Erythrocyte membrane nanodiscs, preparation method thereof and application thereof in assisting phototherapy

Abstract

The present invention discloses an erythrocyte membrane nanodisc, a preparation method, and its application in assisted phototherapy. The erythrocyte membrane nanodisc comprises an erythrocyte membrane, a photosensitizing agent loaded onto the erythrocyte membrane, and a restrictor wrapped around the erythrocyte membrane, the restrictor being used to stabilize the morphology of the erythrocyte membrane. The erythrocyte membrane nanodisc is used in assisted phototherapy to enhance the permeability of the photosensitizing agent. The preparation method includes steps S1: obtaining an erythrocyte membrane; S2: loading the photosensitizing agent onto the erythrocyte membrane; and S3: wrapping the restrictor around the erythrocyte membrane to form a nanodisc. This solution can enhance the permeability of the photosensitizing agent by loading the nanodisc with the photosensitizing agent, thereby enhancing the phototherapy effect.

Description

Technical Field

[0001] The present invention relates to biomedicine, in particular to erythrocyte membrane nanodiscs, a preparation method and application thereof in auxiliary phototherapy. Background Art

[0002] Photothermal therapy (PTT) uses photosensitizers to convert light energy into heat, thereby treating lesions such as tumors or bacteria. However, the poor penetration of photosensitizers into the diseased area limits the effectiveness of phototherapy.

[0003] Nanodiscs (NDs) are synthetic lipid nanostructures commonly used in membrane protein research, drug delivery, and vaccine development. Generally speaking, nanodiscs are flat discs with a lipid bilayer less than 50 nm in size. The periphery of the lipid bilayer is stabilized by a polymer such as styrene-maleic acid (SMA), enabling their stability in water. Due to their size and morphology, NDs are more effectively concentrated at lesion sites than conventional nanomaterials, improving their penetration. However, traditional lipid-based nanodiscs face potential immunological risks, limiting their clinical application. In recent years, nanodiscs based primarily on cell membranes have been developed. For example, red blood cell membrane nanodiscs have been shown to neutralize bacterial virulence factors; platelet membrane nanodiscs have been shown to neutralize autoantibodies; and neuronal cell membrane nanodiscs have been shown to neutralize neurotoxins. Despite this, cell membrane nanodiscs have yet to be applied in phototherapy. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide an erythrocyte membrane nanodisc, a preparation method and its application in auxiliary phototherapy, which can load photosensitizing agents through the nanodisc, improve the penetration of photosensitizing agents and enhance the phototherapy effect.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] An erythrocyte membrane nanodisk comprises an erythrocyte membrane, a photosensitizing agent loaded on the erythrocyte membrane, and a restrictor wrapped around the erythrocyte membrane, wherein the restrictor is used to stabilize the morphology of the erythrocyte membrane nanodisk.

[0007] As a further improvement of the present invention, the restrictor is a styrene-maleic acid copolymer.

[0008] As a further improvement of the present invention, the photosensitizing agent is IR780.

[0009] An application of an erythrocyte membrane nanodisc in assisted phototherapy, wherein the erythrocyte membrane nanodisc adopts the erythrocyte membrane nanodisc as described in any of the above-mentioned improved solutions, and is used to enhance the penetration of a photosensitizing agent.

[0010] A method for preparing red blood cell membrane nanodiscs comprises the following steps:

[0011] S1 obtains red blood cell membrane;

[0012] S2 loads the red blood cell membrane with a photosensitive reagent;

[0013] S3 wraps the restriction around the red blood cell membrane to form a nanodisc.

[0014] As a further improvement of the present invention, the photosensitizing agent is IR780.

[0015] As a further improvement of the present invention, the restrictor is styrene-maleic acid copolymer SMA.

[0016] As a further improvement of the present invention, S2 comprises IR780 and red blood cell membrane in a mass ratio of IR780: red blood cell membrane protein = 2% w / w, which is then incubated in a 37°C incubator and ultrasonically obtained to obtain RBC(IR)-vesicle.

