A novel targeted drug delivery system based on rose hip extracellular vesicles

Abstract

This invention relates to the fields of biomedical engineering and drug delivery technology, specifically disclosing a novel targeted drug delivery system based on rosehip extracellular vesicles, comprising: preparing rosehip-derived extracellular vesicles; preparing drug-loaded vesicles; and preparing targeted modified drug-loaded vesicles. This invention utilizes the specific downregulation of stem cell-related pathways by rosehip components, while simultaneously leveraging doxorubicin to kill conventional tumor cells, forming a complementary therapeutic mechanism to fundamentally reduce the risk of tumor recurrence. The phenylboronic acid group of DSPE-PEG-PBA specifically recognizes sialic acid overexpressed on the surface of tumor cells, significantly increasing drug accumulation in tumor tissues while reducing distribution in normal tissues. By leveraging the natural biocompatibility and low immunogenicity of RHNVs, combined with the long-circulating properties of the PEG chain, systemic toxicity is minimized while ensuring delivery efficiency, particularly reducing the cardiotoxicity and bone marrow suppression of doxorubicin.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering and drug delivery technology, specifically relating to a novel targeted drug delivery system based on extracellular vesicles of rose fruit. Background Technology

[0002] Tumor stem cells and their dangers:

[0003] Breast cancer (BC) is one of the most common cancers, accounting for approximately 30% of new cancer cases in women and ranking as the second leading cause of cancer-related deaths in annual statistics. Treatment options for breast cancer, including mastectomy, radiotherapy (RT), chemotherapy (CT), hormone therapy (HT), and other newer therapies, are determined based on individual clinicopathological characteristics. However, many BC patients still experience recurrence within a few years, and the long-term mortality rate remains high, fluctuating between 41.3% and 49.5% over 15 years. Tumor stem cell-like cells, also known as "tumor initiating cells," are considered one of the defining factors of intratumoral heterogeneity in breast cancer, exhibiting characteristics such as self-renewal, high tumorigenicity, invasiveness, and resistance to conventional radiotherapy and chemotherapy. The presence of CSCs has been shown to be a major culprit in breast cancer progression, recurrence, and metastasis. As a typical undifferentiated cancer cell phenotype, the content of CSCs is much higher in highly malignant tumors and poorly differentiated tumors associated with poor prognosis. Extensive research has been conducted to develop promising anti-CSC strategies, including blocking surface biomarkers and inhibiting self-renewal signaling pathways, but the results have been unsatisfactory.

[0004] The shortcomings of traditional doxorubicin treatment:

[0005] Doxorubicin, a highly effective anthracycline chemotherapy drug, remains one of the core treatments for breast cancer. However, its significant clinical benefits are facing severe constraints from two key scientific challenges: tumor stemness induction and systemic toxicity.

[0006] 1. First, mounting evidence suggests that while doxorubicin kills rapidly proliferating tumor cells, it also exerts a strong selective pressure, enriching and inducing a subset of tumor cells with stem cell-like characteristics. These cells typically exhibit CD44+ / CD24- or high expression of stem cell markers such as ALDH1, demonstrating self-renewal, multi-lineage differentiation, and strong treatment resistance. The mechanism involves doxorubicin activating key stem cell signaling pathways such as Wnt / β-catenin and Notch, and upregulating the expression of core stem cell transcription factors such as OCT4, SOX2, and NANOG. These surviving tumor stem cells after treatment are considered a root cause of tumor recurrence and distant metastasis, significantly limiting the long-term efficacy of doxorubicin.

[0007] 2. Secondly, the broad toxicity of doxorubicin to normal human tissues severely limits its clinical application dosage and duration, impacting patients' quality of life. Its most prominent dose-limiting toxicity is irreversible cardiotoxicity, closely related to mechanisms such as doxorubicin inducing reactive oxygen species (ROS) and triggering ferroptosis in cardiomyocytes. Furthermore, it can cause severe bone marrow suppression, leading to neutropenia and thrombocytopenia, increasing the risk of infection and bleeding; as well as other common side effects such as severe nausea and vomiting, hair loss, and mucositis. These widespread toxicities stem from doxorubicin's non-specific killing effect on normal, rapidly proliferating cells and its inherent cytotoxic mechanism, resulting in a narrow therapeutic window.

