Application of an immune homeostasis-regulating nano-formulation in the treatment of sepsis

Abstract

This invention discloses the application of an immune homeostasis-regulating nanoparticle in the treatment of sepsis, belonging to the field of biomedical technology. Using a biomass compound library constructed from natural amino acids and monosaccharides as precursors and water as a solvent, this invention successfully prepared tryptophan-sorbitol carbon quantum dots (WS-CQDs) via a bottom-up one-step hydrothermal method. This nanoparticle formulation is simple to prepare, low in cost, highly biocompatible, and virtually non-toxic. Experiments showed that WS-CQDs effectively alleviated the "first hit" (excessive inflammation) and reduced mortality in the "second hit" (immunosuppression) stage of CLP-induced sepsis mice, exhibiting a dual protective effect. This finding suggests that WS-CQDs are a nanomaterial capable of regulating immune homeostasis, possessing significant potential for sepsis treatment and clinical translational value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of an immune homeostasis regulating nano-formulation in the treatment of sepsis. Background Technology

[0002] Sepsis is a life-threatening organ dysfunction caused by a dysregulated response to infection (RYNNE J, MOSAVIE M, MASSE MH, et al. Sepsis subtypes and differential treatment response to vitamin C: biological sub-study of the LOVIT trial [J]. Intensive Care Med, 2025, 51(1): 82-93.;GALLANT RM, SANCHEZ KK, JOULIA E, et al. Fluoxetine promotes IL-10-dependent metabolic defenses to protect from sepsis-induced lethality [J]. Sci Adv, 2025, 11(7): eadu4034.). According to The Lancet, sepsis causes as many as 11 million deaths annually (BRANDES-LEIBOVITZ R, RIZA A, YANKOVITZ G, et al. Sepsis pathogenesis and outcome are shaped by the balance between the transcriptional states of systemic inflammation and antimicrobial response[J]. Cell Rep Med, 2024, 5(11): 101829.), accounting for 35.5% of all deaths (XIE J, WANG H, KANG Y, et al. The Epidemiology of Sepsis in Chinese ICUs: A National Cross-Sectional Survey [J]. Crit Care Med, 2020, 48(3): e209-e18.).The core pathological features of this disease are, in turn, the initial infection triggers a severe systemic inflammatory response, which constitutes a "first blow" to the body; then the immune system changes from over-activation to continuous suppression, entering a more lethal "second blow" stage, which often manifests clinically as serious complications such as secondary nosocomial infections and colonization of multidrug-resistant bacteria (WILLMANN K, MOITA L F. Physiologic disorder and metabolic reprogramming in infection and sepsis [J]. CellMetab, 2024, 36(5): 927-46.). Current clinical protocols, which mainly consist of supportive care and broad-spectrum antibiotics, can temporarily maintain vital signs, but they are still prone to exacerbating the immunosuppressive process in patients (WILLMANN K, MOITA L F. Physiologic disruption and metabolic reprogramming in infection and sepsis[J]. Cell Metab, 2024, 36(5): 927-46.;NEDEVA C, MENASSA J, DUAN M, et al. TREML4 receptor regulates inflammation and innate immune cell death during polymicrobial sepsis[J]. Nat Immunol, 2020, 21(12): 1585-96.). The latest clinical observations show that 85% of the deaths of sepsis patients are due to the "secondary blow" during the immunosuppression stage (XU J, GAO C, HE Y, et al. NLRC3 expression in macrophage impairs glycolysis and host immune defense by modulating the NF-kappaB-NFAT5 complex during septic immunosuppression [J]. Mol Ther, 2023, 31(1): 154-73.).This discovery not only explains why most previous clinical trials of drugs targeting the inflammatory phase (“first hit”) have failed (NEDEVA C, MENASSA J, DUAN M, et al. TREML4receptor regulates inflammation and innate immune cell death during polymicrobial sepsis [J]. Nat Immunol, 2020, 21(12): 1585-96.;CHEN F, ZOU L, WILLIAMS B, et al. Targeting Toll-Like Receptors in Sepsis: From Bench to Clinical Trials [J]. Antioxid Redox Signal, 2021, 35(15): 1324-39.), but also suggests that developing immune homeostasis modulators that have both the dual effects of intervening in excessive inflammation and improving immunosuppression has become an urgent need in current sepsis treatment research.

