An intracellular delivery protein vector of antibiotics and its synthesis method and application

Abstract

The application provides an antibiotic intracellular delivery protein carrier, a synthesis method and application thereof, and belongs to the technical field of peptide-containing drug preparations. The antibiotic intracellular delivery protein carrier is a ZF5.3-BSA complex, wherein the BSA is a drug carrier, the ZF5.3 is a transmembrane functional unit, and the BSA and the ZF5.3 are connected through biological orthogonal coupling. The antibiotic intracellular delivery protein carrier can not only retain the small molecule loading advantages of the BSA, but also endow the carrier with efficient transmembrane and intracellular delivery capabilities, and can expand the application scenarios of the ZF5.3 delivery platform from functional protein delivery to small molecule anti-infective drug delivery.

Description

Technical Field

[0001] This application relates to an intracellular delivery protein carrier for antibiotics, its synthesis method, and its application, belonging to the field of peptide-containing drug formulation technology. Background Technology

[0002] Intracellular pathogen infection is one of the core challenges in clinical anti-infective treatment. Among them, intracellular parasites, represented by methicillin-resistant Staphylococcus aureus (MRSA), can invade host cells and remain in the internal compartment. They can evade host immune clearance and avoid the killing effect of most antibiotics that are difficult to penetrate the cell membrane. They are an important cause of recurrent chronic infections and persistent sepsis.

[0003] Compared to protein drugs, small molecule antibiotics are generally more chemically stable and have better tissue diffusion capabilities. Some small molecules can enter cells through passive diffusion or transporter-mediated entry. However, not all small molecules can achieve effective therapeutic concentrations in intracellular pathogen-rich areas. In particular, small molecules with strong hydrophilicity and high polarity, such as cephalosporins, have extremely low transmembrane efficiency and are easily pumped out of cells by extracellular and extracellular transporters, resulting in effective intracellular concentrations that are far lower than in vitro inhibitory concentrations. Therefore, their in vitro activity cannot be directly equated to intracellular therapeutic effects. Insufficient intracellular accessibility is the core bottleneck limiting the application of these antibiotics to intracellular infections.

[0004] Therefore, although most antibiotics exhibit ideal antibacterial activity in vitro, their actual clinical efficacy is often limited by problems such as low intracellular delivery efficiency, insufficient effective intracellular concentration, and difficulty in enriching them in the subcellular compartments where pathogens reside. Developing antibiotic delivery systems that can efficiently target intracellular infection sites is of great significance for improving the clinical treatment of intracellular infections. Summary of the Invention

[0005] In view of this, the first objective of this application is to provide an intracellular delivery protein carrier for antibiotics, which enables the intracellular delivery of small molecule anti-infective drugs.

[0006] Specifically, this application is implemented through the following scheme: An intracellular delivery protein carrier for antibiotics is a ZF5.3-BSA complex, wherein BSA is the drug carrier, ZF5.3 is the transmembrane functional unit, and BSA and ZF5.3 are connected by bioorthogonal coupling.

[0007] Natural BSA has low intracellular incorporation efficiency, which cannot meet the requirements for intracellular delivery. By functionalizing BSA with the cell-penetrating peptide ZF5.3, which has efficient cellular uptake and endosome escape capabilities, the small molecule loading advantage of BSA can be retained, while endowing the carrier with efficient transmembrane and intracellular delivery capabilities. This can expand the application scenarios from functional protein delivery to small molecule anti-infective drug delivery.

[0008] The applicant's second objective is to provide a method for synthesizing the intracellular delivery protein carrier of the aforementioned antibiotic, wherein BSA and ZF5.3 are bio-orthogonally coupled via click chemistry.

[0009] More preferably: In the click chemistry, ZF5.3-N3 and DBCO-BSA are used as raw materials, and the two react to obtain a ZF5.3-BSA complex.

[0010] In the click chemistry, the molar ratio of ZF5.3-N3 to DBCO-BSA is 1:1.2 to 1.5.

[0011] As a preferred embodiment, the ZF5.3-N3 is prepared as follows: S1, under nitrogen purging, the 2-chlorotriphenylmethyl chloride resin is pretreated by cleaning. S2, the first C-terminal amino acid protected by Fmoc was dissolved in anhydrous DCM, pretreated 2-chlorotriphenylmethyl chloride resin was added, N,N-diisopropylethylamine was added dropwise, and after reaction under nitrogen protection, the unreacted resin active sites were blocked, piperidine solution was added to react and remove the Fmoc protecting group, dried and washed; according to the ZF5.3 sequence, the Fmoc protected amino acid and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate were dissolved together in anhydrous DMF, DIPEA was added to the solution and reacted with the resin after removing the Fmoc protecting group and drying and washing, and washed with DMF until coupled to the target site; S3, lysine (K(N3)) is modified at the C-terminus with Fmoc-Lys (N3)-OH by azidation. The coupling operation of S2 is repeated, and then the cycle of "deprotection → amino acid coupling" is repeated until the entire ZF5.3 sequence is coupled. The product at this time is denoted as ZF5.3-N3.