[0008] 3. Rose hips have the ability to reduce cell stemness:

[0009] Studies have shown that rosehip extract and its active ingredients have demonstrated the potential to significantly reduce tumor cell stemness in various tumor models. The core mechanism lies in directly targeting key signaling pathways that maintain stem cell characteristics.

[0010] First, polyphenols (such as ellagic acid and proanthocyanidins) in rose hips have been shown to effectively inhibit the activity of classic stem cell-related pathways such as Wnt / β-catenin, Notch, and Hedgehog. By interfering with these pathways, rose hip extract can downregulate the expression of core stem cell transcription factors such as Nanog, Sox2, and Oct-4, thereby weakening the self-renewal capacity of tumor cells.

[0011] In functional experiments, the ability of cancer cells treated with rose hips to form tumor spheroids was significantly reduced, providing direct evidence of suppressed cell stemness. Simultaneously, the differentiation degree of these cells increased, while their malignancy decreased. Further animal experiments also showed that rose hip intervention significantly reduced the in vivo tumorigenicity of cancer cells, meaning that both the efficiency of tumor formation and the tumor growth rate decreased after inoculation.

[0012] 4. Advantages of rosehip-derived extracellular vesicles (RHNVs):

[0013] It is worth noting that in recent years, plant-derived extracellular vesicles have shown unique advantages, providing new avenues for doxorubicin delivery. Compared with animal-derived RHNVs, plant RHNVs have outstanding advantages such as wide availability, low cost, no risk of human pathogen contamination, and inherently good biocompatibility. Studies have shown that plant RHNVs, due to their natural lipid bilayer structure, have high compatibility with mammalian cell membranes, can be effectively internalized, and due to their inherent anti-inflammatory and biocompatibility properties, can further reduce potential immune responses.

[0014] Based on the aforementioned characteristics of RHNVs, using them to load doxorubicin is considered an effective strategy to address the widespread toxicity of doxorubicin. Its core advantages are reflected in the following aspects: First, the lipid bilayer of RHNVs can efficiently encapsulate doxorubicin, forming a "nanoscale protective chamber." This encapsulation not only protects doxorubicin from premature degradation in systemic circulation but also enables passive or active targeted accumulation of the drug at the tumor site through the EPR effect (enhanced permeability and retention effect) or through engineered target modification. This means that, while achieving the same or even better tumor-suppressive effect, the required systemic exposure dose of doxorubicin can be significantly reduced.

[0015] Secondly, this enhanced targeting directly translates into reduced toxicity to normal tissues. Because RHNVs can deliver more doxorubicin to tumor tissues, they reduce non-specific distribution of the drug in critical organs such as the heart and bone marrow, potentially significantly alleviating its dose-limiting cardiotoxicity and bone marrow suppression. The low toxicity and biodegradability of plant-derived RHNVs further avoid the long-term bioaccumulation toxicity risks associated with traditional synthetic nanomaterials, creating a "double attenuation" effect.

[0016] 5. DSPE-PEG-PBA nanocarrier system:

[0017] In the field of targeted cancer therapy, utilizing biomarkers overexpressed on the surface of cancer cells to achieve precise drug delivery is a research hotspot. Sialic acid, a carbohydrate molecule abnormally highly expressed on the surface of various cancer cells, is closely related to tumor malignancy and poor prognosis, making it an ideal target.

[0018] Based on this, researchers developed the DSPE-PEG-PBA nanocarrier system. The core advantage of this system lies in its terminal phenylboronic acid group, which can specifically form a borate ester bond with the cis-dihydroxy structure of sialic acid under the slightly acidic conditions of tumors, achieving active targeting of cancer cells. Simultaneously, the PEG chain of this system effectively prolongs the circulation time of the nanocarrier in the blood, enhancing its accumulation in tumor tissues.

[0019] This design, which combines long-acting circulation with precise targeting, makes DSPE-PEG-PBA a delivery system with great clinical translation potential, providing a new strategy for improving the efficacy and safety of cancer treatment.

[0020] The shortcomings of existing technology:

[0021] Currently, the strategy of combining natural products with chemotherapy drugs to treat breast cancer mainly faces the following technical bottlenecks:

[0022] 1. Insufficient delivery efficiency and targeting: Although the active ingredients of rosehip can inhibit tumor stem cells, they have problems such as poor water solubility, low in vivo stability and lack of tumor targeting, resulting in limited bioavailability and difficulty in achieving effective therapeutic concentrations at the tumor site.