[0003] In recent years, several research teams have focused on restoring the immune function of sepsis patients using immunostimulants such as granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-17, interferon (IFN-γ), and PD-1 / PD-L1 inhibitors. However, regrettably, most studies have failed to significantly improve patient survival (LOUAGUENOUNIY, MANSART A, ANNANE D, et al. Nanoparticle-based approaches for sepsis treatment: Current trends and perspectives [J]. J Control Release, 2025, 386:114139.). Despite the great potential of these immunostimulants, their application in sepsis treatment often faces unfavorable pharmacokinetic characteristics due to poor biodegradability and low targeting efficiency.

[0004] Therefore, the application of nanoparticle delivery platforms can significantly improve the therapeutic effects of these compounds. Currently, several nanoparticle-based immunomodulatory strategies have been used in sepsis research, such as superparamagnetic iron oxide nanoparticles (SPIONs) coupled with β-glucan, and lipid nanoparticles loaded with the apolipoprotein A1-IL-4 complex. However, these systems still have certain limitations: 1) complex preparation processes and high costs; 2) poor biocompatibility, such as the potential for long-term retention of materials like SPIONs in vivo to induce liver fibrosis or oxidative stress; 3) significant side effects, such as the potential for liposomes to non-specifically activate the complement system; and 4) limited mechanisms of action, often targeting a specific pathological stage of sepsis—either inhibiting early excessive inflammation ("first hit") or intervening in later immunosuppression ("second hit").

[0005] Carbon quantum dots (CQDs), as an emerging carbon nanomaterial, have become a new generation of nano-preparations for controlling bacterial infections and regulating immune responses due to their low toxicity, good water solubility and easy functionalization, and have attracted widespread attention in the biomedical field. However, achieving a comprehensive combination of properties such as low toxicity, low cost, high purity, high yield, simple preparation, controllable reaction, good water solubility, and inherent pharmacological activity remains a huge challenge in this field (SUNL, ZHAO Y, PENG H, et al. Carbon dots as a novel photosensitizer for photodynamic therapy of cancer and bacterial infectious diseases: recent advances [J]. J Nanobiotechnology, 2024, 22(1): 210.;ZHANG B, FAN X, DU H, et al. Foodborne Carbon Dot Exposure Induces Insulin Resistance through GutMicrobiota Dysbiosis and Damaged Intestinal Mucus Layer [J]. Acs Nano, 2023,17(6): 6081-94.). Summary of the Invention

[0006] To address the aforementioned challenges in existing technologies, this invention utilizes a biomass compound library constructed from natural amino acids and monosaccharides as precursors and water as a solvent to successfully prepare tryptophan-sorbitol carbon quantum dots (WS-CQDs) via a one-step hydrothermal synthesis method. This nanoparticle formulation is simple to prepare, low in cost, highly biocompatible, and virtually non-toxic. Furthermore, experiments unexpectedly revealed that WS-CQDs can effectively alleviate the "first hit" (excessive inflammation) and reduce mortality in the "second hit" (immunosuppression) phase of CLP-induced sepsis in mice, exhibiting a dual protective effect. This discovery suggests that WS-CQDs are nanomaterials capable of regulating immune homeostasis, possessing significant potential for sepsis treatment and clinical translational value.

[0007] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: This invention mainly provides the application of tryptophan-sorbitol carbon quantum dots (WS-CQDs) in the preparation of drugs for treating sepsis.

[0008] Furthermore, the WS-CQDs are synthesized via a one-step hydrothermal method using tryptophan and sorbitol as carbon source precursors.

