Nanoscale drug delivery system containing stabilizer, and preparation method and application thereof

Abstract

The application discloses a nano-drug delivery system containing a stabilizer and a preparation method and application thereof, and belongs to the technical field of medicine preparation. The nano-drug delivery system containing the stabilizer comprises a cationic polypeptide / siRNA complex and a particle size stabilizer; the cationic polypeptide / siRNA complex is formed by non-covalent force between a cationic polypeptide and siRNA, and the amino acid sequence of the cationic polypeptide is KKK(RH4RH4RH4RH4R) x wherein x=3 or 4; and the particle size stabilizer is a surfactant or a protein. Experiments prove that the delivery system combined with the cationic polypeptide and the particle size stabilizer has stable nano size in pure water and biological matrix, and has high gene silencing efficiency at the cell level, and does not significantly reduce the cell transfection or knockout efficiency. Therefore, the nano-drug delivery system can keep nano size in the biological matrix for a long time, and has particle size stability.

Description

Technical Field

[0001] This invention belongs to the field of drug preparation technology, specifically relating to a nano-drug delivery system containing stabilizers, its preparation method, and its application. Background Technology

[0002] Cationic peptide / siRNA nanocomposites (including cell-penetrating peptide / siRNA systems) are an important small nucleic acid delivery platform, widely used in recent years for in vitro and in vivo delivery of small interfering RNA (siRNA) due to their excellent transfection efficiency. Although this strategy has shown good transfection efficiency and gene silencing effects at the cellular level, its stability and biocompatibility still pose significant challenges from the perspective of in vivo research and clinical translation. In particular, the non-specific adsorption and rapid aggregation of cationic peptide / siRNA nanocomposites in complex physiological environments has become one of the key bottlenecks hindering its clinical application.

[0003] Cationic peptide / siRNA nanocomposites can maintain uniform nanoscale size and high colloidal stability in low ionic strength environments such as deionized water or ultrapure water. However, once exposed to high ionic strength and complex protein-rich systems such as physiological saline, PBS buffer, cell culture medium, plasma, or serum, the electrical double layer on the surface of the composite is compressed, the electrostatic shielding effect is enhanced, leading to charge neutralization of the nanosystem and a significant decrease in surface potential (zeta potential), or even destruction of the electrical double layer. Simultaneously, the non-specific adsorption of biomacromolecules such as plasma proteins (protein crown formation) further alters the surface properties of the nanocomposites, inducing phenomena such as increased particle size, aggregation and precipitation, and component dissociation. This process not only leads to inconsistent in vivo and in vitro characterization of peptide / siRNA nanocomposites but also results in highly uncertain pharmacokinetic behavior and biodistribution characteristics in blood or interstitial fluid, significantly increasing safety risks such as immune responses, vascular occlusion, and hepatosplenic clearance. Therefore, most current cationic peptide / siRNA nanosystems are difficult to administer systemically (e.g., intravenously), and their applications are limited to in vitro transfection tools or local delivery. Summary of the Invention

[0004] In order to overcome the shortcomings of the prior art, the present invention aims to provide a nano-drug delivery system containing stabilizers, its preparation method and application, so as to solve the technical problem that it is difficult to achieve systemic drug delivery in cationic peptide / siRNA nanosystems.

[0005] To achieve the above objectives, the present invention employs the following technical solution: A first aspect of the present invention discloses a nano-drug delivery system containing a stabilizer, comprising: a cationic peptide / siRNA complex and a particle size stabilizer; wherein the cationic peptide / siRNA complex is formed by the interaction of a cationic peptide and siRNA through non-covalent forces, and the amino acid sequence of the cationic peptide is KKK(RH4RH4RH4RH4R). x The particle size stabilizer is a surfactant or a protein. Where x = 3 or 4.

[0006] Preferably, the amino acid sequence of the cationic polypeptide is KKK(RHHHHRHHHHRHHHHRHHHR)4.