[0012] The sequence of ZF5.3 is YSCNVCGKAFVLSRHLNRHLRVHRRATK, and the complete sequence after coupling is Ac-YSCNVCGKAFVLSRHLNRHLRVHRRATK(N3)-NH2.

[0013] In the above process, in S2, a mixture of methanol and DCM is added to block the unreacted resin active sites. The volume ratio of methanol to DCM is 1:9 to 10.

[0014] As a preferred embodiment, the DBCO-BSA is prepared as follows: S1, Dissolve BSA in PBS buffer to obtain stock solution one; S2, DBCO-NHS were dissolved in DMSO to prepare Mother Solution II; S3, mix mother liquor one and mother liquor two, stir and react overnight at room temperature in the dark to obtain DBCO-BSA.

[0015] In the above process, the molar ratio of BSA to DBCO-NHS is 1:2.5 to 3.0.

[0016] The applicant's second objective is to provide an application of the aforementioned intracellular delivery protein carrier for antibiotics in the preparation of intracellular infection agents. In this application, the carrier is loaded or assembled with an antibiotic, which is any one of ceftriaxone (CRO), cephalexin, or ceftiofur. Ceftriaxone is a first-line third-generation cephalosporin clinically used to treat Staphylococcus aureus infections. It has a broad antibacterial spectrum and strong bactericidal activity. However, when used conventionally, this drug is highly hydrophilic and has a very weak ability to cross the membrane and enter the host cell, failing to effectively accumulate in the lysosomal compartment where the intracellular pathogen resides, thus limiting its therapeutic effect on intracellular infections. Using the delivery carrier of this application for loading and delivery of ceftriaxone eliminates the need for chemical structural modification of ceftriaxone, thereby enhancing its transmembrane ability and effectively maintaining its antibacterial activity. More preferably, the molar ratio of BSA to the antibiotic is 1:1 to 3.

[0017] This application describes an intracellular delivery system for bovine serum albumin (BSA) based on ZF5.3 modification. Through a DBCO-mediated click chemistry reaction, ZF5.3 is stably and efficiently bound to BSA to form a protein carrier. In intracellular infection applications using this carrier, ZF5.3 significantly enhances the intracellular internalization and transport efficiency of BSA, ensuring that the drug-loaded molecules are mainly distributed in the cytoplasm, effectively escaping lysosomes and achieving sustained release. This significantly enhances the therapeutic effect against infections such as Staphylococcus aureus. This delivery system is simple to construct, exhibits excellent biocompatibility, and provides a promising delivery strategy for treating intracellular bacterial infections and achieving intracellular targeted therapy, possessing broad clinical potential. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application.

[0019] Figure 1 This is a schematic diagram of the synthesis process of the vector in this application.

[0020] Figure 2 The figure shows the characterization results of ZF5.3 in this application. Part (A) is the high performance liquid chromatography (HPLC) analysis; part (B) is the mass spectrometry (MS) analysis.

[0021] Figure 3The figure shows the characterization of ZF5.3-BSA. Part (A) shows the UV-Vis absorption spectra of BSA, DBCO-NHS, and DBCO-BSA; Part (B) shows the SDS-PAGE electrophoresis results of BSA, DBCO-BSA, and ZF5.3-BSA.

[0022] Figure 4 Native-PAGE analysis of BSA-CRO and ZF5.3-BSA-CRO complexes.

[0023] Figure 5 The synthesis and FITC labeling characterization of ZF5.3-BSA-FITC conjugates are shown in Figure (A), which displays the fluorescence emission spectra of DBCO-BSA, FITC, and DBCO-BSA-FITC; and (B), which displays the SDS-PAGE electrophoresis results and fluorescence imaging of BSA, DBCO-BSA-FITC, and ZF5.3-BSA-FITC.

[0024] Figure 6 To evaluate the efficiency of ZF5.3-mediated BSA-FITC internalization in cells, part (A) of the figure shows laser confocal microscopy imaging of HeLa cells in different treatment groups (blue: Hoechst 33342 nuclear staining; green: FITC-labeled protein); part (B) shows the intracellular fluorescence intensity distribution detected by flow cytometry; and part (C) shows the quantitative statistics of mean fluorescence intensity (MFI) in different groups. Data are expressed as mean ± standard deviation (SD), with n = 3 biological replicates. ***P < 0.001; ns, no statistically significant difference.

