Erythrocyte drug delivery system modified with indocyanine green derivative dyes

Abstract

The application discloses a red blood cell drug delivery system of a modified indocyanine green derivative dye, the drug delivery system is loaded with a photosensitizer IR808 on the surface of red blood cells and is wrapped with hematin chloride in the interior of the red blood cells. The application also discloses a use of the near-infrared light response red blood cell drug delivery system of the modified indocyanine green derivative dye in preparation of a drug for treating periodontitis. The drug delivery system can be used for PDT anti-infection treatment in the assistance of NIR laser according to the infection condition of a clinical periodontal disease, and can promote the release of hemin and eliminate potential intracellular bacterial infection. The drug delivery system can be used for intrabag administration in a periodontal pocket according to the clinical requirement of different stages of periodontitis, so that a patient can be free from the trauma of periodontal surgery.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and relates to a red blood cell drug delivery system modified with indocyanine green derivative dyes and its use in the preparation of antibacterial drugs for periodontitis. Specifically, it relates to red blood cell photoresponsive drug delivery particles encapsulating heme chloride and their use in the preparation of antibacterial drugs for periodontitis. Background Technology

[0002] Periodontitis is a nonspecific inflammatory disease of the normal oral flora, which can cause destruction of tooth-supporting tissues, leading to tooth loosening and loss. Periodontitis has a high incidence rate, affecting not only oral function but also threatening overall health. The primary task in treating periodontitis is to thoroughly eliminate the source of infection within the tissues. Clinically, this is mainly achieved through non-invasive treatment methods that combine basic periodontal therapy with antibiotics and photosensitizers to assist in sterilization. However, antibiotic use carries the potential risk of drug resistance, and addressing resistance is costly and time-consuming. Secondly, the oral cavity is a unique bacterial environment, posing a risk of reinfection during tissue healing, requiring repeated intracellular administration, which is detrimental to periodontal tissue healing. Furthermore, the survival of intracellular pathogens is a major factor in chronic or recurrent infections, potentially leading to the failure of host defense and anti-infective therapy. Therefore, in periodontal treatment, simple antibacterial therapy cannot effectively regulate local immunity, nor can it eliminate intracellular pathogens, thus failing to provide a suitable regenerative microenvironment for stem cells and enabling them to functionally reconstruct periodontal tissues.

[0003] To design ideal periodontal drugs that achieve periodontal tissue regeneration while controlling infection, the following factors must be considered simultaneously: ① Biosafety properties: good biocompatibility and biodegradability, and long retention time in local tissues; ② Antibacterial properties: non-specific and thorough elimination of pathogens, and non-invasive bactericidal response based on subsequent infection conditions; ③ Chronic inflammatory microenvironment regulation properties: drug delivery promotes the death of intracellular bacteria, thereby activating cellular antioxidant signaling pathways to clear excess ROS generated during antibacterial processes, regulating immune imbalance, and eliminating the adverse effects of endogenous and exogenous toxins on the tissue environment.

[0004] Photoresponsive antibacterial therapy is a newly emerging non-specific antibacterial treatment method in recent years. It utilizes the photodynamic properties of photosensitizers under near-infrared laser irradiation to kill bacteria. It has advantages such as low toxicity, high spatiotemporal controllability, and low invasiveness, and has been used in research on the treatment of periodontal pathogens, drug-resistant bacteria, and bacterial biofilm-related infections. The photosensitizer IR808 is a near-infrared indole cyanine dye. As a derivative of ICG, it is water-soluble, photostable, and has a broad absorption band. It can trigger photodynamic therapy (PDT) modal treatment and can be considered a potential photosensitizer for effectively controlling periodontal pathogen infections.

[0005] Hemin chloride is an FDA-approved drug for the treatment of acute intermittent porphyria attacks, with mild side effects and no impact on liver, kidney, or coagulation function. Currently, repurposing existing drugs is an extremely attractive strategy, offering patients more effective options and providing faster, safer, and cheaper preclinical and clinical validation pathways. Hemin is an iron-binding porphyrin, and its induction of ferroptosis stress in tumor cells and bacteria as an iron overload agent has been considered an effective approach for antitumor and antibacterial purposes. Hemin possesses catalase-mimicking activity, which can enhance the efficacy of photodynamic therapy. Furthermore, low levels of hemin are an inducer and substrate of heme oxygenase-1 (HO-1), which breaks it down into biliverdin, bilirubin, carbon monoxide, and Fe. 2+ By controlling the dosage of Hemin through delivery methods, it has potential periodontal therapeutic effects.

[0006] As a small molecule compound, IR808 is difficult to retain in periodontal pockets when used directly, exhibits poor tissue infiltration, and is easily metabolized. While Hemin is chemically stable, its water solubility is extremely low, and it readily polymerizes, limiting its bioavailability. Therefore, a suitable biological delivery system is crucial. The unique characteristics of the oral cavity (such as friction from chewing food against the gums and rinsing with saliva) impose special requirements on drug delivery systems used in the oral cavity. Summary of the Invention

[0007] The inventors combined the advantages of erythrocyte (ER) drug delivery and photoresponsive drug-switching systems to construct erythrocytes loaded with Hemin and attach IR808 to the surface of the erythrocytes, obtaining a drug delivery system (denoted as Hemin@ER-IR808). This drug delivery system can perform PDT (photodynamic therapy) with NIR laser assistance, based on the clinical periodontal infection status, while simultaneously promoting the release of Hemin and eliminating potential intracellular bacterial infections. This drug delivery system enables intrapocket drug delivery according to the clinical needs of different stages of periodontitis, avoiding the trauma of periodontal surgery for patients. The implementation of this invention is expected to promote technological advancements in intelligent periodontal drug delivery systems.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A red blood cell drug delivery system modified with indocyanine green derivative dye (denoted as Hemin@ER-IR808) encapsulates heme chloride inside red blood cells and loads the photosensitizer IR808 on the surface of red blood cells.