[0023] 2. Significant limitations of single therapy: Although the DSPE-PEG-PBA system can achieve targeted delivery, it mainly delivers traditional chemotherapy drugs and has insufficient specific killing ability against tumor stem cells, thus failing to fundamentally solve the problem of tumor recurrence.

[0024] 3. The biocompatibility of existing carriers needs to be improved: Synthetic nanocarriers (such as polymer micelles) still pose potential risks of immunogenicity and long-term toxicity, while the natural targeting ability of plant exovesicles (RHNVs) is limited, making it difficult to achieve precise drug delivery.

[0025] The present invention aims to overcome the above-mentioned technical defects by constructing a novel targeted delivery system that co-loads the active ingredients of rosehip with doxorubicin into rosehip-derived extracellular vesicles (RHNVs) modified with DSPE-PEG-PBA.

[0026] In response, this application proposes a novel targeted drug delivery system based on extracellular vesicles of rose fruit to address the aforementioned problems. Summary of the Invention

[0027] The purpose of this invention is to provide a novel targeted drug delivery system based on extracellular vesicles of rose fruit to solve the problems mentioned in the background art.

[0028] To achieve the above objectives, the present invention provides the following technical solution:

[0029] A novel targeted drug delivery system based on rosehip extracellular vesicles includes:

[0030] Preparation of extracellular vesicles derived from rose fruit: Rose fruit was treated, dissolved in buffer solution, and subjected to differential centrifugation and ultracentrifugation. The precipitate was collected and resuspended to obtain extracellular vesicles (RHNVs) derived from rose fruit.

[0031] Preparation of drug-loaded vesicles: The RHNVs and drugs were mixed in proportion, incubated, and then centrifuged at high speed. The precipitate was collected and resuspended to obtain drug-loaded rosehip-derived extracellular vesicle RHNVs.

[0032] Preparation of targeted modified drug-loaded vesicles: The targeted modification material was prepared into micelles, mixed with the RHNVs drug in a certain proportion, and after incubation, it was subjected to ultracentrifugation, the precipitate was collected and resuspended to obtain the targeted modified drug-loaded extracellular vesicle TRHNVs drug derived from rose fruit.

[0033] Preferably, the method for processing the rose hips is as follows: after washing and accurately weighing the rose hips, they are crushed using a juicer.

[0034] Preferably, the buffer solution is PBS buffer;

[0035] The differential centrifugation conditions are as follows: at 4℃, centrifuge at 1200 g for 20 min, 3000 g for 30 min, and 10000 g for 60 min in sequence, and collect the supernatant after each centrifugation.

[0036] Preferably, the ultracentrifugation conditions are: centrifuging the supernatant after differential centrifugation at 4°C and 100,000 g for 90 min; and the storage conditions for the RHNVs are: storage at -20°C for 6 months or long-term storage at -80°C.

[0037] Preferably, the drug is DOX;

[0038] The mixing of RHNVs and drugs is carried out in the following proportion: the ratio of RHNVs protein concentration to DOX drug concentration is 1:1, and the same volume is mixed in a round-bottom flask.

[0039] Preferably, the incubation conditions are: 2 h at 37°C and 200 rpm in an intelligent constant temperature incubator; the ultracentrifugation is specifically: centrifugation at 4°C and 130000×g for 90 min.

[0040] Preferably, the targeted modification material is DSPE-PEG-PBA; the method for preparing micelles is as follows: accurately weigh 10.0 mg of DSPE-PEG-PBA, dissolve it in 1×HEPES buffer, incubate it in a 60°C water bath for 15-30 minutes and vortex intermittently until the solution is clear to form micelles.

[0041] Preferably, the micelles are further subjected to ultrasonic treatment after preparation: under ice bath conditions, ultrasonic treatment is performed using a probe ultrasonic instrument with an amplitude of 10 μM for 2 × 5 s.

[0042] Preferably, the conditions for preparing the targeted modification material into micelles and mixing it with the RHNVs drug in a certain proportion are: the mass ratio of DSPE-PEG-PBA to the total RHNVs protein is 1:10; the conditions for the incubation reaction are: incubation in a 40°C water bath for 1-2 hours.