[0017] As a further improvement of the present invention, S3 comprises mixing SMA with RBCM(IR)-vesicle at a mass ratio of SMA: red blood cell membrane protein = 5:1 (w / w) to prepare RBC(IR)-ND.

[0018] As a further improvement of the present invention, the process of preparing RBC-IR-ND by mixing SMA with RBCM-IR-vesicle includes:

[0019] The final concentration of red blood cell membrane was 1 mg / mL, and the final concentration of SMA was 5 mg / mL. After sonication with a probe and stirring at 4°C overnight, the supernatant was concentrated using an ultrafiltration tube after ultracentrifugation to obtain RBC-IR-ND.

[0020] The beneficial effect of the present invention is that the red blood cell membrane is wrapped with a restrictor to form a nanodisc, and the lipid hydrophobic part of the nanodisc is used to encapsulate the photosensitive reagent, thereby forming a new red blood cell membrane phototherapy nanodisc system with enhanced biological permeability and phototherapy effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The particle size and surface potential diagram of RBC(IR)-vesicle and RBC(IR)-ND;

[0022] Figure 2This is the photothermal conversion efficiency diagram of RBC(IR)-vesicle and RBC(IR)-ND;

[0023] Figure 3 Figure 2 is the enrichment diagram of RBC(IR)-vesicle and RBC(IR)-ND in tumors;

[0024] Figure 4 Figure 2 is the therapeutic effect of RBC(IR)-vesicle and RBC(IR)-ND on tumors;

[0025] Figure 5 Figure 2 is the enrichment diagram of RBC(IR)-vesicle and RBC(IR)-ND in MRSA infection;

[0026] Figure 6 The figure shows the therapeutic effects of RBC(IR)-vesicle and RBC(IR)-ND on MRSA infection in mice;

[0027] Figure 7 Schematic diagram of the preparation process of RBCM(IR)-ND. DETAILED DESCRIPTION

[0028] The present invention will be further described below with reference to the embodiments shown in the accompanying drawings.

[0029] 1. Preparation of RBCM(IR)-NDs:

[0030] Reference Figure 7 As shown, IR780 and RBCs were mixed at a mass ratio of IR780:RBC membrane = 2% (w / w) and incubated at 37°C for 30 min to obtain RBC(IR)-ghost. This was followed by sonication with a 100 W probe for 30 s to obtain RBC(IR)-vesicles. SMA was then mixed with RBCM(IR)-vesicles at a mass ratio of SMA:RBC = 5:1 (w / w) to achieve a final RBC concentration of 1 mg / mL and a final SMA concentration of 5 mg / mL. The mixture was sonicated with a 100 W probe for 30 s, stirred overnight at 4°C, and ultracentrifuged at 150,000 × g for 6 h. The supernatant was concentrated using a 100 k filtration tube to obtain RBC(IR)-ND. RBC protein concentration was measured using the BCA assay and used for later use. Before in vivo experiments, BSA was added to the nanodiscs at a mass ratio of RBC membrane: BSA = 1: 64 (w / w), incubated at 37°C for 30 min, and adjusted to isotonicity with 20× PBS before use.

[0031] 2. Basic Characterization of RBC(IR)-ND:

[0032] The particle size and surface potential of RBC(IR780)-ND and RBC(IR)-vesicle were measured using a Zetasizer Nano instrument (Litesizer™ 500, Anton Paar, Austria).

[0033] Figure 1 Particle size and potential results showed that the particle size of RBC(IR)-vesicles was approximately 120 nm, which decreased to 20 nm after being prepared into RBC(IR)-NDs. The surface potential of RBC(IR)-vesicles was approximately -15 mV, but decreased to approximately -30 mV after being prepared into RBC(IR)-NDs.