[0007] Preferably, the surfactant is poloxamer 188, poloxamer 407, polyoxyethylene castor oil EL35, or distearate phosphatidylethanolamine-polyethylene glycol.

[0008] Preferably, the protein is bovine serum albumin, human serum albumin, or human transferrin.

[0009] Preferably, it also includes a freeze-drying protectant.

[0010] More preferably, the freeze-drying protectant is mannitol, glucose, or sucrose.

[0011] Preferably, in the nano-drug delivery system containing stabilizers, the surfactant has a mass-volume percentage concentration of 0.1% to 1%, and the protein has a mass-volume percentage concentration of 0.5% to 5%.

[0012] Preferably, the molar ratio of cationic polypeptide to siRNA is (3~24):1.

[0013] In a second aspect, the present invention discloses a method for preparing the above-mentioned nano-drug delivery system containing a stabilizer, wherein a cationic peptide and siRNA are mixed through a microfluidic system to obtain a cationic peptide / siRNA nanocomposite; the cationic peptide / siRNA nanocomposite is then mixed with a particle size stabilizer to obtain a nano-drug delivery system containing a stabilizer.

[0014] A third aspect of the present invention discloses the application of the above-described nano-drug delivery system containing stabilizers in drug delivery.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a nano-drug delivery system containing stabilizers, wherein: 1) in a cationic polypeptide with the amino acid sequence KKK(RH4RH4RH4RH4R)x, lysine (K) provides the core structure of the branched peptide, arginine (R) provides the positive charge for binding siRNA, and histidine (H) provides pH response characteristics to promote endosome escape of siRNA in acidic endosomes. The branched structure (x=3 or 4) is beneficial to improving the rate of polypeptide binding to siRNA and the encapsulation efficiency; 2) on the basis of the cationic polypeptide / siRNA complex, a surfactant or protein is added as a particle size stabilizer, which enables the polypeptide / siRNA nanocomplex to exist in biological matrices (including: physiological buffer, cell culture medium, plasma or serum) for a longer period of time (48 h) with a particle size of 150~500 nm, thereby improving the particle size stability of the cationic polypeptide / siRNA complex in biological matrices and preventing its non-specific adsorption and rapid aggregation in biological media. Experiments have demonstrated that the stabilizer-containing nano-drug delivery system can mitigate rapid aggregation and particle size increase in different biological media, maintain relative stability in biological matrices, and exhibit high target gene silencing efficiency at the cellular level without significantly reducing cell transfection or knockout efficiency. Therefore, this nano-drug delivery system can maintain its nanoscale size and particle size stability in biological matrices for extended periods, enabling systemic drug delivery.

[0016] Furthermore, a lyophilization protectant is added to the particle size stabilizer to facilitate the freeze-drying and preservation of the peptide / siRNA complex, and it can be quickly reconstituted when needed. After reconstitution, the particle size and potential remain basically unchanged from the corresponding indicators before freeze-drying. Attached Figure Description