[0043] Preferably, the ultracentrifugation specifically refers to centrifugation at 4℃ and 130000×g for 90 min.

[0044] Compared with the prior art, the beneficial effects of the present invention are:

[0045] (1) This invention uses rosehip components to specifically downregulate stem-related pathways, while using doxorubicin to kill conventional tumor cells, forming a complementary therapeutic mechanism to fundamentally reduce the risk of tumor recurrence.

[0046] (2) The present invention utilizes the phenylboronic acid group of DSPE-PEG-PBA to specifically recognize sialic acid overexpressed on the surface of tumor cells, which significantly improves the enrichment of drugs in tumor tissues and reduces the distribution to normal tissues.

[0047] (3) This invention utilizes the natural biocompatibility and low immunogenicity of RHNVs, combined with the long-cycle characteristics of PEG chains, to minimize systemic toxicity while ensuring delivery efficiency, especially reducing the cardiotoxicity and bone marrow suppression of doxorubicin. Attached Figure Description

[0048] Figure 1 This is a scanning electron microscope image of RHNVs of the present invention;

[0049] Figure 2 Here are RHNVs / DOX scanning electron microscope images of the present invention;

[0050] Figure 3 Zeta potential images of RHNVs, RHNVs / DOX, and TRHNVs / DOX of the present invention;

[0051] Figure 4 Malvern particle size distributions of RHNVs, RHNVs / DOX, and TRHNVs / DOX of this invention;

[0052] Figure 5 The size and zeta changes of the RHNV of the present invention within one week are represented as average ± SD (n = 3);

[0053] Figure 6 The following are UV-vis absorption spectra of DOX at different concentrations according to the present invention;

[0054] Figure 7 This is the DOX standard curve diagram of the present invention;

[0055] Figure 8 The FT-IR plots of DOX, RHNVs, DSPE-PEG-PBA, and TRHNVs / DOX of this invention are shown below.

[0056] Figure 9 The in vitro release curve data of DOX of the present invention are represented as mean ± SD (n = 3) plot;

[0057] Figure 10 This is a diagram showing the uptake of 4T1 cells by different experimental groups using laser confocal analysis according to the present invention (blue: cell nucleus, red: DOX);

[0058] Figure 11 The image shows the results of the CCK-8 evaluation of the toxic effects of each experimental group (DOX, RHNVs / DOX, TRHNVs / DOX) on 4T1 cells (left) and stem cells (right) in vitro.

[0059] Figure 12 This is a comparative graph showing the results of the CCK-8 evaluation of the toxic effects of RHNVs on 4T1 cells and 4T1 stem cells in vitro.

[0060] Figure 13 The diagram shows the WB experimental results of this invention;

[0061] Figure 14 Optical images of mouse tumors according to the present invention;

[0062] Figure 15 This is a graph showing the change in mouse tumor volume according to the present invention;

[0063] Figure 16 The tumor quality in mice after treatment according to the present invention is shown in the figure as mean ± SD (n = 7).

[0064] Figure 17 The changes in mouse body weight during the entire treatment period of this invention are represented as mean ± SD (n = 7);

[0065] Figure 18 This is a diagram showing the in vivo biosafety evaluation of the intravenous injection groups of the present invention.

[0066] Figure 19 The images show H&E staining of the main organs (including heart, liver, spleen, lung and kidney) in each experimental group of the present invention (scale bar: 100 μm). Detailed Implementation

[0067] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0068] Example 1:

[0069] A novel targeted drug delivery system based on rosehip extracellular vesicles includes:

[0070] Preparation of RHNVs, RHNVs / DOX, and TRHNVs / DOX:

[0071] 1. Preparation of RHNVs: Rose hips were washed, accurately weighed, and crushed using a juicer and dissolved in PBS. The collected rose hip phosphate buffer was centrifuged at 1200 g for 20 min, 3000 g for 30 min, and 10000 g for 60 min at 4 ℃, following a differential centrifugation process. The supernatant was collected after each centrifugation. The supernatant was then ultracentrifuged at 100000 g for 90 min at 4 ℃. The precipitate was collected and resuspended in PBS to obtain rose hip-derived extracellular vesicles (RHNVs). RHNVs can be stored at -20 ℃ for 6 months or at -80 ℃ for long-term storage.

