Exosome composite nanohydrogel for targeted treatment of psoriasis and preparation method thereof

Abstract

The present invention discloses an exosome composite nanohydrogel for the targeted treatment of psoriasis and its preparation method. The composite nanohydrogel is prepared by embedding the drug tripterine analogue celastrol in a gel to form nanoparticles, loading them into exosomes of cells with high PD-L1 expression, and then encapsulating them in an F127 thermosensitive hydrogel. The hydrogel, through the specific action of PD-1 / PD-L1, delivers drugs to the affected areas to exert their effects, achieving targeted treatment of psoriasis, improving the disease microenvironment, reducing drug toxicity and side effects, and enhancing therapeutic efficacy.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical preparations, and specifically relates to an exosome composite nanohydrogel for targeted treatment of psoriasis and a preparation method thereof. Background Art

[0002] Psoriasis is a chronic inflammatory disease characterized by T-cell-mediated inflammation and abnormal keratinocyte differentiation. Its primary clinical manifestations are localized or generalized scaly, erythematous plaques. It is difficult to treat and prone to relapse, placing a significant financial and psychological burden on patients. The prevalence and incidence of the disease have increased annually in recent years. The pathogenesis of this disease is complex and is currently believed to be related to genetics, stress, mental stress, and immune disorders. Current medical treatments for psoriasis include systemic use of medications such as acitretin and methotrexate, as well as topical glucocorticoids and vitamin D3 derivatives. However, while these traditional medications achieve therapeutic effects, they inevitably cause damage to other organs, such as the liver and kidneys. Furthermore, these traditional medications have a short half-life, are easily metabolized by the body, require frequent administration, and are associated with poor patient compliance.

[0003] Exosomes are vesicles that are actively secreted outside the cell. They have a phospholipid bilayer structure, are 30-150 nm in diameter, and contain proteins, RNA, and lipids. Exosomes represent a novel long-range mode of action that can further regulate immune responses and intercellular signaling. Exosomes have a complex composition, and the composition of each exosome depends on the cell of origin and its production mechanism. Studies have found that exosomes uniformly express certain proteins and related proteins closely related to their cell of origin. Literature reports that exosomes derived from melanoma cells highly express programmed cell death ligand 1 (PDL1), which can suppress immunity and promote wound healing. Experimental results by the present inventors demonstrate that PD-1 expression is significantly increased in skin lesions, CD4+ T cells, and keratinocytes in a psoriasis mouse model. The present invention is completed by utilizing exosomes that highly express PD-L1 as drug carriers and, through the specific interaction of PD-1 / PD-L1, enabling targeted drug delivery to psoriasis lesions, thereby achieving targeted treatment of psoriasis. Summary of the Invention

[0004] The present invention aims to provide an exosome-based composite nanohydrogel for the targeted treatment of psoriasis and its preparation method. The composite nanohydrogel is prepared by extracting and isolating exosomes from cells with high PD-L1 expression, loading them with nanoparticles of psoriasis drugs (such as tripterine and its analogs), and finally encapsulating them in a Pluronic F127 (also known as Pluronic F-127 or PF-127) thermosensitive hydrogel. This composite nanohydrogel can deliver drugs to psoriasis lesions through the specific action of PD-1 / PD-L1, achieving targeted treatment of psoriasis while reducing drug toxicity and side effects.

[0005] To achieve the purpose of the present invention, the following embodiments are provided:

[0006] In one embodiment, the present invention provides an exosome composite nanohydrogel for the targeted treatment of psoriasis, comprising a psoriasis drug, exosomes that highly express PD-L1, and a gel matrix F127. The psoriasis drug is first embedded into nanoparticles with a portion of the F127 gel matrix, then loaded into the exosomes to form a hydrogel with the remaining portion of the F127 gel matrix.

[0007] In the composite nano-hydrogel of the present invention, the psoriasis drugs include but are not limited to triptolide or its analogue celastrol or its pharmaceutically acceptable salt.

[0008] In another embodiment, the present invention provides a method for preparing an exosome composite nanohydrogel for targeted treatment of psoriasis, comprising the following steps:

[0009] 1) Preparation of psoriasis drug nanoparticles

[0010] The psoriasis drug was dissolved in dichloromethane, mixed with a 1.0% F127 aqueous solution, ultrasonically mixed to form an O / W emulsion, and freeze-dried to form nanoparticles;

[0011] 2) Isolation and preparation of exosomes with high surface expression of PD-L1

[0012] The exosome-derived cells that highly express PD-L1 were cultured in DMEM complete culture medium. After passage and culturing for 48 hours, the supernatant was collected, and dead cells and cell debris were removed, and then the vesicles were removed to obtain exosomes.