[0010] Preferably, the erythrocyte drug delivery system modified with indocyanine green derivative dye is prepared by the following method: first, erythrocytes and heme chloride are used as raw materials, and erythrocyte hypotonic-reblocking method is used to prepare erythrocytes loaded with heme chloride (Hemin@ER); Hemin@ER is resuspended in PBS buffer to obtain Hemin@ER suspension; Hemin@ER suspension is mixed with IR808 solution and stirred at 0-4°C; after the reaction is completed, IR808 that has not been modified to the surface of erythrocytes is washed with PBS buffer to remove it, thereby obtaining the erythrocyte drug delivery system modified with indocyanine green derivative dye.

[0011] Another object of the present invention is to provide a method for preparing the erythrocyte drug delivery system of the modified indocyanine green derivative dye, comprising:

[0012] Step (1): Prepare heme chloride solution; prepare 50% v / v red blood cell suspension using PBS buffer;

[0013] Step (2): Prepare hemin-loaded red blood cells (Hemin@ER) using the hypotonic-reblocking method:

[0014] Hypotonic treatment: Red blood cells were resuspended in heme chloride solution at a volume ratio of 1:1 to obtain a cell suspension mixed with heme chloride; the cell suspension mixed with heme chloride was placed in a dialysis bag, and the dialysis bag was placed in the hypotonic buffer at a volume ratio of 1:1 to 1:2 to obtain a cell suspension mixed with heme chloride; the cell suspension was placed in a dialysis bag, and the bag was placed in the hypotonic buffer at a volume ratio of 1:1 to 1:2 to be hypotonic for 12 to 24 hours at 0 to 4°C.

[0015] Hypertonic: Transfer the dialysis bag to a resealed buffer solution for hypertonic treatment and place it at 36–40°C for 30–45 minutes;

[0016] Isotonic: Wash with PBS buffer to remove unencapsulated heme chloride, obtaining heme-encapsulated red blood cells;

[0017] Step (3): Resuspend the heme-coated red blood cells in PBS buffer to obtain a heme-coated red blood cell suspension; dissolve IR808 in PBS buffer to obtain an IR808 solution; mix the IR808 solution with the heme-coated red blood cell suspension and stir at 200-300 rpm for 1-2 hours at 0-4°C; wash with PBS buffer to remove IR808 that failed to be modified onto the surface of the red blood cells to obtain a red blood cell drug delivery system modified with indocyanine green derivative dye.

[0018] In step (1), the concentration of the heme chloride solution is 2-3 mg / mL, preferably 3 mg / mL.

[0019] The preparation of the heme chloride solution includes: dissolving heme chloride in DMSO to obtain a heme chloride stock solution with a concentration of 30 mg / mL, then adding PBS buffer (pH = 7.4) for dilution, and adjusting the pH to 7.2-7.4 using Na2HPO4 to obtain a heme chloride solution with a concentration of 3 mg / mL.

[0020] Specifically, dissolve 3 mg of heme chloride in 100 μL of DMSO, then add 900 μL of PBS buffer (pH = 7.4) to dilute, and adjust the pH to 7.2-7.4 using Na2HPO4 to obtain a heme chloride solution with a concentration of 3 mg / mL.

[0021] The preparation of the red blood cell suspension includes: centrifuging SD rat venous blood at 1200 rpm for 10 minutes, discarding the upper plasma layer, collecting the bottom red blood cells, and preparing a red blood cell suspension with a concentration of 50% v / v using 500 μL PBS buffer.

[0022] In step (2), preferably, the volume ratio of the mixed heme chloride cell suspension to the hypotonic buffer is 1:1.

[0023] Preferably, the temperature of the hypoosmotic treatment is 4°C; and the duration of the hypoosmotic treatment is 12 hours.

[0024] The molecular cutoff of the dialysis bag is 14 kDa.

[0025] The hypotonic buffer solution is: 15mM NaH2PO4·2H2O, 15mM NaHCO3, 2mM ATP, 3mM reduced glutathione, 20mM glucose, 5mM NaCl, 72mOsm / kg, pH 8.

[0026] The volume ratio of the mixed heme chloride cell suspension to the resealing buffer is 1:25.

[0027] The resealing buffer solution is composed of: 250 mM NaCl, 12.5 mM glucose, 12.5 mM sodium pyruvate, 12.5 mM inosine, 12.5 mM NaH2PO4·2H2O, 0.63 mM adenine, 550 mOsm / Kg, pH 8.

[0028] Preferably, the hyperosmotic treatment temperature is 37°C and the hyperosmotic treatment time is 30 minutes.

[0029] In step (3), the amount of red blood cells loaded with heme chloride is calculated based on heme chloride, and the mass ratio of red blood cells loaded with heme chloride to IR808 is 3:2 to 3:3.

[0030] The concentration of the IR808 solution is 2-3 mg / mL, preferably 3 mg / mL.

[0031] The volume ratio of the red blood cell suspension loaded with heme chloride to the IR808 solution is 2:1.

[0032] Preferably, the stirring speed is 300 rpm, the stirring temperature is 4°C, and the stirring time is 2 hours.

[0033] The stirring device is a magnetic stirrer.

[0034] The pH of the PBS buffer in this invention is 7.4.

[0035] Another object of the present invention is to provide the use of the near-infrared light-responsive erythrocyte drug delivery system of the modified indocyanine green derivative dye in the preparation of a drug for treating periodontitis.

[0036] Preferably, the use is the use of the modified indocyanine green derivative dye red blood cell drug delivery system in the preparation of periodontal antibacterial drugs.

[0037] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0038] 1. Hemin@ER-IR808 combines the advantages of ER drug delivery and photoresponsive drug switching system. IR808 achieves NIR sterilization while providing ROS to open ER and control the release of Hemin. This allows for the complete elimination of infection sources in the early stages of treatment, controlling infection and eliminating the risk of reinfection. Subsequently, by controlling the local Hemin concentration, the immune microenvironment can be regulated in the later stages of treatment to improve the level of periodontal regeneration.

[0039] 2. Hemin@ER-IR808 has a near-infrared light (808nm) response effect. When excited by near-infrared light, it generates H2O2, which exerts an antibacterial effect. H2O2 opens the red blood cell membrane and releases the internally encapsulated Hemin, realizing photoresponsive drug delivery.

[0040] 3. Hemin@ER-IR808 can activate ferroptosis stress in bacteria within macrophages, eliminating potential intracellular bacterial infections. Simultaneously, it protects cells by activating macrophage metabolic reprogramming, enhancing the anti-intracellular bacterial effect and providing the possibility for thorough antibacterial treatment of periodontitis.