[0025] Figure 7 Laser confocal microscopy images of 3T3 cells from different treatment groups (blue: Hoechst 33342 nuclear staining; green: FITC-labeled protein; scale bar = 20 μm).

[0026] Figure 8 Lysosome localization experiment and lysosome escape experiment (scale bar = 20 μm).

[0027] Figure 9 To illustrate the binding effect of CRO and BSA, part (A) of the figure shows the UV-Vis absorption spectra of ZF5.3-BSA, CRO, and ZF5.3-BSA-CRO; part (B) shows the molecular docking simulation results of CRO and BSA; part (C) shows the chromatogram of free CRO standard; and part (D) shows the chromatogram of the ultrafiltration flow-through component of the BSA-CRO mixture.

[0028] Figure 10The in vitro release curve of CRO from the ZF5.3-BSA-CRO complex in PBS (pH 7.4, 37 °C) is shown.

[0029] Figure 11 To validate the intracellular Staphylococcus aureus infection model, part (A) in the figure is a confocal microscopy image of Staphylococcus aureus labeled with HADA fluorescent probe, scale bar = 10 μm; part (B) is a confocal microscopy image of 3T3 cells infected with HADA-labeled Staphylococcus aureus, scale bar = 18 μm.

[0030] Figure 12 To evaluate the antibacterial effect of ZF5.3-BSA-CRO, part (A) of the figure shows the plate colony count results of Staphylococcus aureus in different treatment groups; part (B) shows the quantitative statistics of intracellular bacterial survival rate in each group.

[0031] Figure 13 Cell viability of 3T3 cells after treatment with ZF5.3, ZF5.3-BSA, and ZF5.3-BSA-CRO for 24 h, as determined by the CCK-8 assay. Detailed Implementation

[0032] To make the technical problems, technical solutions, and beneficial effects to be solved by this application clearer, the technical solutions in the embodiments of this application will be further described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain this application and are not intended to limit the technical solutions of this application. All other embodiments obtained by those skilled in the art based on the embodiments in this application without creative effort are within the scope of protection of this application.

[0033] Table 1: Reagents used in this embodiment .

[0034] Table 2: Instruments used in this embodiment .

[0035] Table 3: Strains used in this embodiment .

[0036] In the following examples, experimental data were processed, analyzed, and statistically analyzed using SPSS software. Independent samples t-tests were used to analyze differences between two groups, and one-way ANOVA was used to analyze significant differences among multiple groups. Wherein, * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; NS indicates no significant difference.

[0037] This embodiment provides an intracellular antibiotic delivery protein carrier, which is a ZF5.3-BSA complex, wherein BSA is the drug carrier, ZF5.3 is a transmembrane functional unit, and BSA and ZF5.3 are connected by bioorthogonal coupling. The embodiments of this application are described below with reference to the accompanying drawings.

[0038] See Figure 1 , Figure 1 A schematic diagram of the synthesis process of the vector in this embodiment is shown. The specific process is described as follows: Step 1, Preparation of ZF5.3-N3: S1, under nitrogen purging, 2-chlorotriphenylmethyl chloride resin is placed in a solid-phase synthesis tube, soaked in anhydrous dichloromethane, dried, and pretreated by alternating cleaning with N,N-dimethylformamide / DCM.

[0039] S2, the first C-terminal amino acid protected by Fmoc was dissolved in anhydrous DCM, and pretreated 2-chlorotriphenylmethyl chloride resin was added. N,N-diisopropylethylamine (DIPEA) was added dropwise, and the reaction was carried out under nitrogen protection. The unreacted resin active sites were blocked with a mixture of methanol and DCM (volume ratio 1:9), and washed with DMF. Piperidine DMF solution was added to react and remove the Fmoc protecting group. After drying, it was washed with DMF. Then, according to the ZF5.3 sequence, the Fmoc protected amino acid and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate were taken sequentially and dissolved together in anhydrous DMF. The resulting solution was added to DIPEA and reacted with 2-chlorotriphenylmethyl chloride resin, and then washed with DMF until it was coupled to the target site.

[0040] S3, replace ordinary lysine with Fmoc-Lys (N3)-OH, repeat the coupling operation of S2, and then repeat the cycle of "deprotection → amino acid coupling" until the full sequence of ZF5.3 is coupled to obtain ZF5.3-N3.

[0041] Step 2, Preparation of DBCO-BSA: BSA is reacted with DBCO-NHS to introduce orthogonal reactive groups into DBCO. The prepared DBCO-BSA is then coupled with ZF5.3 via a click reaction to obtain ZF5.3-BSA. Specific steps: BSA is dissolved in pH 8.0 PBS buffer to prepare a 10 mg / mL stock solution; DBCO-NHS is dissolved in DMSO to prepare a 5 mM stock solution. BSA and DBCO-NHS are mixed at a molar ratio of 1:3, and the mixture is stirred overnight at room temperature in the dark to generate DBCO-BSA. After the reaction, the mixture is purified by centrifugation using a 10 kDa ultrafiltration tube to remove unreacted small molecules.