[0013] 3) Preparation of drug-loaded exosomes

[0014] a) mixing the exosomes from step 2) with the drug nanoparticles from step 1) at a mass ratio of 2:1 to obtain a mixed solution;

[0015] b) treating the mixed solution obtained in step a with pulsed sonication and incubating at 37° C. for 1 h to restore exosome membrane stability;

[0016] c) removing free drugs to obtain drug-loaded exosomes;

[0017] 4) Preparation of hydrogel

[0018] PF-127 powder was dissolved in a dispersion medium (PBS) to prepare a hydrogel solution with a mass fraction of 20-30%, and a drug-loaded exosome solution was added and mixed to prepare a composite nanohydrogel.

[0019] Preferably, in the above-mentioned preparation method of the present invention, the nanoparticles prepared in step 1) further comprise removing excess F127 in the nanoparticles by dialysis, and the psoriasis drugs include but are not limited to triptolide or its analogue celastrol.

[0020] Preferably, in the preparation method of the present invention, in step 2), the exosome-derived cells that highly express PD-L1 are selected from animal melanoma cells, human melanoma cells, other tumor cells that highly express PD-L1, and genetically modified cells that highly express PD-L1.

[0021] Preferably, in the preparation method of the present invention, in step 3) a), the exosomes are 100 μg and the drug nanoparticles are 50 μg.

[0022] Preferably, in the preparation method of the present invention, in step 3) b), the ultrasonic treatment has an ultrasonic power of 100 W, pulsed ultrasound on / off for 5 seconds each, and is repeated 5 times.

[0023] Preferably, in the above-mentioned preparation method of the present invention, in step 4), the dispersion medium is double distilled water.

[0024] Preferably, in the preparation method of the present invention, in step 4), the mass fraction of PF-127 in the hydrogel solution is 30%.

[0025] Preferably, in the preparation method of the present invention, in step 4), the mass ratio of PF-127 in the composite nano-hydrogel is 25%.

[0026] In a specific embodiment, the method for preparing an exosome composite nano-hydrogel for targeted treatment of psoriasis of the present invention comprises the following steps:

[0027] 1) Preparation of psoriasis drug nanoparticles

[0028] The drug triptolide or celastrol is dissolved in dichloromethane and mixed with a 1.0% F127 aqueous solution, and ultrasonically mixed to form an O / W emulsion. The emulsion is immediately placed in liquid nitrogen for rapid cooling and coagulation, and then placed in a freeze dryer to prepare nanoparticles. Excess F127 is removed by dialysis to obtain drug nanoparticles.

[0029] 2) Isolation and preparation of exosomes with high surface expression of PD-L1

[0030] Cells that highly express PD-L1 were cultured in DMEM complete medium containing exosome-free serum. After passage, the supernatant was collected after 48 hours of culture. Dead cells and cell debris were removed at 2000g for 20 minutes, vesicles were removed at 16500g for 45 minutes, and exosomes were obtained at 110000g for 2 hours.

[0031] 3) Drug-loaded exosomes

[0032] a) preparing a solution: mixing 100 μg of the exosomes prepared in step 2) with 50 μg of the drug nanoparticles prepared in step 1) to obtain a mixed solution;

[0033] b) Pulse drug loading: the mixed solution is subjected to pulse ultrasonic treatment;

[0034] c) Restoration of membrane stability: The mixed solution after ultrasonic treatment was incubated at 37°C for 1 h to restore the stability of the exosome membrane;

[0035] d) Removal of free drugs: The free drugs in the drug-loaded exosome solution were removed by exoEasy spin column;

[0036] 4) Loading of thermosensitive hydrogel

[0037] F127 was dissolved in PBS buffer solution to prepare a hydrogel solution with a F127 mass fraction (mass ratio) of 30%.

[0038] The drug-loaded exosome solution was added to the 30% hydrogel solution and mixed, and then the mass fraction of PF-127 was adjusted to 25% with PBS (buffer) to obtain a composite nanohydrogel.

[0039] In the above specific embodiment, the exosome-derived cells that highly express PD-L1 in step 2) include but are not limited to animal and human melanoma cells and other tumor cells that highly express PD-L1, as well as genetically modified cells that highly express PD-L1.