[0034] 3. Photothermal conversion efficiency of RBC(IR)-ND:

[0035] To evaluate the cyclic photothermal performance of RBC(IR)-ND and calculate the photothermal conversion efficiency, 1 mL of RBC(IR)-ND containing 15 μg / mL IR780 was irradiated with an 808 nm near-infrared laser at a power of 2.0 W / cm² for 5 minutes. After irradiation, the near-infrared laser was turned off, and the sample was allowed to cool naturally to room temperature. The temperature was recorded every 15 seconds using a Fotric 322pro thermal imager. This heating-cooling process was repeated three times to evaluate the photothermal stability. The value of the first cycle was used to calculate the photothermal conversion efficiency (η) using the following formula:

[0036] η= [hA×(Tmax-Tamb) -Qdis] / I×(1-10-A)

[0037] h: heat transfer coefficient (W·m⁻²·K⁻¹); A: surface area of the sample exposed to the laser (m²); Tmax: maximum temperature under 808 nm irradiation (°C); Tamb: ambient temperature (°C); Qdis: heat dissipated by the absorption system in the absence of light (W); I: incident laser power density (W·m⁻²); A: absorbance of the RBC(IR)-vesicle or RBC(IR)-ND at 808 nm.

[0038] Figure 2 The results showed that both RBC(IR)-ND and RBC(IR)-vesicle could undergo photothermal cycling in an on-off manner, with the peak temperature of RBC(IR)-ND being higher than that of RBC(IR)-vesicle during each cycle. The photothermal conversion efficiency (η) of RBC(IR)-ND was 36.06%, higher than the 21.20% of RBC(IR)-vesicle.

[0039] 4. Enrichment of RBC(IR)-ND in mouse 4T1 breast cancer:

[0040] Use 6-8 week old Balb / c female mice and place 5×10 5 4T1 tumor cells were implanted in the fifth mammary fat pad on the left side of mice. Five days after implantation, the mice were divided equally into two groups: the RBC(IR)-vesicle group and the RBC(IR)-ND group. An equal dose of RBC (50 mg / kg) was injected via the tail vein. Fluorescence intensity at the tumor site was measured using a small animal in vivo imaging device at different times (0, 1, 4, 8, 12, 24, 48, and 72 hours), and the fluorescence at the tumor site was statistically analyzed.

[0041] Figure 3 The results showed that IR-ND accumulated faster in the tumor site than IR-vesicle 1 hour after intravenous injection, and the accumulation reached a peak at 24 hours.

[0042] 5. The therapeutic effect of RBC(IR)-ND on 4T1 breast cancer in mice:

[0043] Use 6-8 week old Balb / c female mice and place 5×10 5 4T1 tumor cells were implanted into the fifth mammary fat pad on the left side of the mouse. When the volume of 4T1 breast cancer was about 100 mm 3 At 14:00, RBC(IR)-ND (50 mg / kg) was intravenously injected. 24 h later, NIR near infrared (2.0 W / cm 2 ) irradiated the tumor area for 5 minutes. Tumor temperature was recorded using a Fotric 322pro thermal imager at 0, 60, 180, and 300 seconds. Tumor volume was subsequently measured every 2 days for 14 days. At the end of the experiment, tumor tissue was harvested, photographed, and weighed. An equal dose of RBC(IR)-vesicles and PBS combined with near-infrared irradiation served as a control group. Tumor tissue was stained for apoptosis using a TUNEL kit, and the percentage of apoptotic cells was calculated.

[0044] Figure 4The results showed that compared with the control group, RBC(IR)-ND combined with NIR irradiation significantly inhibited tumor growth, with four of five mice achieving complete tumor regression, while tumors in the RBC(IR)-vesicle and PBS combined with NIR irradiation groups continued to grow. The average tumor weight in the RBC(IR)-ND group was significantly lower than that in the other two groups. In terms of mechanism, tumors treated with RBC(IR)-ND combined with NIR showed significantly increased apoptotic signals, mainly due to RBC(IR)-ND enhancing the accumulation and penetration of IR780 in the tumor and further improving the photothermal conversion efficiency under near-infrared laser.

[0045] 6. Enrichment of RBC(IR)-NDs in MRSA Infection:

[0046] 100 µL of 5 × 10 7 A MRSA skin infection model was established by subcutaneously injecting MRSA (CFU / mL) into male 6-8-week-old ICR mice. Twenty-four hours after bacterial inoculation, the mice were divided equally into two groups: the RBC(IR)-vesicle group and the RBC(IR)-ND group. An equal dose of RBC (50 mg / kg) was injected via the tail vein. Fluorescence intensity at the infected site was measured using a small animal imaging device at different times (0, 1, 4, 8, 12, 24, 48, and 72 hours), and the fluorescence at the infected site was statistically analyzed.