[0017] Figure 1 The particle size distribution of the cationic polypeptide / siRNA complex of the present invention in pure aqueous solution (a) and the trend of rapid particle size increase of different concentrations of complex (siRNA content of 2.97 µM and 5 µM, respectively) in 0.9% NaCl or Opti-MEM cell culture medium (b) are shown. Figure 2 This is a graph showing the trend of rapid particle size increase after lyophilization and reconstitution of different concentrations of the nanocomposite of the present invention (containing only 5% mannitol lyophilization protectant) in 0.9% NaCl and cell culture medium. Figure 3 The figure shows the particle size stability test results of the cationic polypeptide / siRNA complex combination of mannitol and different concentrations of F68 (after freeze-drying and reconstitution) in 0.9% NaCl (a) and Opti-MEM medium (b). Figure 4The figure shows the particle size stability test results of the combined mannitol and cationic polypeptide / siRNA complexes of different concentrations of F127 (after freeze-drying and reconstitution) of the present invention in 0.9% NaCl (a) and Opti-MEM medium (b); Figure 5 The images show the clear appearance of the combined mannitol and cationic polypeptide / siRNA complexes of different concentrations of F127 after incubation in plasma or serum (60%, v / v) for 30 minutes; wherein, a and c do not contain stabilizers. Figure 6 shows the particle size distribution of the combined mannitol and cationic polypeptide / siRNA complexes with different concentrations of EL35 in this invention (a, before freeze-drying) and the particle size change trend in Opti-MEM medium after freeze-drying (b). Figure 7 This is a particle size distribution diagram of the cationic polypeptide / siRNA nanocomplexes of different concentrations of proteins (HSA) of the present invention. Figure 8 The graph shows the zeta potential detection results of the cationic polypeptide / siRNA nanocomposite containing the protein concentration shown in this invention. Figure 9 The cationic polypeptide / siRNA nanocomposite containing the indicated protein (HSA) concentration of the present invention was tested in different biological media (37). o Figure showing the particle size change detection results after incubation at C (containing 10% fetal bovine serum (FBS)); Figure 10 The particle size distribution of the cationic polypeptide / siRNA nanocomplex of the combined human transferrin (Tf, 1% w / v) of the present invention and the results of particle size stability (after incubation for 20 h) in 0.9% NaCl and Opti-MEM medium (containing 10% fetal bovine serum) are shown. Figure 11 shows the cytotoxicity detection results of the b-HKR peptide and nano-drug delivery system of the present invention; where a represents the cell viability and IC50 of the b-HKR peptides (A549, MCF-7). 50 Value; b represents cell viability of b-HKR / siRNA and b-HKR / siRNA (with 0.25% HAS and 200 nM siRNA). Figure 12 The graph shows the hemolysis rate detection results of the free b-HKR peptide, b-HKR / siRNA nanocomposite, and b-HKR / siRNA (with 0.25% HSA added) of the present invention. Figure 13The images show microscopic images of free b-HKR peptide, b-HKR / siRNA (containing 200 nMsiRNA) nanocomposite and b-HKR / siRNA (containing 200 nMsiRNA, with 0.25% HSA added) nanocomposite incubated with blood red blood cells at pH 7.2. Detailed Implementation

[0018] To enable those skilled in the art to understand the features and effects of the present invention, the following description and definitions are only general descriptions of the terms and expressions mentioned in the specification. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in case of conflict, the definitions in this specification shall prevail.

[0019] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0020] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0021] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”

[0022] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0023] In this article, "H" in the amino acid sequence represents histidine, "K" represents lysine, and "R" represents arginine. Unless otherwise specified, "PdI" represents polydispersity index, and "DLS" represents dynamic light scattering.

[0024] This invention provides a nano-drug delivery system containing a stabilizer, comprising a cationic peptide / siRNA complex and a particle size stabilizer; The cationic polypeptide / siRNA complex is formed by the interaction of a cationic polypeptide and siRNA in a molar ratio of (3~24):1 through non-covalent forces (electrostatic and hydrogen bonding), and the amino acid sequence of the cationic polypeptide is KKK(RH4RH4RH4RH4R). x Wherein, -KKK- is a branched peptide center composed of three lysine residues (RH4RH4RH4RH4R). x The siRNA is a branched peptide chain with -KKK- as the core (H represents histidine, R represents arginine; x represents the number of branches), x=3 or 4; the amino acid sequence of the cationic polypeptide is preferably KKK(RH4RH4RH4RH4R)4; the molar ratio of the cationic polypeptide to siRNA is preferably (6~24):1, more preferably (6~15):1; the final concentration of siRNA is 5~20 μM; the particle size of the complex in deionized water is 100~150 nm, for example, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm or a range between the two, 100~150 nm, 150~200 nm; the zeta potential is 15~40 mV, for example, 15 mV, 16 mV, 17 mV, 18 mV, 19 mV, 20 mV, 21 mV. mV, 22 mV, 23 mV, 24 mV, 25 mV, 26 mV, 27 mV, 28mV, 29 mV, 30 mV, 31 mV, 32 mV, 33 mV, 34 mV, 35 mV, 36 mV, 37 mV, 38 mV, 39 mV, 40 mV or range value 15~30 mV, 20~40 mV; The particle size stabilizer is a surfactant, albumin, and / or transferrin. The surfactant is F68 (poloxamer 188 or Pranic F-68), F127 (poloxamer 407 or Pranic F-127), EL (35) (polyoxyethylene ether (35) castor oil), or DSPE-PEG (distearate phosphatidylethanolamine-polyethylene glycol), with a surfactant concentration of 0.1% to 1% (w / v). The albumin is bovine serum albumin (BSA) or human serum albumin (HSA), with an albumin concentration of 0.5% to 5% (w / v) and a transferrin concentration of 1% to 5%. The transferrin is preferably human transferrin (Tf). The particle size stabilizer is any one or more combinations of poloxamer, castor oil, DSPE-PEG, albumin, and transferrin, such as a combination of albumin and transferrin, albumin and poloxamer, or poloxamer and castor oil. The particle size stabilizer accounts for 0.1% to 5% of the mass-volume percentage of the nano-drug delivery system containing the stabilizer.