[0040] Compared with the prior art, the composite nano-hydrogel of the present invention has the following advantages: the composite nano-hydrogel of the present invention, the hydrogel-encapsulated exosome delivery carrier with high expression of PD-L1, the inventor's preliminary experimental results show that the expression of PD-1 on target cells in the psoriasis model is increased, and it is proved that the drug is delivered to the overactivated T cells and keratinocytes in the psoriasis lesion area through the PD-1 / PD-L1 axis, reducing the inflammatory response caused by the overactivation of T cells and inhibiting the abnormal proliferation of keratinocytes. Therefore, the exosome delivery carrier of the present invention can be used to load psoriasis drugs to achieve targeted treatment. At the same time, because the exosomes are loaded on the hydrogel, the biological half-life of the targeted delivery system is extended, providing the effect of long-term drug treatment, which can greatly improve patient compliance. In addition, due to the natural biofilm structure of the exosomes, it has the characteristics of low immunogenicity. At the same time, the method is simple and easy to implement and has good medicinal prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Transmission electron microscopy images of celastrol nanoparticles (A), melanoma cell exosomes (B), and drug-loaded melanoma exosomes (C);

[0042] Figure 2 The distribution of melanoma cell exosomes and the particle size of melanoma exosomes after drug loading;

[0043] Figure 3 For the identification of exosome marker proteins;

[0044] Figure 4 Schematic diagram of the temperature sensitivity of the hydrogel. Side A shows that the hydrogel is solid at room temperature (37 degrees Celsius), and side B shows that the hydrogel is liquid at 4 degrees Celsius.

[0045] Figure 5 The expression of PD-1 on CD4+T cells;

[0046] Figure 6 The expression level of PD-1 on HaCaT cells changes;

[0047] Figure 7 The changes in the average fluorescence intensity of Cy5.5 on CD4+T cells in the control group and the model group;

[0048] Figure 8 The changes in the average fluorescence intensity of Cy5.5 on HaCaT cells in the control group and model group;

[0049] Figure 9 The changes of the mouse back skin under different treatments;

[0050] Figure 10 These are pathological sections of the back skin of mice under different treatments;

[0051] Figure 11 The figure shows the statistical histogram of epidermal thickness changes under different treatments and the Baker score (psoriasis pathological scoring system);

[0052] Figure 12 The changes of PASI scores (psoriasis severity scoring system) under different treatments. DETAILED DESCRIPTION

[0053] In order to make the purpose, technical solution and effect of the present invention more clear, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific implementation described here is only used to explain the present invention and is not intended to limit the present invention.

[0054] The exosomes in the following examples were derived from B16F10 cells (melanoma).

[0055] Example 1 Preparation of exosome composite nanohydrogel

[0056] Step 1: Preparation of drug nanoparticles

[0057] Since prismaticin has poor water solubility, prismaticin was first prepared into nanoparticles.

[0058] Pristimerin was dissolved in dichloromethane and mixed with a 1.0% aqueous solution of F127. The mixture was ultrasonically mixed to form an O / W emulsion. The emulsion was immediately placed in liquid nitrogen to rapidly cool and solidify, and then placed in a freeze dryer to prepare nanoparticles. Finally, the excess F127 was removed by dialysis to obtain pristimerin nanoparticles. The nanoparticles were observed under transmission electron microscopy. The results are shown in Figure 2. Figure 1 A, Figure 1 The leftmost picture (A) is a transmission electron microscope picture of the obtained nanoparticles.

[0059] Step 2: Preparation of exosomes with high expression of PD-L1

[0060] Serum (BI) was ultracentrifuged at 100,000 g for 16 hours, and the supernatant was collected to obtain exosome-free serum. B16F10 cells (exosomes expressing PD-L1) were cultured in DMEM High Glucose (BI) complete medium prepared with exosome-free serum. After passage and culturing for 48 hours, the supernatant was collected and centrifuged at 2,000 g for 20 minutes to remove dead cells and cell debris, at 16,500 g for 45 minutes to remove vesicles, and at 110,000 g for 2 hours to obtain exosomes expressing PD-L1.

[0061] Step 3: Loading drug to express PD-L1

[0062] 1) Prepare solution: Add 50 μg of drug nanoparticles prepared in step 1) to 100 μg of exosomes prepared in the previous step to obtain a mixed solution.

[0063] 2) Pulse-assisted drug loading: Pulse-assisted sonication of the mixed solution was performed using an ultrasonic pulsator. The specific procedure was as follows: insert the EP tube into ice, immerse the probe in the solution, and pulse-assisted sonication (500 V, 2 kHz, 20% power) was repeated five times, with each cycle lasting 5 seconds. The entire process was performed on ice.

[0064] 3) Restoring membrane stability: After the end of ultrasound, place the EP tube in a water bath thermostat, adjust the temperature to 37°C, and incubate for 1 hour to restore the stability of the exosome membrane.