[0042] Step 3, carrier synthesis: ZF5.3-N3 obtained in Step 1 was added to DBCO-BSA obtained in Step 2, and the reaction was carried out at room temperature for 1 h at a molar ratio of ZF5.3-N3 to DBCO-BSA of 1:1.2 to obtain ZF5.3-BSA complex.

[0043] The products of the above process are characterized as follows: (1) Characterization of ZF5.3-BSA coupling

[0044] ZF5.3 was prepared via Fmoc solid-phase peptide synthesis. To facilitate bioorthogonal coupling with bovine serum albumin (BSA), an azide-modified lysine residue was introduced at the C-terminus during synthesis. The purified ZF5.3-N3 was characterized by analytical HPLC and LC-MS, with results as follows: Figure 2 As shown: HPLC chromatogram (see...) Figure 2 Part (A) of the spectrum shows a single main peak at a retention time of 13.82 minutes, with a peak area ratio of 95.86%, indicating high product purity; the mass spectrum (see [reference]) Figure 2 In part (B) of the diagram, multiple charged ions were observed at m / z 1130.90, 848.50, and 679.00, corresponding to [M+3H], respectively. 3+ [M+4H] 4+ and [M+5H] 5+ The calculated molecular weight of 3389.87 Da is in high agreement with the theoretical value of 3389.93 Da, confirming that the synthesized small protein is consistent with the designed sequence. Combined HPLC and mass spectrometry results indicate that the above process successfully prepared high-purity ZF5.3 suitable for subsequent experiments.

[0045] Before coupling with ZF5.3, BSA was first modified with DBCO. DBCO-modified BSA (i.e., DBCO-BSA) was prepared by reacting BSA with DBCO-NHS and then ultrafiltration. The UV-Vis absorption spectroscopy characterization results are as follows: Figure 3 As shown: Pure BSA exhibits a typical protein absorption peak at 280 nm, while DBCO-NHS shows two absorption peaks at 295 and 310 nm. The absorption spectrum of BSA after DBCO-NHS coupling shows the typical absorption peaks of the protein core DBCO, indicating the successful preparation of the coupling compound DBCO-BSA. Subsequently, ZF5.3-BSA was prepared by coupling ZF5.3-N3 with DBCO-BSA using the click reaction of DBCO with azide (see...). Figure 3 (Part A) of the text). The SDS-PAGE electrophoresis results of the protein are as follows: Figure 3As shown in section (B), BSA exhibits a single, clear band at approximately 66 kDa. After modification with DBCO, the DBCO-BSA band shifts slightly upward compared to natural BSA. However, after coupling with ZF5.3 (theoretical molecular weight 3.38 kDa), the ZF5.3-BSA complex band shifts upward to approximately 70 kDa, exhibiting a single band without obvious impurities, indicating that the coupling reaction between ZF5.3 and DBCO-BSA proceeds efficiently.

[0046] Furthermore, the homogeneity of the complex was further verified by natural polyacrylamide gel electrophoresis combined with silver staining. SDS-PAGE electrophoresis was performed using a 12% separating gel and a 5% stacking gel, with the stacking gel at 80 V and the separating gel at 120 V. After electrophoresis, fluorescence imaging or Coomassie brilliant blue staining was performed. Results are as follows: Figure 4 As shown, both BSA-CRO and ZF5.3-BSA-CRO migrated as single, clear bands without tailing, indicating that these complexes have intact and homogeneous structures.

[0047] (2) Assessment of intracellular endocytosis efficiency

[0048] To facilitate observation of endocytosis efficiency via fluorescence signals, we first prepared DBCO-BSA-FITC by modifying BSA with FITC. Subsequently, we further conjugated this to obtain ZF5.3-BSA-FITC: FITC was dissolved in DMSO to prepare a 5 mM stock solution, which was then added at a BSA to FITC molar ratio of 1:3. The mixture was stirred at room temperature in the dark for 6 h. After the reaction, unreacted FITC was removed by centrifugation using a 10 kDa ultrafiltration tube, yielding purified DBCO-BSA-FITC. DBCO-BSA-FITC was then reacted with ZF5.3-N3 protein at a molar ratio of 1.2:1 at room temperature for 1 h to form the ZF5.3-BSA-FITC complex.

[0049] Figure 5 The fluorescence emission spectrum and SDS-PAGE electrophoresis results confirmed the successful preparation of the fluorescently labeled protein.