[0041] 4. Red blood cells (ER) are an excellent drug delivery system with sufficient lifespan (the circulation time of human ER is about 120±20 days), large internal capacity and good biocompatibility. The Hemin@ER-IR808 can encapsulate up to 1.982 mg / 10¹⁰ RBCs, and the encapsulation of drugs can significantly improve their pharmacokinetic characteristics. Attached Figure Description

[0042] Figure 1 The conditions and results of the Hemin loading process are shown; where A is the volume ratio of red blood cell suspension to hypotonic solution in the dialysis bag, B is the hypotonic temperature, C is the hypotonic time, and D is the effect of the initial concentration of Hemin.

[0043] Figure 2 The images show scanning electron microscope (SEM) images of Hemin@ER during different permeation processes; from left to right, they are SEM images of Hemin@ER during hypotonic, hypertonic, and isotonic processes.

[0044] Figure 3 The particle size of Hemin@ER is shown in the figure for different permeation processes.

[0045] Figure 4 Microscopic images of Hemin@ER-IR808.

[0046] Figure 5 Fluorescence image of FITC-labeled Hemin@ER-IR808.

[0047] Figure 6 The hematocrit (HCT), hemoglobin content (HGB), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCH), and mean corpuscular hemoglobin content (MCHC) of Hemin@ER-IR808 were measured.

[0048] Figure 7 The images show scanning electron microscope (SEM) images of Hemin@ER-IR808 under near-infrared light irradiation for different durations; from left to right, these are SEM images of Hemin@ER-IR808 under near-infrared light irradiation for 10 seconds, 40 seconds, 60 seconds, and without irradiation.

[0049] Figure 8 This shows the drug release of Hemin@ER-IR808 before and after near-infrared light irradiation.

[0050] Figure 9 This shows the release of H2O2 from Hemin@ER-IR808 after near-infrared light irradiation.

[0051] Figure 10 This represents the amount of Hemin released by Hemin@ER-IR808 after near-infrared light irradiation.

[0052] Figure 11 For the stability of Hemin@ER-IR808.

[0053] Figure 12 To determine the safe and effective concentration of Hemin@ER-IR808 using CCK-8 assay.

[0054] Figure 13 The effect of Hemin@ER-IR808 on the proliferation of periodontal pathogens Porphyromonas gingivalis (Pg) and Fusobacterium nucleatum (Fn).

[0055] Figure 14 The results of crystal violet staining of single and double biofilms of periodontal pathogens Porphyromonas gingivalis (Pg) and Fusobacterium nucleatum (Fn) by Hemin@ER-IR808.

[0056] Figure 15 This study aimed to quantitatively analyze the crystal violet staining results of Hemin@ER-IR808 on single and double biofilms of periodontal pathogens Porphyromonas gingivalis (Pg) and Fusobacterium nucleatum (Fn).

[0057] Figure 16 To investigate the effect of Hemin@ER-IR808 on the live / dead ratio of mono- and di-species biofilms of periodontal pathogens Porphyromonas gingivalis (Pg) and Fusobacterium nucleatum (Fn).

[0058] Figure 17 Intracellular Fe in each group after 6 h and 24 h of intracellular bacterial infection 2+ Level; where A is intracellular Fe 2+ Fluorescent staining image; B represents intracellular Fe. 2+ The results of flow cytometry quantitative detection were as follows: (1) Blank control group; (2) Intracellular Pg infection group; (3) Intracellular Pg infection + NIR treatment group; (4) Intracellular Pg infection + NIR + Hemin@ER treatment group; (5) Intracellular Pg infection + NIR + Hemin@ER-IR808 treatment group.

[0059] Figure 18 The intracellular lipid peroxidation levels of each group after 6h and 24h of intracellular bacterial infection were measured. Among them, (1) blank control group; (2) intracellular Pg infection group; (3) intracellular Pg infection + NIR treatment group; (4) intracellular Pg infection + NIR + Hemin@ER treatment group; (5) intracellular Pg infection + NIR + Hemin@ER-IR808 treatment group.

[0060] Figure 19 The results of intracellular Pg count (A) and semi-quantitative analysis (B) after 6 hours of intracellular bacterial infection and treatment in each group.

[0061] Figure 20 Transmission electron microscopy images of cells after 6 hours of intracellular bacterial infection in each group.

[0062] Figure 21 The intracellular Pg sequence was quantified 6 h after infection with intracellular bacteria in each group. Detailed Implementation

[0063] Example 1

[0064] Condition selection during Hemin loading process

[0065] Red blood cell collection: Fresh blood was collected from the heart of SD rats and collected into anticoagulant tubes. The blood was centrifuged at 1200 rpm for 10 minutes and washed three times with PBS buffer (pH=7.4) to remove cells other than red blood cells. The red blood cells were collected and a 50% v / v red blood cell suspension was prepared with 500 μL PBS buffer (pH=7.4) for storage and later use.

[0066] Different masses (1, 2, 3, 5 mg) of Hemin powder were dissolved in 100 μL of DMSO, and then 900 μL of PBS buffer (pH = 7.4) were added to prepare Hemin solutions with different final concentrations (1, 2, 3, 5 mg / mL, respectively). The pH was adjusted to 7.2-7.4 using Na2HPO4 powder to obtain the Hemin solutions.

[0067] The Hemin encapsulated in the ER was detected using a UV spectrophotometer. The UV absorption curve of the sample was measured in the wavelength range of 300–500 nm, and the maximum UV absorbance value (around 400 nm) was analyzed to determine the optimal conditions for material synthesis, including the volume ratio (V / L). 红细胞 / V 低渗缓冲液 V RBC / V Hypotonic fluid Dialysis time, hypotonic temperature, and initial Hemin concentration.

[0068] I. Investigate the volume ratio of cell suspension to hypotonic buffer.