[0065] 4) Removal of free drugs: The excess free drugs in the drug-loaded exosome solution were removed using QIAGEN exoEasy spin column to obtain drug-loaded exosomes (i.e., exosomes loaded with prismaticin). The shape and particle size distribution of the exosomes before and after drug loading were observed using transmission electron microscopy. Figure 1-3 . Figure 1 B and C are transmission electron microscopy images of exosome shapes, Figure 2 is the distribution of exosome particle size before and after drug loading, Figure 3 For the identification of exosome marker proteins.

[0066] Step 4: Determination of exosome drug loading

[0067] The drug loading capacity of drug-loaded exosomes was determined by HPLC.

[0068] Prior to assay, an equal volume of acetonitrile was added to the drug-loaded exosome solution, mixed thoroughly, and the membranes were disrupted by ultrasonication. The solution was then centrifuged at 13,000 × g for 10 minutes to remove impurities. The supernatant was removed, filtered through a 0.22 μm syringe filter, and diluted appropriately with mobile phase. The vancomycin-containing solution was transferred to an HPLC injection vial, and 20 μL of the sample was injected into each vial. Chromatographic analysis was performed according to the standard curve conditions. The drug loading efficiency of the drug system was measured using the following formula: drug loading efficiency (%) = (mass of encapsulated drug) / (mass of exosomes) * 100%. The drug loading efficiency of the drug-loaded exosomes was determined to be 10.73 ± 0.45%.

[0069] Step 5: Loading of thermosensitive hydrogel

[0070] Weigh 3g of Pluronic F-127 powder and dissolve it in 10ml of PBS. Stir thoroughly until a paste forms. Place in a magnetic rotor and stir overnight in a 4°C refrigerator until a clear, transparent hydrogel forms (30% F-127 by weight). The drug-loaded exosome solution is added to the 30% PF-127 hydrogel, and an appropriate volume of PBS is added to adjust the PF-127 by weight fraction to 25%. This yields the composite nanohydrogel (hereafter referred to as Pri@exo).

[0071] The schematic diagram of the thermosensitivity of the prepared hydrogel is shown in Figure 4 As shown in Figure 2, side a shows the hydrogel in a solid form at room temperature (37°C). Side b shows the hydrogel in a liquid form at 4°C. This demonstrates the hydrogel's temperature-sensitive properties.

[0072] The following examples are targeted and therapeutic in vivo experiments of the exosome composite nano-hydrogel of the present invention, using the Pristimerin exosome composite nano-hydrogel (codenamed Pri@exo) prepared in Example 1 as the experimental sample.

[0073] Example 2 Targeting of composite nanohydrogel

[0074] Pri@exo targeted anti-psoriasis effect detection

[0075] 1) In the psoriasis model, PD-1 expression was upregulated on CD4+ T cells and HaCaT cells. Figure 5 , is the expression of PD-1 on CD4+ T cells. The model group (IMQ) was higher than the control group (Control). An in vitro hyperproliferative model of psoriasis was established by stimulating human immortalized keratinocytes (HaCaT) with TNF-α. Changes in PD-1 expression on HaCaT cells were detected by flow cytometry. Figure 6 , is the expression of PD-1 on HaCaT cells. The expression of PD-1 in the model group (TNF-α) was significantly increased compared with the control group (Control).

[0076] 2) Pri@exo can be used for targeted anti-psoriatic effects: Using CD4+ T cells extracted from primary mice, we demonstrated that the addition of Cy5.5-labeled Pri@exo resulted in changes in the mean fluorescence values ​​of CD4+ T cells in the model group and the control group, demonstrating that the highly expressed PD-L1 on Pri@exo can bind to the PD-L1 / PD-1 ligand receptor, allowing targeted drug delivery to T cells that play an important role in the pathogenesis of psoriasis (preliminary studies have shown that PD-1 expression is increased in psoriasis). Figure 7 , is the expression change of mean fluorescence value in control group and model group (IMQ). Similarly, the change of mean fluorescence value of HaCaT cells was detected. Figure 8It can be seen that the average fluorescence value of the model group (TNF-α) was significantly increased compared with the control group (Control), indicating that Pri@exo can target keratinocytes in the psoriasis state.

[0077] Example 3: Evaluation of Pri@exo's in vivo therapeutic efficacy

[0078] Pri@exo was administered subcutaneously to psoriasis-prone mice. The anti-psoriatic effect of Pri@exo was compared using skin appearance, Psoriasis Severity Index (PASI), epidermal thickness, and pathological sections. Animal selection: Male BALB / C mice, 6-8 weeks old, with good dorsal skin condition and weighing 20-22 g, were used to establish the animal model.