[0025] Furthermore, the above composition also contains a lyophilization protectant, which is mannitol (5%, w / v), glucose (5%, w / v) and / or sucrose (10%, w / v).

[0026] The present invention also provides a method for preparing the above-mentioned composition containing a cationic polypeptide / siRNA complex, wherein the cationic polypeptide / siRNA complex is mixed with a particle size stabilizer, or mixed using microfluidic technology at a flow rate of 100~1000 µL / min.

[0027] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading this description, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined in this application.

[0028] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. The cationic polypeptide branched-HKR (b-HKR) used in the following examples was synthesized and purified by a commercial synthetic company using solid-phase synthesis based on its amino acid sequence. All other raw materials used, unless otherwise stated, are conventional commercially available products with specifications standard in the art.

[0029] I. Preparation and structural characterization of cationic peptide / siRNA nanocomposites 1. Preparation of cationic peptide / siRNA nanocomposites Table 1. Characterization results of b-HKR / siRNA at different molar ratios

[0030] As shown in Table 1, cationic peptides branched-HKR (b-HKR) with amino acid sequences of KKK(RHHHHRHHHHHHHHHRHHHR)4 and a final concentration of 20 μM GAPDH-targeting siRNA (nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2 in Table 2) were mixed in equal volumes using a microfluidic system (Ignite, NIN0002, Precision NanoSystems) at a flow rate of 200 µL / min to obtain cationic peptide / siRNA nanocomposites (containing 10 μM siRNA).

[0031] Table 2 Nucleotide Sequence List

[0032] 2. Structural characterization of cationic peptide / siRNA nanocomposites 1) Particle size and zeta potential detection The particle size and zeta potential of the prepared cationic peptide / siRNA nanocomposites (molar ratios of 1:1, 3:1, 6:1, 9:1, and 12:1) in a pure water (deionized water) system were determined using dynamic light scattering (DLS) technique on a Malvern particle size analyzer (Zetasizer Lab).

[0033] The results are shown in Table 1 and Figure 1 As shown in Figure a, the particle size range of cationic polypeptide / siRNA nanocomposites with different molar ratios ((3~12):1) in pure water (deionized water) system is 100~200 nm, and the Zeta potential is 30~45 mV.

[0034] 2) Encapsulation efficiency test The encapsulation efficiency (EE%) of the cationic peptide / siRNA nanocomplex prepared in step 1 was determined using a Ribogreen kit and quantitative real-time fluorescence method. The specific steps are as follows: Following the Ribogreen (Invitrogen) kit instructions, cationic peptide / siRNA nanocomposites with different molar ratios were incubated with Ribogreen working solution. The total amount of siRNA in the nanocomposites and the content of siRNA in the unencapsulated fraction were determined using fluorescence spectroscopy (excitation wavelength: 500 nm, emission wavelength: 525 nm). For the determination of the total siRNA in the nanocomposites: appropriate amounts of heparin sodium and dilute hydrochloric acid solution were added to fully release the siRNA, and then the siRNA concentration was tested according to the Ribogreen method. For the determination of the concentration of unencapsulated siRNA: the nanocomposite samples were directly tested according to the Ribogreen method (without the addition of heparin sodium and dilute hydrochloric acid).