[0069] Red blood cells were resuspended in Hemin solution at a volume ratio of 1:1 (red blood cell suspension to Hemin solution (3 mg / mL)) to obtain a cell suspension. The cell suspension was placed in a dialysis bag (molecular cutoff 14 kDa). The dialysis bag was placed in hypotonic buffer (15 mM NaH2PO4·2H2O, 15 mM NaHCO3, 2 mM ATP, 3 mM reduced glutathione, 20 mM glucose, 5 mM NaCl, 72 mOsm / kg, pH 8) at 4°C for 12 hours according to the volume ratio of cell suspension to hypotonic buffer of 1:0.5, 1:1, and 1:2.

[0070] II. Examining different dialysis times

[0071] Red blood cells were resuspended in Hemin solution at a volume ratio of 1:1 (red blood cell suspension to Hemin solution (3 mg / mL)) to obtain a cell suspension. The cell suspension was placed in a dialysis bag (molecular cutoff 14 kDa). The dialysis bag was then placed in a hypotonic buffer solution (15 mM NaH2PO4·2H2O, 15 mM NaHCO3, 2 mM ATP, 3 mM reduced glutathione, 20 mM glucose, 5 mM NaCl, 72 mOsm / kg, pH 8) at a volume ratio of 1:1 and incubated at 4°C for 1, 6, 12, 18, and 24 hours, respectively.

[0072] III. Investigation of different hypotonic temperatures

[0073] Red blood cells were resuspended in Hemin solution at a volume ratio of 1:1 (red blood cell suspension to Hemin solution (3 mg / mL)) to obtain a cell suspension. The cell suspension was placed in a dialysis bag (molecular cutoff 14 kDa). The dialysis bag was then placed in a hypotonic buffer solution (15 mM NaH2PO4·2H2O, 15 mM NaHCO3, 2 mM ATP, 3 mM reduced glutathione, 20 mM glucose, 5 mM NaCl, 72 mOsm / kg, pH 8) at a volume ratio of 1:1 and incubated at 0, 4, 25, and 37 °C for 12 hours.

[0074] IV. Examination of initial Hemin concentration

[0075] Red blood cells were resuspended in Hemin solution at a volume ratio of 1:1 (1, 2, 3, and 5 mg / mL, respectively) to obtain cell suspensions. The cell suspensions were placed in dialysis bags (molecular cutoff 14 kDa). The dialysis bags were then placed in hypotonic buffer (15 mM NaH2PO4·2H2O, 15 mM NaHCO3, 2 mM ATP, 3 mM reduced glutathione, 20 mM glucose, 5 mM NaCl, 72 mOsm / kg, pH 8) at a volume ratio of 1:1 and incubated at 4°C for 12 hours.

[0076] The results of the condition selection during the Hemin loading process are shown below. Figure 1When the volume ratio of red blood cell suspension to hypotonic buffer in the dialysis bag is 1:1 to 1:2, the initial concentration of Hemin is 2 to 3 mg / mL, the hypotonic temperature is 0 to 4℃, and the hypotonic time is 12 to 24 h, the loading capacity of Hemin is relatively high. The optimal conditions are a 1:1 volume ratio of red blood cell suspension to hypotonic buffer in the dialysis bag, an initial concentration of Hemin of 3 mg / mL, a hypotonic temperature of 4℃, and a hypotonic time of 12 h. Under these optimal conditions, the loading capacity of Hemin is the highest, reaching 1.982 mg / 10¹⁰ RBC.

[0077] Example 2

[0078] Construction of hemin@erythrocyte-indocyanine dye (Hemin@ER-IR808)

[0079] ① Red blood cell collection: Fresh blood was collected from the heart of SD rats and collected into anticoagulant tubes. The blood was centrifuged at 1200 rpm for 10 minutes and washed three times with PBS buffer (pH=7.4) to remove cells other than red blood cells. Red blood cells were collected. 500 μL of red blood cells were taken and 500 μL of PBS (pH=7.4) was used to prepare a 50% v / v red blood cell suspension. The suspension was stored for later use.

[0080] ② Hemin encapsulation using the hypotonic-reblocking method (hypotonic pre-expansion method):

[0081] Dissolve 3 mg of Hemin powder in 100 μL of DMSO, then add 900 μL of PBS buffer (pH = 7.4), and adjust the pH to 7.2-7.4 using Na2HPO4 powder to obtain 1 mL of Hemin solution with a concentration of 3 mg / mL.

[0082] Red blood cell encapsulation was performed using a hypotonic-re-blocking method, as follows:

[0083] ① Hypotonic: Mix 1 mL of red blood cell suspension with 1 mL of Hemin solution to obtain 2 mL of mixed Hemin cell suspension; place the mixed Hemin cell suspension in a dialysis bag (molecular cutoff 14 kDa), and place the dialysis bag in hypotonic buffer (15 mM NaH2PO4·2H2O, 15 mM NaHCO3, 2 mM ATP, 3 mM reduced glutathione, 20 mM glucose, 5 mM NaCl, 72 mOsm / kg, pH 8) at 4 °C for 12 hours;

[0084] ② Hypertonic: Transfer the dialysis bag to 50 mL of resealed buffer (250 mM NaCl, 12.5 mM glucose, 12.5 mM sodium pyruvate, 12.5 mM inosine, 12.5 mM NaH2PO4·2H2O, 0.63 mM adenine, 550 mOsm / Kg, pH 8) and incubate at 37°C for 30 minutes;

[0085] ③Isotonic: Wash twice with PBS buffer to remove unencapsulated Hemin and obtain Hemin-encapsulated red blood cells (Hemin@ER).

[0086] ③ IR808-modified Hemin-loaded erythrocytes (Hemin@ER-IR808): The Hemin@ER prepared above was resuspended in PBS buffer to obtain 2 mL of Hemin@ER suspension; 3 mg of IR808 was dissolved in 1 mL of PBS buffer to obtain IR808 solution; the IR808 aqueous solution was mixed with the Hemin@ER suspension and stirred at 300 rpm for 2 hours at 4°C using a magnetic stirrer; the IR808 that failed to be modified onto the surface of the erythrocytes was washed with PBS buffer to remove the IR808 that failed to be modified onto the surface of the erythrocytes, and the erythrocyte drug delivery system modified with indocyanine green derivative dye was obtained (denoted as Hemin@ER-IR808).

