and the monomer feed ratio (see table below), while keeping other conditions unchanged, PPLB and PPLN series polymers with different monomer ratios and molecular weights were synthesized. The yield of this series of polymers was 75%-90%.

[0076] polymer The molecular weight of mPEG monomer LA to corresponding monomer feed ratio PPLB6 6k BnVL 3:1 PPLN5 5k NaVL 1:1 PPLN6 6k NaVL 1:1 PPLV4 4k VL 1:1

[0077] Example 4: Preparation of siRNA-drug complex (taking IR as an example, the complex is denoted as: [siRNA&IR]).

[0078] Prepare IR·HCl (0.15 mL) or DOX·HCl (0.15 mL) of different concentrations using DMSO, with different drug:siRNA nucleotide (siRNA) molar ratios (see [reference]). Figure 9 and Figure 10As shown in the image, gradually add 0.15 mL (3.0 nmol) of siRNA (small interfering RNA, purchased from Gene Pharma (Shanghai, China)) to an aqueous solution. After incubating at room temperature for 10 minutes, place the mixture in a refrigerated centrifuge and centrifuge at 12,000 rpm for 15 minutes at 4°C. Collect the precipitate and supernatant; the precipitate is the target drug complex.

[0079] Example 5: Determination of siRNA binding rate to drug

[0080] The concentration of residual IR or DOX in the supernatant obtained in Example 4 was determined using an ELISA reader to ascertain the binding rate of IR or DOX to siRNA. Using pure water as a reference solution, the UV absorption intensity of IR at 354 nm and the UV absorption intensity of DOX at 480 nm were measured in the supernatant. The results are as follows: Figure 9 , 10 As shown in Figure 11 ( Figure 9 and Figure 10 In the text, N / P represents the molar ratio of drug to siRNA. Figure 11 In the middle: PPLB6@[siRNA&IR] was prepared using the method in Example 7; [siRNA&IR] was a complex with a molar ratio of 3.

[0081] Example 6: Changes in Cy5.5-siRNA fluorescence detection

[0082] A complex of IR·HCl (46 × 3 nmol, 0.15 mL) and Cy5.5-labeled siRNA (1 nmol of siRNA has 46 nmol of phosphate groups, so 46 nmol of irinotecan is required, and it is added in triplicate) and IR:siRNA-nt molar ratio of 3:1 was prepared for 10 minutes. Then, polyanionic heparin sodium (5.0 mg / mL, 0.5 mL) was added to the complex solution. During this process, the fluorescence of Cy5.5-siRNA was tracked using a microplate reader. The fluorescence spectrum at 600-800 nm was scanned at an excitation wavelength of 580 nm at 0 min, 2 min, and 10 min after the addition of heparin sodium. The fluorescence spectrum of free Cy5.5-siRNA at the same concentration was also scanned as a control. Figure 12As shown, without the addition of heparin, most of the Cy5.5 fluorescence was quenched after the formation of the complex. Upon addition of sodium heparin, the fluorescence recovery of Cy5.5-siRNA was clearly observed. Anionic heparin has been widely used to disrupt the electrostatic interaction between siRNA and positively charged materials. Therefore, this result demonstrates the successful preparation of the complex [siRNA&IR] and confirms that electrostatic interactions drive the hydrophobication of siRNA with IR·HCl. Figure 11 As shown, the changes in the UV-Vis absorption spectrum indicate that after the formation of the complex, the IR absorbance is reduced compared to the free IR due to aggregation induction, which further demonstrates the successful formation of the complex.

[0083] Example 7 Preparation of Nanoparticles

[0084] Empty and drug-loaded nanoparticles were prepared using a nanoprecipitation method. Polymer materials (PPLB6, PPLN6, PPLN5, PPLV4) were dissolved in DMSO to prepare a 20 mg / mL solution, and the composite obtained in Example 4 was prepared in a 1 mg / mL DMSO solution. 0.5 mL of each was taken, mixed in equal volumes, and incubated for 30 min to form the oil phase. Separately, 10 mL of ultrapure water was placed in a sample vial to form the aqueous phase. The oil phase was added dropwise to the continuously stirred aqueous phase at an O / W ratio of 1:10 to form a nanoemulsion solution. After the addition was complete, stirring was continued for approximately 20 min. The nanosolution was then transferred to an activated 3500 Da dialysis bag and dialyzed in ultrapure water for 8 h to remove solvent and unencapsulated small molecule drugs, with the water changed every 2 h during this period. Finally, the insoluble suspended matter is removed using a 0.45 μm filter membrane to obtain an aqueous solution of drug-loaded nanoparticles (resulting in nanoparticles (NPs) of PPLB6, PPLN6, PPLN5, PPLV4, and IR or DOX, denoted as PPLB6@[siRNA&drug]NP or PPLB6@siRNA&drugNP). Empty nanoparticles are prepared using the same method. Figure 14 This is a TEM image of the PPLN5@IR complex.