[0009] Furthermore, the preparation method of the WS-CQDs includes the following steps: (1) Dissolve tryptophan and sorbitol in water and mix well to obtain a mixed solution; (2) The mixed solution is subjected to a hydrothermal reaction to obtain crude WS-CQDs; (3) The crude WS-CQDs were purified to obtain purified WS-CQDs.

[0010] Furthermore, the mass ratio of tryptophan to sorbitol in step (1) is 1:(4~6).

[0011] Furthermore, the reaction conditions for the hydrothermal reaction in step (2) are: temperature 150~170 ℃, time 8~12 hours.

[0012] Furthermore, the purification in step (3) includes centrifugation and dialysis.

[0013] Furthermore, the centrifugation conditions are 11000~13000 g, 14~16 minutes.

[0014] Furthermore, the dialysis uses a dialysis bag with a molecular weight cutoff of 8-12 kDa.

[0015] Furthermore, the medicament for treating sepsis contains a therapeutically effective amount of WS-CQDs and a pharmaceutically acceptable carrier or excipient.

[0016] Furthermore, the drug for treating sepsis may be in the form of an injection.

[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention is the first to propose the application of WS-CQDs in the treatment of sepsis. The WS-CQDs are synthesized using a green and economical one-step hydrothermal method with natural amino acids and monosaccharides as precursors, using only water as a solvent. The reaction conditions are mild, and the resulting product has excellent biocompatibility and extremely low cytotoxicity. Compared with existing carbon quantum dot synthesis processes that rely on complex biological sources (such as E. coli cell walls, apoptotic vesicles, etc.), the preparation process of WS-CQDs in this invention uses readily available raw materials, has simple steps, and high yield. It not only avoids cumbersome biological extraction processes and significantly reduces costs, but also has good potential for large-scale and standardized production, laying a solid foundation for clinical medical translation.

[0018] (2) Existing carbon quantum dots in research have relatively singular functions, typically targeting only a single pathological stage of sepsis (such as inhibiting inflammation), making it difficult to address the complex and dynamically evolving pathological states during the disease's progression. The WS-CQDs in this invention overcome the limitations of existing carbon quantum dots with their singular functions, exerting a dual regulatory effect throughout the entire course of sepsis. They can both inhibit the early "first hit" (excessive inflammation) and reduce mortality associated with the later "second hit" (immunosuppression), demonstrating a unique potential for coordinated responses to opposing pathological stages of sepsis. To date, no single formulation has achieved such dual regulation, signifying a significant advantage of the WS-CQDs in this invention in the treatment of sepsis. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the experimental design in this invention.

[0020] Figure 2 This is the result of the cytotoxicity assessment of WS-CQDs on cells in Example 2 of the present invention. Cell viability was detected using CCK8 assay after co-incubation of RAW264.7 cells and Kupffer cells with different concentrations of WS-CQDs for 24 h and 36 h.

[0021] Figure 3 This is the in vivo toxicity assessment result of WS-CQDs on C57 / 6J mice in Example 3 of this invention. A) Blood routine related indicators: white blood cells (WBC), red blood cells (RBC), platelets (PLT), and hemoglobin (HGB); B) Liver and kidney function related indicators: alanine aminotransferase (ALT), total bilirubin (TBIL), lactate dehydrogenase (LDH), urea (UERA), and creatine kinase (CK). The difference was not statistically significant (ns); C) Representative image of tissue H&E staining. Scale bar: 100 μm.

[0022] Figure 4 This describes the effect of WS-CQDs on the release of inflammatory factors from LPS-induced RAW264.7 macrophages in Example 4 of the present invention. A) Western blotting was used to detect changes in the protein levels of inflammatory factors (TNF-α, IL-6, and IL-1β); B) qPCR was used to detect changes in the mRNA levels of inflammatory factors (TNF-α, IL-6, and IL-1β). , P <0.05, , P <0.01, , P <0.001.