[0072] 2. Preparation of RHNVs / DOX: To prepare RHNVs / DOX, a co-incubation method was used for drug loading. RHNVs and DOX were prepared at a protein concentration to drug concentration ratio of 1:1, and equal volumes were mixed in a round-bottom flask. The round-bottom flask was placed in an intelligent thermostatic shaker (Honour HNY-200B, China). The reaction was set at 37℃ and 200 rpm for 2 h to complete drug loading. The resulting RHNVs / DOX and RHNVs / DOX mixtures were centrifuged at 130,000 × g for 90 min at 4℃. The precipitate was collected and resuspended in PBS to obtain RHNVs / DOX.

[0073] 3. Preparation of TRHNVs / DOX: Accurately weigh 10.0 mg DSPE-PEG-PBA and dissolve it in 1× HEPES buffer. Incubate in a 60℃ water bath for 15-30 minutes, intermittently vortexing, until the solution becomes clear and micelles are formed. Sonicate the micelle solution briefly at 10 μM amplitude for 2×5 s using a probe sonicator under ice bath conditions. Add the treated micelle solution to the RHNVs / DOX suspension at a certain ratio (DSPE-PEG-PBA:total RHNVs protein = 1:10, w / w). Incubate the mixture in a 40℃ water bath for 1-2 hours. Centrifuge at 130000×g for 90 min at 4℃, collect the precipitate, and resuspend in PBS to obtain PBA-RHNVs / DOX.

[0074] As can be seen from the above, by specifically downregulating stem-related pathways through rosehip components and simultaneously using doxorubicin to kill conventional tumor cells, a complementary therapeutic mechanism is formed, fundamentally reducing the risk of tumor recurrence.

[0075] Achieving precise targeted delivery: Utilizing the phenylboronic acid group of DSPE-PEG-PBA to specifically recognize sialic acid overexpressed on the surface of tumor cells, significantly improving drug enrichment in tumor tissues while reducing distribution to normal tissues.

[0076] Enhancing treatment safety: By leveraging the natural biocompatibility and low immunogenicity of RHNVs, combined with the long-circulating properties of PEG chains, systemic toxicity is minimized while ensuring delivery efficiency, particularly reducing the cardiotoxicity and bone marrow suppression of doxorubicin.

[0077] Example 2:

[0078] Characterization of RHNVs, RHNVs / DOX, and TRHNVs / DOX:

[0079] 1. For example Figure 1 and Figure 2 As shown, the surface morphology of RHNVs and RHNVs / DOX was characterized by scanning electron microscopy. The images show that they all have a near-spherical structure with a size of 100-200 nm.

[0080] 2. For example Figure 3 As shown, in order to demonstrate the biocompatibility of RHNVs, RHNVs / DOX, and TRHNVs / DOX, potential analysis was then performed on the three. Zeta potential measurements showed that all three had strong negative charges on their surfaces, indicating that they all have good biocompatibility.

[0081] 3. For example Figure 4 As shown, the hydrated particle size of RHNVs, RHNVs / DOX, and TRHNVs / DOX, as determined by Malvern, is within 200 nm, slightly larger than the size captured by scanning electron microscopy. The nanoscale size of both can prolong the circulation time of extracellular vesicles in the blood and facilitate their aggregation at the tumor site through the EPR effect.

[0082] 4. For example Figure 5 As shown, in order to test the in vitro structural stability of RHNVs, particle size was measured for seven consecutive days. The Malvern particle size analysis showed that the particle size of both was between 100-200 nm for seven consecutive days, with no significant change, which proves that RHNVs have good structural stability.

[0083] III. Verification of successful TRHNV preparation:

[0084] 1. For example Figure 6 and Figure 7As shown, during the drug loading process, we used an ultraviolet spectrophotometer to perform spectral analysis on doxorubicin standards of different concentrations, obtained ultraviolet spectra of doxorubicin standards of different concentrations, and thus obtained the standard curve of doxorubicin standards.

[0085] 2. Spectral analysis:

[0086] like Figure 8 The UV-vis absorption spectra of DOX, RHNVs, DSPE-PEG-PBA, and TRHNVs / DOX are shown below.