[0050] The effect of ZF5.3 modification on endocytosis was investigated using laser confocal microscopy. The results are as follows: Figure 6 As shown: Control group ( Figure 6In the Control group (part A), no obvious green fluorescence signal was observed in the cells; only weak punctate fluorescence was visible in the DBCO-BSA-FITC group, indicating that unmodified BSA could not effectively penetrate the cell membrane and enter the cell; while the ZF5.3-BSA-FITC group showed strong and uniform green fluorescence, with the signal mainly distributed in the cytoplasm and perinuclear region, directly confirming that ZF5.3 can significantly enhance the internalization of BSA. This result indicates that ZF5.3 can significantly improve the endocytosis efficiency of BSA. Flow cytometry was used to quantitatively analyze the intracellular fluorescence intensity to determine the ZF5.3-mediated cell delivery efficiency. The results showed that the mean intracellular fluorescence intensity (MFI) of the ZF5.3-BSA-FITC treatment group was significantly higher than that of the DBCO-BSA-FITC group, with an enhancement efficiency of more than 5 times, indicating that ZF5.3 modification significantly enhances the cellular uptake of BSA, laying a key foundation for subsequent intracellular antibiotic delivery. Figure 6 (Parts (B) and (C)).

[0051] The fluorescently labeled ZF5.3-BSA-FITC complex was used for subsequent cellular uptake experiments to trace the cellular uptake of BSA, and the intracellular distribution of BSA protein was directly reflected by the fluorescence signal: The following samples were diluted with PBS (pH 7.4) to appropriate concentrations (DBCO-BSA-FITC and DBCO-BSA were 15 μM, and 45 μM free FITC was used as a control), and 150 μL of each was added to the microplate reader. The emission spectrum of 500–600 nm was recorded using the microplate reader under the conditions of excitation wavelength of 488 nm and slit width of 20 nm.

[0052] Taking the cell uptake capacity of ZF5.3 in 3T3 cells as an example: HeLa cells were seeded in confocal culture dishes (for confocal imaging) and 12-well plates (for flow cytometry). When the cell confluence reached 70%, DBCO-BSA-FITC and ZF5.3-BSA-FITC diluted with DMEM medium were added to a final concentration of 8 μmol / L (based on protein). The cells were incubated at 37°C for 4 hours. After incubation, the cells were washed three times with PBS, stained with Hoechst 33342 staining solution in the dark for 15 minutes, and then washed twice with PBS. The cells in the confocal culture dishes were directly placed under a laser confocal microscope to observe the fluorescence distribution of FITC (green) and Hoechst (blue). The cells in the 12-well plates were digested with trypsin, collected by centrifugation, resuspended in PBS, and the fluorescence intensity of the FITC channel was detected using flow cytometry. Using untreated cells as a negative control, the mean fluorescence intensity (MFI) of each treatment group was calculated to evaluate the enhancement of BSA intracellular delivery efficiency by ZF5.3. 3T3 cells were treated using the same method. Results are as follows: Figure 7As shown, the ZF5.3-BSA-FITC complex group significantly enhanced cellular uptake capacity, consistent with the results obtained in HeLa cells.

[0053] To directly verify its lysosomal escape ability, a colocalization experiment was performed in 3T3 cells. 3T3 cells were plated, sample was added, incubated, washed with PBS, and fluorescence was observed using confocal microscopy. Then, 1× live cell staining solution and 50 nM red lysosomal fluorescent probe were added and co-incubated with the cells for 30 min, and fluorescence was observed again. The results of the fluorescence colocalization analysis are as follows: Figure 8 As shown, 3T3 cells treated with ZF5.3-BSA-FITC exhibit diffuse green fluorescence in the cytoplasm, and the colocalization with lysosomal markers is incomplete, indicating that they have the ability to effectively escape lysosomes.

[0054] In summary, these findings reflect the level of protein carrier internalization and demonstrate the versatility of this delivery system across cell types, which has important implications for future treatment of infections in different host cells.

[0055] (3) Preparation of BSA complex loaded with CRO and determination of drug loading

[0056] After verifying the efficient endocytosis capacity of ZF5.3-BSA, we further investigated its drug loading capacity. Ceftriaxone (CRO), a third-generation cephalosporin antibiotic, was selected as the model antibiotic, and a drug loading system was constructed by co-incubating CRO with ZF5.3-BSA: BSA and CRO were dissolved separately in PBS buffer. They were then mixed at a molar ratio of BSA:CRO = 1:3 and incubated overnight in the dark to allow for sufficient binding of CRO and BSA. After incubation, the BSA-CRO mixture was transferred to a 10 kDa ultrafiltration centrifuge tube for ultrafiltration separation. Equal concentrations of BSA and BSA-CRO were diluted with PBS, and the absorbance at 241 nm was measured. BSA served as a background control. The concentration of CRO in the complex was calculated based on the CRO standard curve, thus determining the drug loading amount. Simultaneously, HPLC was used to determine the total CRO load, with a free CRO solution prepared with the same initial total CRO amount as a total CRO control. The BSA-CRO ultrafiltration flow-through solution and the 3x free CRO control were diluted with PBS or brought to the same volume. All samples were filtered through a filter membrane before being injected into HPLC for analysis, and the peak areas of the CRO characteristic peaks were recorded.