[0085] Example 8: Study on the storage stability of nanoparticles

[0086] The prepared nanoparticle solution was stored in a cool, dark place, and any changes were observed visually at 0, 1, 2, 4, and 7 days after preparation. Samples were also taken for DLS analysis to detect changes in particle size and PDI. Figure 15 and 16 This indicates that the drug-loaded nanoparticles prepared by this hydrophilic-hydrophobic interaction and π-π stacking can maintain good stability over a period of time.

[0087] Example 9: In vitro release of drug-loaded nanoparticles

[0088] 2.5 mL of the prepared nanoparticle aqueous solution was placed in a suitable dialysis bag, and both ends of the dialysis bag were tied tightly. The dialysis bag was then immersed in a centrifuge tube containing 15 mL of phosphate buffer (pH 5.5 or 7.4), and placed in a constant temperature water bath shaker at 37°C and a cycle frequency of 120 rpm for three days in the dark. 0.5 mL samples were collected at 0 h, 1 h, 3 h, 6 h, 12 h, 24 h, 36 h, 48 h, and 72 h. Fresh PB solution was added to the centrifuge tube after each sampling. The drug content in the samples was detected using an ELISA reader. All in vitro release experiments of the nanoparticles were repeated three times, and the drug release amount was the average of the three parallel experiments. The in vitro release of the drug-loaded nanoparticles is as follows: Figure 17 and 18 As shown in the figure, the results indicate that the complex can be effectively released from the drug-loaded nanoparticles, and the complex-loaded nanoparticles prepared using the four materials PPLB6, PPLN5, PPLN6 and PPLV4 all showed good pH responsiveness, which is beneficial for achieving efficient co-delivery of siRNA and chemical drugs and generating strong chemical gene combination therapy against tumors.

[0089] Example 10: Cytotoxicity Experiment of Drug-Loaded Nanoparticles

[0090] This invention evaluates the oncogene silencing efficiency of NP by selecting the oncogene targeting gene Polo-like kinase 1 (siPLK1). The MTT assay was used to assess the killing effect of polymer@siPLK1&Drug NP (preparation method as described in Example 7, the molar ratio of siPLK1 to drug in the complex is 1:2) on tumor cells. Hela-Luc cells were seeded at a density of 5000 cells / well in 96-well clear plates and incubated at 37°C for 24 hours. The culture medium was then replaced with fresh medium containing different concentrations of the drug. After 48 hours of incubation, 20 μL of MTT reagent (5 mg / mL) was added to each well, and incubation continued for another 4 hours. Subsequently, the culture medium was aspirated, and 150 μL of DMSO was added to dissolve the resulting blue crystals in dimethyl sulfoxide. After shaking for 10 minutes, the absorbance was measured at 490 nm using a microplate reader. Normalized analysis was performed using the untreated group as a negative control. Experimental results are as follows Figure 19 and 20 As shown (in) Figure 19Taking the results corresponding to PPLB6 as an example, the four columns at each concentration, from left to right, are PPLB6 NP (PPLB6 nanoparticles), Free IR (free IP), PPLB6@IR NP (PPLB6-loaded IP), and PPLB6@siPLK1&IR NP (prepared according to the method in Example 7). This shows that the IR-loaded and DOX-loaded nanoparticles prepared using PPLB6, PPLN5, PPLN6, and PPLV4 materials exhibited dose-dependent cytotoxicity after co-culturing with HeLa cells. The combined administration showed stronger cell-killing activity compared to single administration, validating the feasibility of these non-cationic amphiphilic polymers as co-delivery carriers for siRNA and small molecule drugs.