[0087] 3. Infrared spectral analysis showed that TRHNVs / DOX contained amino groups (3525 cm⁻¹). -1 ), hydroxyl (3329 cm) -1 ), carbonyl (1730 cm) -1 ), benzene ring (1523 cm) -1 Characteristic peaks such as -CH2- and -CH3 (2918 cm⁻¹) were observed in TRHNVs / DOX, similar to those found in the FT-IR spectra of RHNVs and doxorubicin functional groups. These results indicate that DOX was successfully loaded into RHNVs. Simultaneously, -CH2- and -CH3 (2918 cm⁻¹) peaks were observed in TRHNVs / DOX. -1 The characteristic peaks indicate that DSPE-PEG-PBA has been successfully modified onto the surface of nanoparticles.

[0088] IV. TRHNVs / DOX in vitro drug release assay:

[0089] like Figure 9 As shown, in vitro drug release experiments clarified the drug release pattern of TRHNVs / DOX under different pH conditions (pH 5.3, 6.5, and 7.4). The drug release curves showed that the DOX release rate increased continuously with decreasing pH. When pH=5.3, the DOX release rate was 68.59%; when pH=6.5, the DOX release rate was 51.46%; and when pH=7.4, the DOX release rate was 38.02%. TRHNVs / DOX has a good drug release rate, and the drug release rate is higher in the tumor microenvironment than in the normal physiological environment, providing data support for subsequent in vivo dosing design.

[0090] In vitro DOX release curves of TRHNVs / DOX in PBS at pH 7.4, pH 6.5, and pH 5.3.

[0091] V. In vivo experiments:

[0092] like Figure 10As shown, through laser confocal microscopy experiments, it was directly observed that TRHNVs / DOX was more effectively taken up by breast cancer cells and tumor stem cells than RHNVs / DOX, confirming the cell delivery capability of the carrier.

[0093] 2. For example Figure 11 and Figure 12 As shown in the CCK-8 cytotoxicity assay, TRHNVS / DOX preliminarily verified that it had the most significant killing effect on ordinary 4T1 cells and 4T1 stem cells. Comparing the two figures, it can be seen that with increasing drug concentration, TRHNVs / DOX and RHNVs / DOX were more effective at killing 4T1 stem cells than ordinary breast cancer cells. Furthermore, compared to free DOX, TRHNVs / DOX and RHNVs / DOX are safer, reducing the widespread toxicity of DOX.

[0094] Furthermore, with increasing RHNV dosage, RHNVs were more effective at killing 4T1 stem cells (CSCs) than ordinary breast cancer cells (4T1).

[0095] 3. For example Figure 13 As shown in the Western blot results, the expression levels of CD133 and ALDH1, positively correlated markers of stemness in DOX, RHNVs / DOX, and TRHNVs / DOX, gradually decreased, while the expression level of CD24, a negatively correlated regulator, gradually increased. These results indicate that TRHNVs / DOX can significantly reduce the stemness of 4T1 stem cells compared to RHNVs / DOX and DOX alone.

[0096] The six holes from left to right are:

[0097] 1.4T1 cells + PBS

[0098] 2.4T1 CSC (4T1 stem cells) + PBS

[0099] 3.4T1 CSC (4T1 stem cells) + RHNVs

[0100] 4.4T1 CSC (4T1 stem cells) + DOX

[0101] 5.4T1 CSC (4T1 stem cells) + RHNVs / DOX

[0102] 6.4T1 CSC (4T1 stem cells) + TRHNVs / DOX

[0103] In comparison, DOX has almost no ability to reduce stemness, while rosehip extracellular vesicles (RHNVs) have the ability to reduce stemness. The ability to reduce stemness decreases from TRHNVs / DOX to RHNVs / DOX to RHNVs.

[0104] VI. In vitro experiments:

[0105] 1. For example Figure 14 As shown in the optical images, compared to the PBS group, RHNVS, DOX, RHNVS / DOX, and TRHNVs / DOX all reduced tumor size, but the TRHNVs / DOX group showed the strongest ability to reduce tumor size. In the PBS group, due to the lack of drug treatment, the tumor volume naturally increased. Compared to the PBS group, the RHNVs group showed reduced tumor growth, but the tumors remained large. Compared to the PBS group, the DOX group showed reduced tumor growth, but the tumors were slightly larger but smaller than those in the RHNVS group. RHNVS / DOX and TRHNVs / DOX groups effectively inhibited tumor growth, but the TRHNVs / DOX effect was more significant, thanks to the combined targeted killing of tumor cells and inhibition of stemness by TRHNVs loaded with doxorubicin.