[0035] Encapsulation efficiency (EE%) = (Total siRNA measured - Unencapsulated siRNA) / (Total siRNA measured) × 100%.

[0036] The test results are shown in Table 1. When the molar ratio of cationic peptide / siRNA nanocomplex (b-HKR:siRNA) is ≥6, the encapsulation efficiency of siRNA by the nanocomplex is ≥95%.

[0037] II. Preparation and Structural Characterization of Nanoparticle Drug Delivery Systems Containing Stabilizers 1. Preparation of nano-drug delivery systems containing stabilizers Example 1 1) b-HKR (concentration 120 μM) and GAPDH-targeted siRNA (concentration 20 μM) with a molar ratio of 6:1 were mixed in equal volumes through a microfluidic system (Ignite, NIN0002, Precision NanoSystems) at a flow rate of 200 µL / min to obtain a cationic peptide / siRNA nanocomposite (final concentration of siRNA 10 μM).

[0038] 2) The cationic peptide / siRNA nanocomposite obtained in step 1 is mixed with F68 (final concentration in the mixture is 0.2% (w / v)) to obtain a nano-drug delivery system containing F68 stabilizer.

[0039] Examples 2 to 4 The difference from Example 1 is that the final concentrations of F68 are different, namely 0.1% (w / v), 0.05% (w / v) and 0.025% (w / v).

[0040] Example 5 The difference from Example 1 is that F68 is replaced with F127 (final concentration of 0.05% (w / v)).

[0041] Example 6 The difference from Example 1 is that F68 is replaced with EL35 (final concentration of 0.05% (w / v)).

[0042] Example 7 The difference from Example 1 is that F68 is replaced with DSPE-PEG (final concentration of 0.05% (w / v)).

[0043] Example 8 The difference from Example 1 is that F68 is replaced with HSA (final concentration of 0.5% (w / v)).

[0044] Examples 9 and 10 The difference from Example 8 is that the final concentration of HSA is different, being 0.25% (w / v) and 1% (w / v), respectively.

[0045] Example 11 The difference from Example 10 is that HSA is replaced with Tf.

[0046] Example 12 The difference from Example 10 is that HSA is replaced with BSA.

[0047] Example 13 The difference from Example 12 is that the concentration of GAPDH-targeting siRNA is 200 nM.

[0048] Example 14 1) b-HKR (concentration 120 μM) and GAPDH-targeting siRNA (concentration 20 μM) with a molar ratio of 6:1 were mixed in equal volumes through a microfluidic system (Ignite, NIN0002, Precision NanoSystems) at a flow rate of 200 µL / min to obtain a cationic peptide / siRNA nanocomposite (final concentration of GAPDH-targeting siRNA was 10 μM).

[0049] 2) The cationic peptide / siRNA nanocomposite obtained in step 1 was mixed with F127 (final concentration of 0.1% (w / v) in the mixture) and mannitol (final concentration of 5% (w / v) in the mixture), and then pre-cooled to -20°C. o After temperature C, it was transferred to the cold trap of a freeze dryer (FD-12A-50T) and programmed to cool to -50°C. o Below C, a vacuum program is then initiated to reduce the vacuum level to below 125 mTorr. The mixture is freeze-dried overnight to remove the solvent, resulting in a nano-drug delivery system containing a stabilizer.

[0050] Examples 15-18 The difference from Example 14 is that the final concentrations of F127 are different, namely 0.075% (w / v), 0.05% (w / v), 0.0125% (w / v), and 0.25% (w / v).

[0051] Example 19 The difference from Example 14 is that F127 is replaced with EL (35).