[0091] Example 11 Cellular uptake experiment of drug-loaded nanoparticles

[0092] This invention uses fluorescence microscopy to determine in vitro cell uptake efficiency. Hela-Luc cells were seeded at a density of 1×10⁵ cells / well in six-well plates and incubated for 24 hours. 1 mL of fresh DMEM was replaced, and then different volumes (10 μL, 30 μL, 40 μL, 50 μL) of PPLB6@cy5-siRNA & DOX nanoparticle solution (DOX: 35.6 μg / mL and cy5-siRNA: 12.86 μg / mL) were added to treat the cells for 4 hours. Then, the old culture medium was aspirated, and the cells were rinsed 2–3 times with phosphate-buffered saline (PBS). 1 mL / well of 4% paraformaldehyde was added, and the cells were fixed at 4°C for 30 min. The fixative was then aspirated, and the cells were rinsed 2–3 times with PBS. 1 mL / well of 5 μg / mL DAPI dilution was added to stain the cell nuclei, and the cells were incubated for 8 minutes before the DAPI was aspirated. The cells were then washed twice with PBS, and 1 mL of PBS was added to each well to prevent cell lysis. During the test, PBS was aspirated, and the sample was observed under a fluorescence microscope. For example... Figure 21 As shown, the results indicated that after 4 hours of incubation, the cells generally survived well, with the cell nuclei stained blue. DOX exhibited red fluorescence distributed within the cell nucleus, while Cy5 was distributed in the cytoplasm. The fluorescence signal intensified with increasing drug concentration, indicating good drug uptake by the cells. Cy5-siRNA and DOX dissociated in tumor cells, and the red and blue fluorescence overlapped to form purple, indicating that DOX entered the cell nucleus, while Cy5-siRNA remained in the cytoplasm, each playing its respective role. This demonstrates that the polymer material exhibits excellent capabilities in cellular uptake and drug release, effectively inhibiting tumor cell growth.

[0093] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A non-cationic polymeric siRNA and chemical drug co-delivery system, characterized in that, This includes non-cationic polymers and complexes of siRNA and drugs loaded on the polymer; The complex is composed of the target siRNA and the corresponding drug; The non-cationic polymer contains several branches with aromatic rings, wherein the aromatic rings are selected from one or more of 5- to 15-membered aromatic rings or 5- to 15-membered heteroaromatic rings. The drug is selected from one or more of gemcitabine, mitoxantrone, topotecan, belotecone, irinotecan, doxorubicin, camptothecin, and derivatives of any of the above drugs.

2. The non-cationic polymer siRNA and chemical drug co-delivery system according to claim 1, characterized in that, The non-cationic polymer is one or more of the following structures: R1, R2, R3, and R4 are the branched chains with aromatic rings, and each of R1, R2, R3, and R4 is independently selected from one or more of 5- to 15-membered aromatic rings or 5- to 15-membered heteroaromatic rings; n, x, y, a, and b represent the degree of polymerization, where n is 1-5000, x is 1-4000, y is 1-4000, a is 1-4000, and b is 1-4000.

3. The non-cationic polymer siRNA and chemical drug co-delivery system according to claim 1 or 2, characterized in that, The aromatic ring-bearing side chain is selected from one or more of the following structures:

4. The non-cationic polymer siRNA and chemical drug co-delivery system according to claim 1 or 2, characterized in that, The main chain molecular weight of the non-cationic polymer is 4000-20000 g / mol.

5. The non-cationic polymer siRNA and chemical drug co-delivery system according to claim 1 or 2, characterized in that, The non-cationic polymer is obtained by random polymerization of polyethylene glycol monomethyl ether, lactide and / or glycolide, δ-valerolactone and / or aromatic ring-substituted δ-valerolactone.

6. The non-cationic polymer siRNA and chemical drug co-delivery system according to claim 1 or 2, characterized in that, The delivery system comprises nanoparticles autonomously packaged from a complex of siRNA and a drug with the non-cationic polymer.

7. A drug-loaded nanoparticle, characterized in that, It includes the non-cationic polymer as described in any one of claims 1 to 6 and the complex of siRNA and drug loaded on the carrier.

8. A method for preparing drug-loaded nanoparticles according to claim 7, characterized in that, The non-cationic polymer material based on π-π stacking was prepared into solution I at a concentration of 1-20 mg / mL, and the complex of siRNA and drug was prepared into drug solution II at a concentration of 1-2 mg / mL. The two solutions were mixed and incubated for 20-40 minutes to form the oil phase. Water is used as the aqueous phase. The oil phase is added dropwise to the continuously stirred aqueous phase to form a milky white nanoemulsion solution. After the addition is complete, stirring continues for 15–30 minutes. Then, the nano solution is transferred to an activated dialysis bag and dialyzed in ultrapure water for 6–10 hours. Finally, insoluble suspended solids are removed using a filter membrane to obtain the drug-loaded nanoparticle aqueous solution.

9. The method for preparing drug-loaded nanoparticles according to claim 8, characterized in that, The mass ratio of the non-cationic polymer material to the complex is 10:1-30:1; the volume ratio of solution I to solution II is 1:20-30:1; in the complex, the molar ratio of drug to target siRNA is 0.1:1-5:1; and the volume ratio of oil phase to water phase is 1:1-30:

1.

10. The use of a non-cationic polymer as described in any one of claims 1 to 5 in the delivery of siRNA and drug complexes.

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