[0106] like Figure 15 As shown in the figure, the tumor volume change over 14 consecutive days showed that the tumor volume in each group continued to increase, but compared with the tumor volume in the PBS group, TRHNVs / DOX showed the strongest tumor growth inhibition ability.

[0107] 3. For example Figure 16 As shown, the tumor weight results after mouse sacrifice are consistent with the results of tumor optical images and tumor volume;

[0108] 4. In vivo safety evaluation:

[0109] (1). For example Figure 17 As shown, the potential toxicity of MTVs in vivo following intravenous administration was then assessed. No significant abnormalities in feeding, grooming, activity, or excretion were observed in mice during the experiment. There were no significant changes in body weight in any group of mice over the 14 days of treatment.

[0110] (2) Collect mouse blood for routine blood analysis and biochemical examination:

[0111] like Figure 18 As shown, biochemical test results indicated that there were no significant differences in key blood parameters such as red blood cells, HGB, and PLT between the mice in each group and the control group (PBS group), indicating that no adverse symptoms such as anemia were observed in any group of mice. Liver damage indicators (AST, ALT) and kidney damage indicators (BUN) in each treatment group were within safe ranges, indicating that neither treatment had any effect on liver and kidney function in the mice.

[0112] (3) H&E staining images:

[0113] like Figure 19As shown, histological analysis of the major organs (heart, liver, spleen, lung, and kidney) of mice collected by H&E staining showed that the heart tissue of the DOX group showed cardiotoxicity, while the major organs of the other groups (PBS, RHNVs, RHNVs / DOX, and TRHNVs / DOX) maintained normal physiological morphology and no inflammatory response was observed. These results indicate that at the tested dose, TRHNVs / DOX has almost no toxicity in vivo and has excellent biosafety.

[0114] As can be seen from the above, the present invention has the following advantages:

[0115] 1. Significantly improved synergistic treatment effects:

[0116] In the 4T1 breast cancer mouse model, the tumor inhibition rate of the TRHNVs / DOX group was significantly higher than that of the control groups, and the tumor volume was reduced.

[0117] Western blotting and immunofluorescence confirmed that TRHNVs / DOX significantly downregulated the expression of CD133 and ALDH1, positively correlated markers of stem cell function, upregulated the expression of CD24, negatively correlated marker, and reduced tumor stem cells.

[0118] 2. Systemic toxicity is significantly reduced:

[0119] Blood biochemistry analysis showed that liver injury markers (AST, ALT) and kidney injury markers (BUN) in the TRHNVs / DOX treatment group mice were all within safe ranges.

[0120] H&E staining confirmed that the cardiac tissue in the TRHNVs / DOX group maintained normal physiological morphology, and no typical cardiotoxic lesions typical of doxorubicin were observed.

[0121] During the treatment, the mice maintained a stable weight, showed no obvious toxic reactions, and their hematological parameters were not significantly different from those of the control group.

[0122] The CCK8 experiment confirmed that loading doxorubicin with RHNVs can significantly reduce the cytotoxic effects of doxorubicin.

[0123] 3. Optimization of targeting and drug release characteristics:

[0124] Laser confocal experiments confirmed that TRHNVs / DOX are taken up more effectively by breast cancer cells and tumor stem cells than RHNVs / DOX.

[0125] In vitro release experiments showed that TRHNVs / DOX had pH-sensitive drug release characteristics. Under the condition of tumor microenvironment pH=5.3, the DOX release rate reached 68.59%, while under the condition of physiological pH=7.4, it was only 38.02%.

[0126] In vivo distribution studies showed that drug accumulation was increased at tumor sites and decreased in the heart.

[0127] 4. Significant advantages in production technology and economic efficiency:

[0128] By using rosehips as both a source of active ingredients and a natural carrier material, the cost of raw materials is reduced.

[0129] The preparation steps for carriers based on natural exovesicles are reduced, the production cycle is shortened, and energy consumption is lowered.