[0087] Characterization of Hemin@ER-IR808:

[0088] The surface morphology of Hemin@ER was observed using scanning electron microscopy (SEM), and the changes in Hemin@ER particle size were observed when it was hypotonic in hypotonic buffer, hypertonic in resealed buffer, and isotonic in PBS. Figure 2 Scanning electron microscope images of Hemin@ER during different permeation processes. Figure 3 The particle size of Hemin@ER varies when it is hypotonic in hypotonic buffer (left), hypertonic in resealed buffer (middle), and isotonic in PBS (right). The particle size of Hemin@ER is the largest in hypotonic buffer and the smallest in hypertonic buffer.

[0089] Hemin@ER-IR808 was observed under a microscope. Figure 4 It is evident that Hemin@ER-IR808 possesses a complete erythrocyte morphology.

[0090] An equimolar amount of FITC-labeled (green) was added to the Hemin solution, and FITC-labeled Hemin@ER-IR808 (i.e., erythrocyte hypotonic-reblocking method for Hemin loading) was prepared according to the method of "IR808 modification of Hemin-loaded erythrocytes" to obtain FITC-labeled Hemin@ER-IR808. Figure 5(FITC-Hemin). IR808 exhibits inherent red fluorescence. FITC-labeled Hemin@ER-IR808 was prepared and images were obtained using a confocal microscope to observe whether Hemin@ER-IR808 was successfully synthesized. Fluorescence microscopy was used to detect the loading of Hemin and IR808 (FITC green fluorescent labeling for Hemin, IR808 exhibiting inherent red fluorescence). The ER fluorescence image after IR808 binding is shown in [image missing]. Figure 5 This proves that Hemin and IR808 have been successfully loaded onto red blood cells.

[0091] A fully automated blood analyzer was used to analyze freshly prepared Hemin@ER-IR808 blood samples, measuring hematocrit (HCT), hemoglobin content (HGB), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCH), and mean corpuscular hemoglobin content (MCHC), and compared with normal red blood cells. Results are shown below. Figure 6 This indicates that the drug loading process has no significant effect on the erythrocytes themselves.

[0092] NIR switch function of Hemin@ER-IR808 and detection of Hemin controlled release capability

[0093] The morphological changes of Hemin@ER-IR808 under NIR irradiation for 10, 40, and 60 seconds and with NIR off were observed using scanning electron microscopy (SEM). The NIR irradiation intensity was 1 W / cm². 2 The wavelength is 808nm. Figure 7 The images show SEM images of Hemin@ER-IR808 after NIR irradiation for different times. SEM images at different stages of the release process show significant changes in the morphology of the ER. The shrinkage of the ER worsens with prolonged NIR irradiation time. After 40 seconds of irradiation, protrusions appear on the ER surface. With further irradiation (60 seconds), more protrusions appear, representing the burst release of Hemin. Intermittent NIR irradiation demonstrates that the ER has a switching function.

[0094] The release of Hemin from Hemin@ER and Hemin@ER-IR808 before and after NIR irradiation was detected using a microplate reader at a wavelength of 420 nm. The NIR irradiation intensity was 1 W / cm². 2 The irradiation time was 60 seconds and the wavelength was 808nm. Figure 8 The results showed that Hemin@ER-IR808 only released Hemin under NIR irradiation; regardless of whether there was NIR irradiation, Hemin@ER did not release Hemin for a short period of time.

[0095] At 0.5, 1, and 1.5 W / cm 2After NIR (808nm) irradiation for different times, 50μL of Hemin@ER-IR808 suspension was mixed with... Hydrogen peroxide detection kit The O16 detection working solution was mixed and incubated at room temperature in the dark for 30 min. Detection was performed using a fluorescence microplate reader with excitation wavelength of 570±15 nm and emission wavelength of 585±20 nm. The concentration of H2O2 was calculated. Results are shown below. Figure 9 It can be seen that as lasers of different power irradiate, the concentration of H2O2 increases, thereby achieving a bactericidal effect.

[0096] At 0.5, 1, and 1.5 W / cm 2 After NIR (808nm) irradiation for different times, the release rate of Hemin was detected using a UV spectrophotometer. Since Hemin is located inside the ER, it needs to be released after the ER cell membrane opens, such as... Figure 10 The display shows 0.5 W / cm 2 Hemin was released 50 seconds after irradiation, at a rate of 1 W / cm². 2 Hemin was released 1.5 W / cm² after 40 seconds of irradiation. 2 Hemin was released 30 seconds after irradiation.

[0097] The prepared Hemin@ER-IR808 was stored in PBS buffer solution and incubated at 4°C. The concentration of Hemin in erythrocytes was measured using a UV spectrophotometer at 0, 1, 3, 5, and 7 days to observe its stability. Stability indicators are as follows: Figure 11 As shown, Hemin@ER-IR808 remained stable for 5 days after synthesis, and began to degrade after 7 days.

[0098] Example 3

[0099] Take 1W / cm 2 Irradiation for 60 seconds was used as the illumination parameter for NIR in subsequent experiments.

[0100] Investigation into the safe and effective concentration of Hemin@ER-IR808

[0101] THP-1 cells were cultured in RPMI 1640 medium. THP-1 cells were cultured at a rate of 4 × 10⁶ cells / year. 3 THP-1 cells were seeded per well in 96-well plates and induced to differentiate into macrophages for 24 h with phorbol ester (PMA) at 100 ng / mL. The safe concentrations of Hemin@ER-IR808 (1% v / v and 2% v / v) were observed in the presence and absence of intracellular Pg infection to ensure NIR activation (laser intensity 1 W / cm²). 2After the photosensitizer IR808 generates ROS (residue on cells 60s), it does not cause excessive cell death.