[0047] Figure 5 The results showed that compared with RBC(IR)-vesicle, RBC(IR)-ND could accumulate rapidly at the site of MRSA infection and reached a peak 24 hours after injection.

[0048] 7. Therapeutic effect of RBC(IR)-ND on MRSA infection in mice:

[0049] A MRSA skin infection model was established by subcutaneously injecting 100 μL of 5 × 107 CFU / mL MRSA into male ICR mice aged 6-8 weeks. Twenty-four hours after bacterial inoculation, RBC(IR)-ND was intravenously injected at a dose of 50 mg / kg. Twenty-four hours later, the mice were exposed to near-infrared (NIR) light (2.0 W / cm 2 ) irradiated the infected lesions for 5 minutes. Temperature at the infected site was recorded using a Fotric 322pro thermal imager at 0, 60, 180, and 300 seconds. Lesion size and body weight were subsequently measured every two days for 13 days. After the experiment, the skin was removed, the tissue homogenized, and cultured on TSB agar. MRSA colonies were counted after 24 hours of incubation. An equal dose of RBC(IR)-vesicles and PBS combined with near-infrared irradiation served as a control group.

[0050] Figure 6 Results showed that RBC(IR)-NDs achieved a higher local temperature (50°C) under NIR irradiation compared to RBC(IR)-vesicles. Furthermore, lesion damage in the RBC(IR)-ND-treated group recovered more rapidly compared to the control group. Furthermore, the body weight of mice in all groups remained stable throughout the study. Furthermore, compared to the other two groups, mice treated with RBC(IR)-NDs combined with NIR irradiation showed significantly lower bacterial loads in infected tissues, further demonstrating the enhanced therapeutic effect of RBC(IR)-NDs on bacterial infections.

[0051] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiment. All technical solutions based on the concept of the present invention are within the scope of protection of the present invention. It should be noted that for those skilled in the art, various improvements and modifications that do not depart from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing red blood cell membrane nanodiscs, characterized in that: The steps include: S1 obtains red blood cell membrane; S2 loads the red blood cell membrane with a photosensitive reagent; S3 wraps the restriction to the RBC-IR-vesicle to form a nanodisc; The photosensitizer is IR780; The restrictor is styrene-maleic acid copolymer SMA; S2 comprises IR780 and erythrocyte membrane at a mass ratio of IR780: erythrocyte membrane protein = 2% w / w, and then incubated in a 37°C incubator, and RBC-IR-vesicles were obtained by sonication.

2. The preparation method according to claim 1, characterized in that S3 includes SMA and RBCM-IR-vesicle mixed at a mass ratio of SMA: red blood cell membrane protein = 5:1 w / w to prepare RBC-IR-ND.

3. The preparation method according to claim 2, characterized in that The process of preparing RBC-IR-ND by mixing SMA with RBCM-IR-vesicle includes: The final concentration of red blood cell membrane was 1 mg / mL, and the final concentration of SMA was 5 mg / mL. After sonication with a probe and stirring at 4°C overnight, the supernatant was concentrated using an ultrafiltration tube after ultracentrifugation to obtain RBC-IR-ND.

4. An erythrocyte membrane nanodisc prepared by the method according to any one of claims 1 to 3, characterized in that: The invention comprises a red blood cell membrane, a photosensitive reagent loaded on the red blood cell membrane, and a restriction object wrapped around the red blood cell membrane, wherein the restriction object is used to stabilize the morphology of the red blood cell membrane nanodisc.

5. An application of erythrocyte membrane nanodiscs in the preparation of a drug for assisted phototherapy, characterized in that: The erythrocyte membrane nanodisc adopts the erythrocyte membrane nanodisc as claimed in claim 1 and is used to enhance the permeation effect of a photosensitizing agent.

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