[0130] The use of organic solvents is reduced, making the production process more environmentally friendly.

[0131] 5. Excellent stability and biocompatibility:

[0132] The formulation is stable for 6 months at 4°C, which meets the requirements for clinical use.

[0133] Malvern particle size analysis showed that the particle size remained between 100-200 nm for seven consecutive days without significant change. Zeta potential measurements indicated that the surface had a strong negative charge and good biocompatibility.

[0134] This invention achieves a dual improvement in therapeutic efficacy and safety through innovative engineering modifications, while also possessing multiple advantages such as simplified production processes, reduced costs, and enhanced stability.

[0135] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A novel targeted drug delivery system based on extracellular vesicles of rose fruit, characterized in that, include: Preparation of extracellular vesicles derived from rose fruit: Rose fruit was treated, dissolved in buffer solution, and subjected to differential centrifugation and ultracentrifugation. The precipitate was collected and resuspended to obtain extracellular vesicles (RHNVs) derived from rose fruit. Preparation of drug-loaded vesicles: The RHNVs and drugs were mixed in proportion, incubated, and then centrifuged at high speed. The precipitate was collected and resuspended to obtain drug-loaded rosehip-derived extracellular vesicle RHNVs. Preparation of targeted modified drug-loaded vesicles: The targeted modification material was prepared into micelles, mixed with the RHNVs drug in a certain proportion, and after incubation, it was centrifuged at high speed, the precipitate was collected and resuspended to obtain the targeted modified drug-loaded extracellular vesicle TRHNVs drug derived from rose fruit. The method for processing the rose hips is as follows: wash the rose hips, weigh them accurately, and then crush them using a juicer; The drug in question is DOX; The mixing of RHNVs and drugs in the following proportion is as follows: the ratio of protein concentration of RHNVs to drug concentration of DOX is 1:1, and the same volume is mixed in a round-bottom flask. The targeted modification material is DSPE-PEG-PBA; the method for preparing micelles is as follows: accurately weigh 10.0 mg DSPE-PEG-PBA, dissolve it in 1×HEPES buffer, incubate it in a 60℃ water bath for 15-30 minutes and vortex intermittently until the solution is clear to form micelles; The conditions for preparing the targeted modification material into micelles and mixing it with the RHNVs drug in the specified ratio are: the mass ratio of DSPE-PEG-PBA to the total RHNVs protein is 1:10; the conditions for the incubation reaction in the preparation of the targeted modified drug-loaded vesicles are: incubation in a 40°C water bath for 1-2 hours.

2. The novel targeted drug delivery system based on extracellular vesicles of rose fruit according to claim 1, characterized in that, The buffer solution used to prepare the extracellular vesicles derived from rose fruit was PBS buffer. The differential centrifugation conditions are as follows: at 4℃, centrifuge at 1200 g for 20 min, 3000 g for 30 min, and 10000 g for 60 min in sequence, and collect the supernatant after each centrifugation.

3. The novel targeted drug delivery system based on rosehip extracellular vesicles according to claim 1, characterized in that, The conditions for ultracentrifugation in preparing extracellular vesicles from rose fruit are as follows: the supernatant after differential centrifugation is centrifuged at 4°C and 100,000 g for 90 min; the storage conditions for the RHNVs are -20°C for 6 months or -80°C for long-term storage.

4. A novel targeted drug delivery system based on extracellular vesicles of rosehips according to claim 1, characterized in that, The incubation conditions for preparing drug-loaded vesicles are as follows: reaction at 37°C and 200 rpm for 2 h in an intelligent constant temperature incubator; the ultracentrifugation for preparing drug-loaded vesicles is as follows: centrifugation at 4°C and 130,000 × g for 90 min.

5. A novel targeted drug delivery system based on extracellular vesicles of rosehips according to claim 1, characterized in that, The micelles are prepared by ultrasonic treatment: under ice bath conditions, ultrasonic treatment is performed for 2 × 5 s using a probe ultrasonic instrument with an amplitude of 10 μM.

6. A novel targeted drug delivery system based on extracellular vesicles of rosehips according to claim 1, characterized in that, The ultracentrifugation in the preparation of targeted modified drug-loaded vesicles specifically involves centrifugation at 4°C and 130,000 × g for 90 min.

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