[0102] In the absence of intracellular Pg infection, THP-1 cells were cultured at 4 × 10⁻⁶ cells / cells. 3 THP-1 cells were seeded per well in 96-well plates and induced to differentiate into macrophages for 24 h with 100 ng / mL phorbol ester (PMA). Cells were then divided into two groups: Control group (blank control): macrophages were cultured in fresh medium and no NIR laser irradiation was performed after 1, 2, and 3 days; NIR group: macrophages were cultured in fresh medium and irradiated with 1 W / cm² laser for 1, 2, and 3 days. 2 Macrophages were irradiated with NIR laser for 60 seconds; Hemin@ER-IR808 1% v / v group: macrophages were cultured in a medium containing 1% v / v Hemin@ER-IR808, and were not irradiated with NIR laser after 1, 2, and 3 days; Hemin@ER-IR808 2% v / v group: macrophages were cultured in a medium containing 2% v / v Hemin@ER-IR808, and were not irradiated with NIR laser after 1, 2, and 3 days; NIR+Hemin@ER-IR808 1% v / v group: macrophages were cultured in a medium containing 1% v / v Hemin@ER-IR808, and were treated with 1W / cm² laser after 1, 2, and 3 days. 2 Macrophages were irradiated with NIR laser for 60 seconds; NIR+Hemin@ER-IR808 2% v / v group: macrophages were cultured in a medium containing 2% v / v Hemin@ER-IR808, and after 1, 2, and 3 days of culture, they were treated with 1W / cm 2 Macrophages were irradiated with NIR laser for 60 seconds.

[0103] Cells were cultured for 1, 2, and 3 days using media containing 1% v / v and 2% v / v Hemin@ER-IR808, respectively.

[0104] In the presence of intracellular Pg infection, THP-1 cells were infected at a rate of 4 × 10⁻⁶. 3THP-1 cells were seeded per well in 96-well plates and induced to differentiate into macrophages for 24 h with 100 ng / mL phorbol ester (PMA). The culture medium was then replaced with *Porphyromonas gingivalis* (Pg) to infect macrophages for 3 h (MOI = 50:1). After 3 h of infection, the cells were washed three times with PBS to remove extracellular Pg. Cells were then divided into three groups: Control group (blank control): macrophages were cultured in fresh medium for 1, 2, and 3 days without NIR laser irradiation; Pg group: macrophages were infected with intracellular Pg and then cultured in fresh medium for 1, 2, and 3 days without NIR laser irradiation; NIR group: macrophages were infected with intracellular Pg and then cultured in fresh medium for 1, 2, and 3 days with 1 W / cm² of PBS. 2 Macrophages were irradiated with NIR laser for 60 seconds; Hemin@ER-IR808 1% v / v group: After intracellular Pg infection, macrophages were cultured in a medium containing 1% v / v Hemin@ER-IR808, and NIR laser irradiation was not performed after 1, 2, and 3 days; Hemin@ER-IR808 2% v / v group: After intracellular Pg infection, macrophages were cultured in a medium containing 2% v / v Hemin@ER-IR808, and NIR laser irradiation was not performed after 1, 2, and 3 days; NIR+Hemin@ER-IR808 1% v / v group: After intracellular Pg infection, macrophages were cultured in a medium containing 1% v / v Hemin@ER-IR808, and NIR laser irradiation was not performed after 1, 2, and 3 days; 2 Macrophages were irradiated with NIR laser for 60 seconds; NIR + Hemin@ER-IR808 2% v / v group (macrophages were infected with intracellular Pg, and then cultured in a medium containing 2% v / v Hemin@ER-IR808; after 1, 2, and 3 days of culture, they were treated with 1W / cm² of NIR laser). 2 Macrophages were irradiated with NIR laser for 60 seconds.

[0105] The experimental results are shown in Figure 12 The results indicate that intracellular Pg infection of macrophages for 3 days inhibited cell proliferation, and Hemin treatment could restore the cell proliferation activity inhibited by intracellular Pg. 1% v / v Hemin@ER-IR808 caused partial cell death after NIR stimulation, with a cell survival rate of approximately 75%, while 2% v / v Hemin@ER-IR808 significantly inhibited cell proliferation activity after NIR stimulation.

[0106] Investigating the inhibitory effect of NIR-responsive Hemin@ER-IR808 on the growth of periodontal pathogens and its disruptive effect on biofilm.

[0107] ① Detection of airborne bacterial proliferation: *Porphyromonas gingivalis* (Pg) and *Fusobacterium nucleatum* (Fn) were cultured on BHI medium (38.5 g / L BHI, 0.5 g / L L-cysteine, 5 g / L yeast, 2 mg / L vitamin K1, 5 mg / L heme chloride). After culturing, the initial concentrations of both Pg and Fn cultures were adjusted to 10⁻⁶. 8 CFU / mL was used to group the cells into two groups: Control group: bacterial culture; NIR group: using 1 W / cm³. 2 NIR laser irradiation of bacterial culture for 60 seconds; NIR+Hemin@ER group: Hemin@ER was added to the bacterial culture to make the volume ratio of Hemin@ER to bacterial culture 1% v / v, using 1W / cm 2 The bacterial culture was irradiated with a NIR laser for 60 seconds, followed by bacterial culture for 0, 6, 12, 24, and 48 hours to observe bacterial proliferation. The NIR+Hemin@ER-IR808 group: Hemin@ER-IR808 was added to the bacterial culture to achieve a Hemin@ER-IR808 to bacterial culture volume ratio of 1% v / v, using 1W / cm² laser. 2 Pg bacterial culture was irradiated with NIR laser for 60 seconds, and then the bacteria were cultured for 0, 6, 12, 24 and 48 hours to observe the bacterial proliferation in the culture.

[0108] ② Detection of single / double bacterial biofilm disruption: Place 0.8 cm diameter hydroxyapatite sheets at the bottom of a 24-well plate, and add 1 mL of each 10% concentration of hydroxyapatite. 8 Pg and Fn bacterial suspensions at CFU / mL were added to well plates, and single-species biofilms formed on hydroxyapatite plates after 24 hours. (The text then abruptly shifts to a different topic: "...with concentrations of 10...") 8 CFU / mL Bacillus filtrate and Fn Bacillus filtrate were mixed at a volume ratio of 100:1. 1 mL of the mixed bacterial solution was added to a 24-well plate with a hydroxyapatite sheet at the bottom. After 24 hours, a dual-species biofilm formed. Once single-species and dual-species biofilms had formed, the bacterial culture medium was gently aspirated, and the cells were divided into groups: Control group: no NIR laser irradiation was used for the bacterial biofilm, and the cells were cultured for another 24 hours; NIR group: 1 W / cm² NIR laser irradiation was used for the NIR group. 2 Bacterial biofilm was irradiated with NIR laser for 60 seconds and cultured for another 24 hours; NIR+Hemin@ER group: Hemin@ER was added to the bacterial biofilm supernatant to achieve a concentration of 1% v / v, and 1 W / cm² was used. 2NIR laser irradiation for 60 seconds, followed by culturing for 24 hours; NIR + Hemin@ER-IR808 group: Hemin@ER-IR808 was added to the bacterial biofilm supernatant to achieve a concentration of 1% v / v, using 1 W / cm 2 The bacterial culture was irradiated with a NIR laser for 60 seconds and cultured for another 24 hours. Hydroxyapatite slides were collected and stained with crystal violet to observe the biofilm damage. The ratio of live to dead bacteria in the biofilm was observed by staining with live and dead bacteria.