[0057] The combined effect of ZF5.3-BSA-CRO is as follows: Figure 9As shown in Figure 3.9 (CD), UV-Vis absorption spectroscopy revealed a significant increase in absorbance of ZF5.3-BSA (ZF5.3-BSA-CRO) with adsorbed CRO in the 250-290 nm range, attributed to the superposition effect of the CRO absorption spectrum. The binding ratio of ZF5.3-BSA to CRO was calculated based on the changes in the absorption spectrum. The results showed that the ratio of CRO to BSA in ZF5.3-BSA-CRO was approximately 1.02 ± 0.05, meaning that each BSA molecule bound to approximately one CRO molecule. To further determine the binding ratio of BSA to CRO, the content of unbound CRO was detected using ultrafiltration combined with high-performance liquid chromatography (HPLC). The HPLC results, shown in Figure 3.9 (CD), indicate that CRO exhibited a characteristic peak at the same retention time. The peak area of ​​the 3x free CRO control was 977.204, while the peak area of ​​CRO in the BSA–CRO ultrafiltration flow-through was 598.350. Based on the peak area ratio, unbound CRO accounts for approximately 61.2% of the total CRO, therefore approximately 38.8% of the CRO is bound to BSA. Since the initial molar ratio of BSA:CRO = 1:3, it is calculated that each BSA molecule binds to an average of approximately 1.16 CRO molecules, meaning the binding ratio of BSA to CRO is approximately 1:1.

[0058] Next, molecular docking was used to analyze the binding of BSA and CRO. The crystal structure of BSA was downloaded from the RCSB PDB database (PDB ID: 8wdd, resolution 2.47 Å). PyMOL was used to remove water of crystallization, small ligands, and heteroatoms, and hydrogen atoms were added to the protein using AutoDockTools. The two-dimensional structure of CRO was drawn using ChemDraw and converted to PDB format using Open Babel. Based on the BSA structure (PDB ID: 8wdd), a 16 Å docking box was set centered on the hydrophobic cavity of BSA, and semi-flexible molecular docking was performed using AutoDock Vina. After docking, the predicted binding affinity (unit: kcal / mol) was extracted to determine the optimal binding conformation. PyMOL and Discovery Studio were used to analyze the non-covalent interactions between BSA and CRO, including hydrogen bonds, hydrophobic interactions, and van der Waals forces.

[0059] The molecular docking simulation results between CRO and BSA are as follows: Figure 9As shown in section (B), CRO can stably bind to the hydrophobic cavity of the BSA subdomain. The predicted binding free energy calculated by AutoDock Vina is -3.98 kcal / mol, indicating that the two can spontaneously form a relatively stable complex with good conformational matching and no obvious steric hindrance. Detailed interaction analysis shows that CRO can form multiple interactions with several key amino acid residues of BSA. Among them, the side chain of CRO forms a hydrogen bond network with residues such as TRP132, GLU128, GLU15, and LYS129, while the cephalosporin nucleus forms extensive hydrophobic interactions with residues such as LEU281 and PRO279. The above synergistic effect drives the stable loading of CRO into the BSA cavity, further confirming at the molecular level that CRO and BSA bind in a 1:1 stoichiometric ratio. These results indicate that the delivery strategy constructed in this application has the potential to become a universal platform for intracellular antibiotic delivery.

[0060] The in vitro CRO release curve of the ZF5.3-BSA-CRO complex in PBS (pH 7.4, 37 °C) is as follows: Figure 10 As shown, CRO exhibits sustained release characteristics from the ZF5.3-BSA-CRO complex, with a cumulative release rate of approximately 70% after 8 hours. This release rate is beneficial for maintaining effective intracellular drug concentrations. Therefore, the CRO-BSA binding results in this application demonstrate the feasibility of drug delivery using the proposed vector, indicating that the constructed delivery strategy can serve as a universal platform for intracellular antibiotic delivery.