[0052] Examples 20-22 The difference from Example 18 is that the final concentration of EL (35) is different, being 0.5% (w / v), 0.25% (w / v), and 0.175% (w / v), respectively.

[0053] Example 23 The difference from Example 8 is that it also contains Tf (final concentration of 0.5% (w / v)).

[0054] Examples 24-27 The difference from Example 14 is that F127 is replaced with F68, and the final concentrations of F68 are 0.2% (w / v), 0.1% (w / v), 0.05% (w / v), and 0.025% (w / v).

[0055] Comparative Example 1 The difference from Example 1 is that it does not contain F68.

[0056] Comparative Example 2 The difference from Example 14 is that it does not contain F127.

[0057] Comparative Examples 3-7 The difference between this example and Examples 19-22 and Comparative Example 2 is that freeze drying was not performed.

[0058] Comparative Example 8 The difference from Example 13 is that the GAPDH-targeting siRNA is replaced with Scramble siRNA.

[0059] 2. Stability characterization of nano-drug delivery system The nano-drug delivery systems prepared in the above examples and comparative examples were placed in Opti-MEM medium, physiological saline (0.9% NaCl solution), serum or plasma, and incubated at 37°C for different times. Then, the particle size changes and Zeta potentials under different pH conditions (pH 7.2 and pH 5.0) were measured using a Malvern Zetasizer.

[0060] The results showed that: 1) the b-HKR / siRNA nanocomposites prepared in Example 1 (without any stabilizer or lyophilization protectant) and Example 14 (with 5% mannitol added as a lyophilization protectant, without any stabilizer) rapidly increased in particle size (5-15 min) in physiological saline and cell culture medium (Opti-MEM), reaching a particle size of 500-1500 nm. Figure 1 and Figure 2 Meanwhile, the b-HKR / siRNA nanocomposite prepared in Example 14 is prone to causing aggregation in plasma or serum ( ); Figure 5 The above results indicate that the cationic peptide / siRNA nanocomposites without any added stabilizers (the surfactants or proteins) exhibit extremely unstable particle size in biological media. 2) Adding any surfactant at a concentration of 0.1%–1% (w / v) or any albumin at a concentration of 0.5%–5% (w / v) can alleviate the rapid aggregation and particle size increase of the cationic peptide / siRNA nanocomposites in biological media to varying degrees. Figure 3 , Figure 4 and Figure 9 3) The lyophilized cationic peptides / siRNA containing stabilizers (taking the addition of EL(35) as an example) can be rapidly reconstituted, and the particle size after reconstitution (EL(35) addition amount ≥0.25%) can remain relatively stable in the biological matrix (as shown in Figure 6). 4) Under the condition of pH 7.2, the addition of protein stabilizers does not affect the original particle size of cationic peptides / siRNA ( Figure 7 ), can reverse the Zeta potential of cationic polypeptide nanocomposites (the potential is reversed from positive to negative). Figure 8 ); However, under pH 5.0 (weakly acidic) conditions, the b-HKR / siRNA nanocomposite with added protein stabilizers recovered from a negative potential to a positive potential. This result indicates that protein stabilizers endow cationic peptide nanocomposites with a pH-responsive property; that is, under normal physiological conditions (pH 7.2), protein stabilizers can shield the positive charge of cationic peptides (which often causes toxicity and immune responses), while intracellularly (e.g., in acidic endosomes / lysosomes, pH 4.5–6.0), they restore the positive charge of the peptides to promote siRNA endosome escape. 5) Adding HSA (≥0.5%) or Tf alone can maintain the particle size of the nanocomposite within various biological media. o The incubation conditions under C remained essentially unchanged for 24–48 hours. Figure 7 and Figure 10 6) A mixture of multiple protein stabilizers (mixed concentration ≥1%) can also maintain the stability of the nanocomposite. Figure 8 ).