[0109] Under NIR excitation, Hemin@ER-IR808 significantly inhibited the growth of Pg and Fn. Figure 13 Crystal violet staining and semi-quantitative results indicated that the biofilm of bacteria in the Hemin@ER-IR808 group was significantly damaged under NIR excitation, and the degree of damage was greater than that in the Hemin@ER group. Figure 14 and Figure 15 In the bacterial live / dead staining experiment, the Hemin@ER-IR808 group showed a higher proportion of bacterial death and a reduced biofilm thickness. Figure 16 In conclusion, 1% v / v Hemin@ER-IR808 exhibited a significant antibacterial effect under NIR excitation.

[0110] Hemin@ER-IR808 controls the release of Hemin, eliminating intracellular pathogenic bacteria by inducing ferroptosis stress in intracellular bacteria.

[0111] ①Detection of intracellular ferroptosis markers: THP-1 cells were induced with 100 ng / mL PMA for 48 h. After THP-1 cells differentiated into macrophages, they were infected with Pg at MOI=50:1 for 3 h. Extracellular Pg was removed by washing with PBS 3 times, and the macrophage intracellular Pg infection model was successfully constructed. Group settings: (1) Blank control group: Macrophages without intracellular Pg infection were not irradiated with NIR. Cells were collected after 6 h and 24 h of normal culture medium and intracellular Fe was detected. 2+ (2) Intracellular Pg infection group: Macrophages infected with intracellular Pg were not subjected to NIR irradiation. Cells were collected after 6 h and 24 h of normal culture medium, and intracellular Fe was detected. 2+ Level detection and cellular lipid peroxidation level; (3) Intracellular Pg infection + NIR treatment group: NIR light irradiation for 1 min, normal culture medium for 6 h and 24 h followed by cell collection, and intracellular Fe was detected. 2+Level detection and cellular lipid peroxidation level; (4) Intracellular Pg infection + NIR + Hemin@ER treatment group: Hemin@ER was added to the cell culture supernatant to make the drug-to-culture medium volume ratio 1% v / v, and NIR light was used with light parameters of 1W / cm 2 Cells were irradiated with NIR for 1 min, and collected 6 h and 24 h after irradiation to detect intracellular Fe. 2+ Level detection and cellular lipid peroxidation level; (5) Intracellular Pg infection + NIR + Hemin@ER-IR808 treatment group; Hemin@ER-IR808 was added to the cell culture supernatant to make the drug-to-culture medium volume ratio 1% v / v, and NIR light was used with light parameters of 1W / cm 2 Cells were irradiated with NIR for 1 min, and collected 6 h and 24 h after irradiation to detect intracellular Fe. 2+ Level detection and cellular lipid peroxidation levels.

[0112] Six hours after intracellular Pg stimulation, intracellular Fe 2+ Upregulation of Hemin@ER can reduce Pg-induced Fe 2+ Upregulation; Hemin@ER-IR808, due to the early release of large amounts of the iron overload agent Hemin, increases intracellular Fe... 2+ The upregulation was more significant than in the Pg group, but after 24 hours, intracellular Fe... 2+ The intracellular Pg infection group showed a significant downregulation compared to 6 hours; intracellular bacteria persisted in the cells of the Pg infection group, and Fe2+ was significantly reduced after 24 hours. 2+ The level was higher than at 6 h; Hemin@ER continuously released Hemin, and intracellular Fe 2+ The levels remained consistently high. Cells in the intracellular Pg infection + NIR + Hemin@ER-IR808 treatment group produced a large amount of ROS and released the iron overload agent Hemin within 6 hours after NIR stimulation, and the cells were in a high-level inflammatory state. Figure 17 ).

[0113] After 6 hours of intracellular Pg stimulation, cellular lipid peroxidation levels significantly increased. Hemin@ER had no significant inhibitory effect on the Pg-induced increase in lipid peroxidation levels. After 6 hours of NIR+Hemin@ER-IR808 treatment, cellular lipid peroxidation levels significantly increased. After 24 hours, lipid peroxidation levels in the Pg and NIR groups were significantly higher than at 6 hours, while lipid peroxidation levels in the Hemin@ER group showed no significant change. However, after 24 hours, the lipid peroxidation level in the Hemin@ER-IR808 group decreased compared to 6 hours, and was significantly lower than that in the Pg and NIR groups. Figure 18 ).

[0114] ② Intracellular bacterial count detection: The grouping settings were the same as those for "Intracellular ferroptosis index detection": (2) Intracellular Pg infection group; (3) Intracellular Pg infection + NIR treatment group; (4) Intracellular Pg infection + NIR + Hemin@ER treatment group; (5) Intracellular Pg infection + NIR + Hemin@ER-IR808 treatment group; After 6 hours of NIR irradiation, the following were used The intracellular Pg was labeled with a probe for detecting Porphyromonas gingivalis, and the number of Pg in the cells was observed. After 6 hours of NIR irradiation, the cells were collected for transmission electron microscopy to observe cell morphology and intracellular bacterial status. After 6 hours of NIR irradiation, the cells were collected, cell mRNA was extracted, and the intracellular Pg sequence content was detected by RT-qPCR.