[0023] Figure 5 This is the result of the effect of WS-CQDs on the immune response of monocytes and macrophages in Example 5 of the present invention. A) Schematic diagram of the construction of the in vitro immunosuppression model; B) qPCR detection of the effect of WS-CQDs on the mRNA level of the pro-inflammatory factor TNF-α in isolated CLP model BMDM cells after LPS secondary stimulation; C, D) Flow cytometry detection of the expression level of MHC-II on the surface of BMDM cells after LPS secondary stimulation and its statistical graph in the in vitro immunosuppression model. P < 0.005; P<0.001.

[0024] Figure 6 This describes the therapeutic effect of WS-CQDs on "one-hit" CLP sepsis mice in Example 6 of this invention. A) Survival analysis; B) Quantitative detection of serum inflammatory factors (TNF-α, IL-6, and IL-1β) in mice in the Sham group, CLP group, and CLP+WS-CQDs (5 mg / kg) group using an ELISA kit. , P <0.05, , P <0.01.

[0025] Figure 7This describes the therapeutic effect of WS-CQDs on the immunosuppressive phase of sepsis in Example 7 of this invention. A) Schematic diagram of the sepsis "second hit" model; B) KM survival curve analysis; C) Bacterial load and statistical graphs in peripheral blood, lung tissue, and bronchoalveolar lavage fluid (BALF) 24 hours after PA infection using the standard plate method; F) Representative images of HE staining of mouse lung, liver, and kidney tissues 24 hours after PA infection; , P <0.01.

[0026] Figure 8 This describes the regulatory effect of WS-CQDs on the immune microenvironment of mice with sepsis secondary infection in Example 8 of this invention. A) Serum inflammatory factor protein levels 12 h after administration (detected by ELISA and CBA); C) Lung tissue inflammatory factor protein levels 4 h after administration; D) Lung tissue cytokine mRNA expression levels 12 h after administration (qPCR). Detailed Implementation

[0027] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. The reagents, products, and instruments used in the following examples are all commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventionally used methods.

[0028] The tryptophan-sorbitol carbon quantum dot (WS-CQDs) nanoformulation of this invention is synthesized via a one-step hydrothermal method using tryptophan (W) and sorbitol (S) as natural carbon source precursors. Based on this, this invention aims to further protect the use of WS-CQDs in the treatment of sepsis. Specifically, it includes the following steps: 1) Preparation of WS-CQDs stock solution: Accurately weigh 100 mg tryptophan (0.49 mmol) and 500 mg sorbitol (2.74 mmol), dissolve in 50 mL double-distilled water, and mix thoroughly. Dispense the mixture into 5 mL ampoules and seal them. Place them in a high-pressure reactor and carry out a hydrothermal reaction at 160 °C for 10 hours.

[0029] 2) Dialysis purification of WS-CQDs: To effectively remove unreacted precursors and small molecule byproducts and accurately explore the intrinsic properties of WS-CQDs, the reaction solution was purified by centrifugation and dialysis to obtain an aqueous solution of WS-CQDs. The obtained solution was autoclaved and stored at 2-8 ℃ for subsequent experiments.

[0030] 3) Quantification of WS-CQDs: Freeze the WS-CQDs aqueous solution in a sterile petri dish at -20°C overnight. Then remove the lid, cover with plastic wrap, prick holes, and freeze in a freeze dryer for 24-48 hours. Accurate quantification can be achieved by weighing the obtained freeze-dried product, which is then dissolved for later use.

[0031] 4) Observe the effect of WS-CQDs on the survival rate of CLP sepsis mice in the "first hit" (excessive inflammation) and "second hit" (immunosuppression) phases: A. Construction of a sepsis "one-hit" model: Eight-week-old male C57BL / 6J mice were selected. After anesthesia and abdominal disinfection, the abdominal cavity was incised along the midline to expose the cecum. The distal end of the cecum was ligated with sterile silk sutures, and a small amount of intestinal contents was squeezed out into the abdominal cavity using a 22-G needle. The cecum was then returned to the abdominal cavity, and the incision was sutured layer by layer to complete the construction of the CLP model, simulating the early excessive inflammatory stage of sepsis.