[0061] III. Exploration of the Delivery Effect of Nanoparticle Drug Delivery Systems Normal skin fibroblasts (NSFB) lines were seeded into 24-well plates and provided with 500 µL of complete growth medium (DMEM containing 10% FBS). Once the cells reached approximately 50% confluence (within 24 h post-seeding), the medium was replaced with Opti-MEM, and NSFB transfection experiments were performed according to the following groupings.

[0062] Experimental group: The nano-drug delivery system obtained in Example 13 was transfected into NSFB cells; Positive control group: NSFB cells were transfected with RNAiMAX transfection reagent and GAPDH-targeting siRNA at a final concentration of 40 nM. Blank control group: NSFB cells were not treated in any way; Negative control group 1 (NT): NSFB cells were transfected with the transfection reagents RNAiMAX and peptide b-HKR (without siRNA); Negative control group 2 (NC): NSFB cells were transfected with the transfection reagent RNAiMAX (containing 40 nM Scramble siRNA) and the nano-drug delivery system obtained in Comparative Example 8, respectively.

[0063] Four to six hours after cell transfection, the culture medium for each group was replaced with DMEM complete medium, and the cells were cultured for another 24 hours before RNA was extracted and detected by qPCR.

[0064] The results showed that the silencing efficiency of the nano-drug delivery system containing BSA (1%) (88% ± 5.9%, n=3) was not significantly different from that of the nano-drug delivery system without BSA (91% ± 6.5%, n=3).

[0065] IV. Safety Evaluation of Nanoparticle Drug Delivery Systems 1. Cytotoxicity test 1) Cytotoxicity detection of b-HKR peptide Following the CCK-8 kit instructions, A549 or MCF-7 cell lines were suspended in complete medium (DMEM, 10% FBS) and seeded into 96-well plates (approximately 10,000 cells per well), then incubated in a CO2 incubator (Thermo Scientific, Forma Series II) for 24 hours. Subsequently, the medium was changed (serum-free or complete medium), and b-HKR peptide dissolved in DMEM was added to the corresponding wells at the final concentration gradient (0.15, 0.3, 0.6, 1.2, 2.4, 4.8, 9.6, 19.2, 38.4 µM, n = 6), and incubated for 18 h. Afterward, 10 µL of CCK-8 solution was added to each well, and the plate was incubated in a CO2 incubator for 2 hours. The absorbance at 450 nm (A450) was measured using a microplate reader (FLUOstar OPTIMA).

[0066] 2) Cytotoxicity detection of nano-drug delivery systems Following the cytotoxicity assay method in step 1), b-HKR / siRNA nanocomposite (containing 200 nMsiRNA, Comparative Example 1) and b-HKR / siRNA (containing 200 nMsiRNA, with 0.25% HSA added, Example 9) were added to the corresponding wells containing A549 or MCF-7 cells, respectively. Cells without any sample treatment were used as a negative control, and cell culture medium without cells and samples was used as a blank control. Each group was incubated for 18 h. Afterwards, 10 µL of CCK-8 solution was added to each well, and the wells were incubated in a CO2 incubator for 2 hours. The absorbance at 450 nm was measured using a microplate reader.

[0067] Under serum-free and serum-containing conditions, the cytotoxicity of free b-HKR peptide against A549 and MCF-7 cells was negligible in the concentration range of 0.15–1.2 µM. SPSS regression analysis was used to determine the half-maximal (50%) inhibitory concentration (IC50) of the peptide against A549 and MCF-7 cells. 50 The data are shown in Figure 11a). For b-HKR / siRNA (containing 200 nMsiRNA) and b-HKR / siRNA (containing 200 nMsiRNA, with 0.25% HSA added), no cytotoxicity was observed in either cell line, even in serum-free medium (Figure 11b).