[0079] Modeling and grouping: Mice were depilated with a depilatory cream one day in advance (Day 0). A 2×3 cm area was removed along the spine. The normal control group (Control) received 62.5 mg of medical petroleum jelly applied daily to the back skin. The model group (IMQ) received 62.5 mg of 5% imiquimod cream applied daily to the back skin. The pristimerin group (Pristimerin) received 62.5 mg of 5% imiquimod cream applied daily to the back skin, followed by a local subcutaneous injection of an equal volume of F127 gel containing pristimerin particles 2 hours later. The pristimerin exosome group (Pri@exo) received 62.5 mg of 5% imiquimod cream applied daily to the back skin, followed by a local subcutaneous injection of 100 μg of pristimerin-loaded melanoma cell exosomes 2 hours later. The experimental period lasted for seven consecutive days, and all mice were killed on the eighth day. Psoriasis model: A psoriasis-like mouse model was established, and the PASI score was used to compare the skin lesions of each group after treatment. Histological examination: The skin tissue of the modeling area on the eighth day was obtained, and HE staining was performed to observe and compare the epidermal length of each group and Baker score was used for pathological sections; Figure 9 As shown in the figure, the IMQ group showed obvious skin erythema, scaling and infiltration, while these symptoms were significantly alleviated in the Pri@exo group. The relief in the Pristimerin group was not as significant as that of Pri@exo. Figure 10 The epidermal thickness of the Pri@exo group was close to that of the normal control group, and was significantly alleviated compared with the model group (IMQ). No obvious acanthosis, parakeratosis and other typical psoriasis pathological manifestations were observed. Figure 11 As shown in the figure, the epidermal thickness of the Pri@exo group was significantly reduced compared with the IMQ group; Baker score is a psoriasis pathology scoring system, and the effect of Pri@exo was better than that of the Pristimerin group. Figure 12As shown, PASI is a psoriasis severity scoring system that determines the severity of psoriasis based on erythema, scaling, infiltration and cumulative scores. The Pri@exo group showed significant relief.

Claims

1. An exosome composite nanohydrogel for targeted treatment of psoriasis, comprising a psoriasis drug, exosomes that highly express PD-L1, and a gel matrix F127, characterized in that: The psoriasis drug is first embedded into nanoparticles using a portion of the F127 gel matrix, which are then loaded into the exosomes to form a hydrogel with another portion of the F127 gel matrix. The psoriasis drug is tripterine or celastrol. The exosome-derived cells that highly express PD-L1 are selected from animal melanoma cells and human melanoma cells. The exosome-composite nanohydrogel is prepared by the following method, which includes the following steps: 1) Preparation of psoriasis drug nanoparticles The psoriasis drug was dissolved in dichloromethane, mixed with a 1.0% F127 aqueous solution, ultrasonically mixed to form an O / W emulsion, and freeze-dried to form nanoparticles; 2) Isolation and preparation of exosomes with high surface expression of PD-L1 Exosome-derived cells that highly express PD-L1 were cultured in DMEM complete medium. After passage and culturing for 48 hours, the supernatant was collected, and dead cells and cell debris were removed. The vesicles were then removed to obtain exosomes. The cells were resuspended in an appropriate amount of PBS and stored at -80°C for later use. 3) Preparation of drug-loaded exosomes a. Mixing the exosomes from step 2) with the drug nanoparticles from step 1) at a mass ratio of 2:1 to obtain a mixed solution; b. Pulse ultrasonic treatment of the mixed solution in step a and incubation at 37°C for 1 h to restore exosome membrane stability; c. Remove free drugs to obtain drug-loaded exosomes; 4) Preparation of hydrogel PF-127 powder is dissolved in a dispersion medium to prepare a hydrogel solution with a mass fraction of 20-30%, and a drug-loaded exosome solution is added and mixed to prepare a composite nanohydrogel.

2. The exosome composite nano-hydrogel according to claim 1, wherein the preparation of the nanoparticles prepared in step 1) further comprises removing excess F127 by dialysis.

3. The exosome composite nano-hydrogel according to claim 1, wherein in step 3) a, the exosomes are 100 μg and the drug nanoparticles are 50 μg.

4. The exosome composite nanohydrogel according to claim 1, wherein in step 3) b, the ultrasonic treatment comprises pulsed ultrasound on / off for 5 seconds each, repeated 5 times.

5. The exosome composite nano-hydrogel according to claim 1, wherein in the preparation, in step 4), the dispersion medium is PBS, and the mass fraction of PF-127 in the hydrogel solution is 30%.

6. The exosome composite nano-hydrogel according to claim 1, wherein in the preparation, in step 4), the mass ratio of PF-127 in the composite nano-hydrogel is 25%.

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