[0115] Cells in the NIR+Hemin@ER-IR808 group produced a large amount of ROS and released the iron overload agent Hemin within 6 hours after NIR stimulation, resulting in a high level of inflammation. Under this state, the cells' ability to resist ferroptosis increased, and the number of intracellular Pg was limited. Intracellular Pg was labeled using a Pg-specific fluorescent probe. It was found that the number of intracellular Pg was significantly reduced in the NIR+Hemin@ER-IR808 treatment group. Figure 19 Transmission electron microscopy results showed that cells in the Control group had regular morphology and intact organelles and nuclei; vesicles phagocytosing bacteria and swollen mitochondria were observed in cells of the Pg and NIR groups. Incompletely released material was observed in the NIR+Hemin@ER group; numerous vacuoles were observed in cells of the NIR+Hemin@ER-IR808 group, and fragmented bacteria were observed encapsulated within these vesicles. Figure 20 Based on RT-qPCR results, the Hemin@ER-IR808 group showed the lowest intracellular Pg sequence content. Figure 21 This demonstrates that the Hemin@ER-IR808 group can eliminate intracellular bacteria by inducing ferroptosis stress in intracellular bacteria.

Claims

1. An erythrocyte drug delivery system modified with an indocyanine green derivative dye, characterized in that: The drug delivery system is loaded with photosensitizer IR808 on the surface of red blood cells and encapsulates hematin chloride inside the red blood cells.

2. The erythrocyte drug delivery system of a modified indocyanine green derivative dye according to claim 1, characterized in that: It is prepared by the following method: first, using red blood cells and hematin chloride as raw materials, adopting the low-osmotic pressure-resealing method of red blood cells, hematin chloride-encapsulated red blood cells are prepared; the hematin chloride-encapsulated red blood cells are resuspended in PBS buffer to obtain hematin chloride-encapsulated red blood cell suspension, and the hematin chloride-encapsulated red blood cell suspension is mixed with IR808 solution, stirred at 0-4°C, washed with PBS buffer after the reaction is completed, and the red blood cell drug delivery system modified with indocyanine green derivative dye is obtained.

3. A method for preparing the erythrocyte drug delivery system of the modified indocyanine green derivative dye according to claim 1, characterized by: It comprises: Step (1), preparing a hematin chloride solution; A 50% v / v red blood cell suspension is prepared using PBS buffer; Step (2), preparing hematin chloride-encapsulated red blood cells using the low-osmotic pressure-resealing method: Low-osmotic pressure: the red blood cells are resuspended in the hematin chloride solution according to the volume ratio of the red blood cell suspension to the hematin chloride solution being 1:1 to obtain a mixed hematin chloride cell suspension; the mixed hematin chloride cell suspension is placed in a dialysis bag, the dialysis bag is placed in a low-osmotic pressure buffer according to the volume ratio of the cell suspension to the low-osmotic pressure buffer being 1:1-1:2, and the dialysis bag is subjected to low-osmotic pressure treatment at 0-4°C for 12-24 hours; High-osmotic pressure: the dialysis bag is transferred to a resealing buffer for high-osmotic pressure treatment, and is placed at 36-40°C for 30-45 minutes; Isotonicity: PBS buffer is used for washing to remove unencapsulated hematin chloride, and hematin chloride-encapsulated red blood cells are obtained; Step (3), resuspending the hematin chloride-encapsulated red blood cells in PBS buffer to obtain hematin chloride-encapsulated red blood cell suspension; IR808 is dissolved in PBS buffer to obtain an IR808 solution; the IR808 solution is mixed with the hematin chloride-encapsulated red blood cell suspension, and stirred at 0-4°C at a rotation speed of 200-300 rpm for 1-2 hours; PBS buffer is used for washing to remove IR808 that fails to be modified to the surface of the red blood cells, and the red blood cell drug delivery system modified with indocyanine green derivative dye is obtained.

4. The method for preparing a red blood cell drug delivery system of a modified indocyanine green derivative dye according to claim 3, characterized by: In step (1), the concentration of the hematin chloride solution is 2-3 mg / mL.

5. The method for preparing a red blood cell drug delivery system of a modified indocyanine green derivative dye according to claim 4, characterized by: In step (1), the concentration of the hematin chloride solution is 3 mg / mL.

6. The method for preparing a red blood cell drug delivery system of a modified indocyanine green derivative dye according to claim 3, characterized by: In step (2), the volume ratio of the mixed hematin chloride cell suspension to the low-osmotic pressure buffer is 1:1; the low-osmotic pressure treatment is carried out at 4°C for 12 hours; the high-osmotic pressure treatment is carried out at 37°C for 30 minutes.

7. The method of claim 3, wherein the preparation of the erythrocyte drug delivery system of the modified indocyanine green derivative dye is characterized by: In step (2), the molecular cut-off of the dialysis bag is 14 kDa; The low-osmotic pressure buffer is: 15 mM NaH2PO4•2H2O, 15 mM NaHCO3, 2 mM ATP, 3 mM reduced glutathione, 20 mM glucose, 5 mM NaCl, 72 mOsm / kg, pH 8; The resealing buffer is: 250 mM NaCl, 12.5 mM glucose, 12.5 mM sodium pyruvate, 12.5 mM inosine, 12.5 mM NaH2PO4·2H2O, 0.63 mM adenine, 550 mOsm / Kg, pH 8.

8. The method for preparing a red blood cell drug delivery system of a modified indocyanine green derivative dye according to claim 3, characterized by: In step (3), the amount of the hematin-loaded red blood cells is calculated based on hematin, the mass ratio of the hematin-loaded red blood cells to IR808 is 3:2-3:3, the concentration of the IR808 solution is 2-3 mg / mL, and the volume ratio of the hematin-loaded red blood cell suspension to the IR808 solution is 2:

1.

9. The method of claim 8, wherein the preparation of the erythrocyte drug delivery system of the modified indocyanine green derivative dye is characterized by: In step (3), the concentration of the IR808 solution is 3 mg / mL.

10. The method of claim 3, wherein the preparation of the erythrocyte drug delivery system of the modified indocyanine green derivative dye is characterized by: In step (3), the stirring speed is 300 rpm, the stirring temperature is 4°C, and the stirring time is 2 hours.

11. Use of the near-infrared light responsive red blood cell drug delivery system of the modified indocyanine green derivative dye in claim 1 in the preparation of a drug for treating periodontitis.

12. Use according to claim 11, characterized in that: The use is the use of the red blood cell drug delivery system of the modified indocyanine green derivative dye in the preparation of an antibacterial drug for periodontitis.

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