[0061] (4) Establishment of an intracellular bacterial infection model

[0062] An intracellular infection model was established using Staphylococcus aureus (ATCC 29213) and 3T3 cells. Staphylococcus aureus was inoculated into LB broth and cultured at 37 °C with shaking at 220 r / min until the logarithmic growth phase (OD2). 600 = 0.6-0.8). After washing twice with PBS, the bacterial culture was resuspended in antibiotic-free DMEM medium for later use. 3T3 cells were seeded in culture plates or confocal culture dishes and cultured at 37 ℃ and 5% CO2 until confluence reached 70%–80%. The medium was discarded, and bacterial culture was added at the desired multiplicity of infection (MOI) of 50, and incubated at 37 ℃ for 1 h. After incubation, the cells were washed three times with PBS to remove unbound extracellular bacteria, and then complete medium containing 100 μg / mL gentamicin was added, and incubation was continued at 37 ℃ for 30 min to kill any remaining extracellular bacteria. Finally, the gentamicin medium was discarded, and the cells were washed three more times with PBS for subsequent experiments.

[0063] In the experiment of observing intracellular infection with fluorescence imaging, Staphylococcus aureus was subjected to metabolic labeling and fluorescence staining using HCC-Amino-D-alanine hydrochloride (HADA) to facilitate the observation of bacterial infection of cells.

[0064] First, Staphylococcus aureus was fluorescently labeled using a metabolic labeling method. The results are as follows: Figure 11 The results show that HADA-labeled Staphylococcus aureus exhibits bright blue fluorescence (see [link]). Figure 11 (Part A)); Subsequently, HADA-labeled Staphylococcus aureus was used to infect 3T3 cells, and the distribution of bacteria within the cells was observed using laser confocal microscopy. A large number of blue fluorescently labeled bacteria were distributed in a dotted pattern within the 3T3 cells, indicating that Staphylococcus aureus had successfully invaded the host cells (see Part A). Figure 11 (Part (B) of the report) confirms the successful construction of the intracellular infection model.

[0065] (5) Intracellular antibacterial experiment

[0066] After constructing the intracellular infection, intracellular surviving bacteria were quantitatively analyzed using plate colony counting. Different samples were added for treatment: PBS, free CRO, DBCO-BSA-CRO, and ZF5.3-BSA-CRO, with a final CRO concentration of 10 μM for each sample. The cells were then incubated at 37°C for 4 h. After incubation, the supernatant was discarded, and the cells were washed three times with PBS. Next, 200 μL of 0.5% Triton X-100 lysis buffer (dissolved in PBS) was added to each well, and the cells were lysed on ice for 15 min, repeatedly pipetting to ensure complete lysis. The lysis buffer was serially diluted, and equal volumes were plated onto LB agar plates and incubated at 37°C for 12–16 h. Plates with colony counts between 100 and 500 were used for counting. Since all samples (PBS control group and each treatment group) used the same dilution factor and plate volume, the inhibition rate could be directly calculated from the average colony count. Each experiment had three replicates, each independently repeated three times.

[0067] .

[0068] The antibacterial effect of ZF5.3-BSA-CRO is as follows: Figure 12 As shown: the blank control group plates showed a large number of colonies growing; the CRO treatment group showed no significant change in colony count compared to the control group; the DBCO-BSA-CRO group had a slightly lower colony count than the free CRO group, while the ZF5.3-BSA-CRO group had a significantly lower colony count (see...). Figure 12 (Part A) of the text. Figure 1The bar chart in section (B) visually demonstrates the above-mentioned antibacterial effect: the intracellular bacterial survival rate of the ZF5.3-BSA-CRO group was significantly lower than that of the free CRO group and the DBCO-BSA-CRO group.

[0069] Intracellular antibacterial experiments validated the functional delivery value of the ZF5.3-BSA-CRO system. The free CRO group showed poor efficacy, highlighting the critical bottleneck of insufficient drug accessibility in the treatment of intracellular infections. The DBCO-BSA-CRO group exhibited some antibacterial effect, indicating that BSA, as a protein carrier, can carry some CRO into cells via endocytosis, but its delivery efficiency remains limited. In contrast, the ZF5.3-BSA-CRO group showed a significant reduction in intracellular bacteria, indicating that ZF5.3 modification significantly enhanced the cellular uptake and intracellular delivery capacity of the BSA-CRO complex, thereby improving the killing effect of CRO on intracellular bacteria. This result corroborates the aforementioned ZF5.3-BSA intracellularization experiments, demonstrating that ZF5.3-functionalized BSA not only improves the level of protein carrier entry into cells but also translates into actual intracellular antibacterial effects.