[0068] 2. Hemolysis test 120 µL of erythrocyte (RBC) suspension and 15 µL of different concentrations (siRNA doses of 100, 200, 300, 400, and 500 nM) of test solution (free peptide b-HKR (siRNA dose corresponding to the concentration of free peptide b-HKR), b-HKR / siRNA nanocomposite, or b-HKR / siRNA nanocomposite with 0.25% HSA) were added to a centrifuge tube and mixed well. Red blood cells incubated in PBS served as a negative control, and red blood cells incubated in Triton X-100 (5%, v / v) served as a positive control. The mixture was incubated at 37°C for 1 h, then centrifuged (3000 rpm, 4°C, 5 min), and 100 µL of the supernatant was transferred to a 96-well plate. Hemoglobin concentration (OD) was measured using a microplate reader (FLUOstar OPTIMA). 544 value).

[0069] To observe changes in erythrocyte morphology and aggregation, erythrocytes were incubated with free peptide b-HKR, b-HKR / siRNA (containing 200 nMsiRNA) nanocomplex, or b-HKR / siRNA (containing 200 nMsiRNA, with 0.25% HSA added) nanocomplex at 37°C for 30 minutes. The samples were then placed on a glass slide and observed under a microscope (EVOS fl, Thermo Fisher) using a transparent model.

[0070] The hemolysis test results showed that, within the test concentration range of pH 7.2, free b-HKR peptides and b-HKR / siRNA exhibited certain hemolytic activity, while b-HKR / siRNA (with 0.25% HSA added) showed no hemolytic activity. Figure 12 Furthermore, free b-HKR peptides and b-HKR / siRNA cause erythrocyte swelling and aggregation. Figure 13 This may be due to the nonspecific binding between cationic peptides and negatively charged erythrocyte membranes. In contrast, b-HKR / siRNA (with 0.25% HSA added) did not cause erythrocyte swelling or aggregation at pH 7.2, indicating that the addition of the stabilizer improved the blood compatibility of b-HKR / siRNA.

[0071] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of this invention.

Claims

1. A nano-drug delivery system containing a stabilizer, characterized in that, include: A cationic peptide / siRNA complex and a particle size stabilizer; the cationic peptide / siRNA complex is formed by the interaction of a cationic peptide and siRNA through non-covalent forces, and the amino acid sequence of the cationic peptide is KKK(RH4RH4RH4RH4R). x The particle size stabilizer is a surfactant or a protein. Where x = 3 or 4.

2. The nano-drug delivery system containing a stabilizer according to claim 1, characterized in that, The amino acid sequence of the cationic polypeptide is KKK(RHHHHRHHHHRHHHHRHHHR)4.

3. The nano-drug delivery system containing a stabilizer according to claim 1, characterized in that, The surfactant is poloxamer 188, poloxamer 407, polyoxyethylene castor oil EL35, or distearate phosphatidylethanolamine-polyethylene glycol.

4. The nano-drug delivery system containing a stabilizer according to claim 1, characterized in that, The protein is bovine serum albumin, human serum albumin, or human transferrin.

5. The nano-drug delivery system containing a stabilizer according to any one of claims 1 to 4, characterized in that, It also includes freeze-drying protectants.

6. The nano-drug delivery system containing a stabilizer according to claim 5, characterized in that, The freeze-drying protectant is mannitol, glucose, or sucrose.

7. The nano-drug delivery system containing a stabilizer according to any one of claims 1 to 4, characterized in that, In nano-drug delivery systems containing stabilizers, the surfactant has a mass-volume percentage concentration of 0.1% to 1%, and the protein has a mass-volume percentage concentration of 0.5% to 5%.

8. The nano-drug delivery system containing a stabilizer according to any one of claims 1 to 4, characterized in that, The molar ratio of cationic polypeptide to siRNA is (3~24):

1.

9. A method for preparing the nano-drug delivery system containing a stabilizer according to any one of claims 1 to 8, characterized in that, A cationic peptide / siRNA nanocomposite was obtained by mixing cationic peptides and siRNAs through a microfluidic system; the cationic peptide / siRNA nanocomposite was then mixed with a particle size stabilizer to obtain a nano-drug delivery system containing the stabilizer.

10. The use of the nano-drug delivery system containing a stabilizer as described in any one of claims 1 to 8 in drug delivery.

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