[0070] (6) Cytotoxicity test

[0071] To further evaluate the biocompatibility of the ZF5.3-BSA-CRO composite system, we used the CCK-8 assay to detect its effect on 3T3 cell viability. 3T3 cells were seeded at a density of 5,000 cells per well in 96-well plates, with 100 μL of DMEM complete medium containing 10% FBS added to each well. After culturing for 24 h at 37°C in a 5% CO2 atmosphere, the original medium was discarded and replaced with fresh DMEM medium containing different samples (including the control group, ZF5.3 group, ZF5.3-BSA group, and ZF5.3-BSA-CRO group). The concentrations of relevant substances were kept consistent across treatment groups, with a final CRO concentration of 20 μM in the CRO-containing group; each experimental group had at least five replicate wells. Blank wells (containing only medium and CCK-8 reagent, without cells) were used for background subtraction. After 24 h of treatment, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated at 37°C for another 1 h. Finally, the absorbance of each well was measured at 450 nm using a microplate reader, and the cell viability (%) was calculated using this formula: .

[0072] The results are as follows Figure 13As shown, compared with the control group, the viability of 3T3 cells remained high after 24 hours of treatment with ZF5.3, ZF5.3-BSA, and ZF5.3-BSA-CRO, which may be attributed to the nutrient-enhancing effect of BSA itself. This indicates that within the tested concentration range, none of the treatment groups significantly inhibited the metabolic or proliferative activity of 3T3 cells. Furthermore, even when the final CRO concentration reached 20 μM, the ZF5.3-BSA-CRO group maintained good cell viability, indicating that CRO loading or BSA modification did not induce significant cytotoxicity. These results demonstrate that ZF5.3-BSA-CRO has good biocompatibility, providing a safety basis for its subsequent biomedical applications.

[0073] The above-described embodiments are merely illustrative of several feasible implementations of the present invention, and their descriptions are relatively specific and detailed. However, they should not be construed as limiting the scope of the present invention, nor are the embodiments intended to limit the scope of protection in the claims of the present invention. For those skilled in the art, various modifications and improvements can be made without departing from the concept of the present invention. All equivalent implementations or changes that do not depart from the present invention should be included in the technology of the present invention.

Claims

1. An intracellular delivery protein carrier for antibiotics, characterized in that: It is a ZF5.3-BSA complex, in which BSA is a drug carrier, ZF5.3 is a transmembrane functional unit, and BSA and ZF5.3 are connected by bioorthogonal coupling.

2. A method for preparing the intracellular delivery protein carrier of the antibiotic according to claim 1, characterized in that: BSA and ZF5.3 achieve bioorthogonal coupling through click chemistry.

3. The preparation method according to claim 2, characterized in that: Using ZF5.3-N3 and DBCO-BSA as raw materials, a ZF5.3-BSA complex was obtained through a click chemical reaction.

4. The preparation method according to claim 3, characterized in that: The molar ratio of ZF5.3-N3 to DBCO-BSA is 1:1.2 to 1.

5.

5. The preparation method according to claim 2, characterized in that, The ZF5.3-N3 is prepared as follows: S1, Under nitrogen purging, the 2-chlorotriphenylmethyl chloride resin is cleaned to complete the pretreatment; S2, the first C-terminal amino acid protected by Fmoc was dissolved in anhydrous DCM, pretreated 2-chlorotriphenylmethyl chloride resin was added, N,N-diisopropylethylamine was added dropwise, and after reaction under nitrogen protection, the unreacted resin active sites were blocked, piperidine solution was added to remove the Fmoc protecting group, the solution was dried and washed; according to the ZF5.3 sequence, the Fmoc protected amino acid and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate were dissolved together in anhydrous DMF, DIPEA was added to the solution and reacted with the resin after removing the Fmoc protecting group and drying and washing, and washed with DMF until coupled to the target site; S3, lysine is modified with Fmoc-Lys-OH at the C-terminus by azidation, and the coupling operation of S2 is repeated. Then the cycle of "deprotection → amino acid coupling" is repeated until the entire ZF5.3 sequence is coupled. The product is denoted as ZF5.3-N3.

6. The preparation method according to claim 2, characterized in that, The DBCO-BSA is prepared as follows: S1, Dissolve BSA in PBS buffer to obtain stock solution one; S2, DBCO-NHS were dissolved in DMSO to prepare Mother Solution II; S3, mix mother liquor one and mother liquor two, stir and react overnight at room temperature in the dark to obtain DBCO-BSA.

7. The preparation method according to claim 6, characterized in that: The molar ratio of BSA to DBCO-NHS is 1:2.5 to 3.

0.

8. The use of the intracellular delivery protein carrier of claim 1 in the preparation of an intracellular infection agent, wherein the carrier is loaded with or assembled with an antibiotic.

9. The application according to claim 8, characterized in that: The antibiotic is any one of ceftriaxone, cefalexin, or ceftiofur.

10. The application according to claim 8, characterized in that: The molar ratio of BSA to antibiotics is 1:1 to 3.

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