[0032] B. Establishment of the sepsis "second hit" model: On the 4th day after CLP, surviving sepsis mice were anesthetized, fixed in a supine position, and their trachea was exposed. An intravenous catheter was inserted through the tracheal ring gap, and Pseudomonas aeruginosa bacterial fluid was slowly injected through a microinjector to induce a secondary infection in the lungs, thereby inducing an immunosuppressive "second hit".

[0033] C. Drug intervention and observation: After the "secondary blow" model was established, WS-CQDs solution was injected via the tail vein, and the survival status of mice in each group was continuously observed and recorded.

[0034] The experimental design diagram in this invention is as follows: Figure 1 As shown.

[0035] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0036] Example 1 This embodiment aims to prepare tryptophan-sorbitol carbon quantum dots (WS-CQDs), and the specific steps are as follows: 1) Preparation of the stock solution for WS-CQDs: Accurately weigh 100 mg tryptophan (0.49 mmol) and 500 mg sorbitol (2.74 mmol), dissolve them in 50 mL double-distilled water, and mix thoroughly. Dispense the mixture into 5 mL ampoules and seal them. Place the ampoules in a high-pressure reactor and carry out a hydrothermal reaction at 160 °C for 10 hours. This synthesis method has been granted a Chinese National Invention Patent (Patent No.: CN115845125B).

[0037] 2) Dialysis purification of WS-CQDs: To effectively remove unreacted precursors and small molecule byproducts and accurately explore the intrinsic properties of WS-CQDs, the reaction solution was purified by centrifugation and dialysis to obtain an aqueous solution of WS-CQDs. The obtained solution was autoclaved and stored at 2-8 °C for subsequent experiments. The centrifugation conditions were 12000 g for 15 minutes, and the dialysis was performed using a dialysis bag with a molecular weight cutoff of 10 kDa.

[0038] 3) Quantification of WS-CQDs: Freeze the WS-CQDs aqueous solution in a sterile petri dish at -20°C overnight. Then remove the lid, cover with plastic wrap, prick holes, and freeze in a freeze dryer for 24-48 hours. Accurate quantification can be achieved by weighing the obtained freeze-dried product, which is then dissolved for later use.

[0039] Example 2 This embodiment aims to evaluate the in vitro toxicity of tryptophan-sorbitol carbon quantum dots (WS-CQDs) using the CCK8 method. The specific steps are as follows: Step (1): RAW264.7 (mouse-derived macrophage cell line) and Kupffer (mouse primary hepatic macrophages) were used at a ratio of 2 x 10⁻⁶ cells / mL. 4 The wells were seeded at a density equal to the density of the wells in a 96-well plate and incubated overnight.

[0040] Step (2): Treatment was performed with different concentrations of WS-CQDs (5, 10, 20, 40, 80, 100 and 200 ug / mL), with PBS as the control group and cell-free culture medium as the blank control. The cells were cultured at 37℃ and 5% CO2 for 24 or 36 hours.

[0041] Step (3): At the endpoint, add 10 μl of CCK8 solution to each well and incubate for 2 h. Finally, determine cell viability by measuring absorbance at 450 nm (OD450 nm), calculated using the following formula: Cell viability (%) = (OD450 nm / (OD450 nm)) / (OD450 nm ... 色氨酸碳量子点 -OD450 空白 ) / (OD450) PBS - OD450 空白 ) x 100%.

[0042] like Figure 2 As shown in the results of the cytotoxicity assay, WS-CQDs did not exhibit significant toxicity to RAW264.7 and Kupffer cells at concentrations of 100 μg / mL and below. Therefore, WS-CQDs demonstrated good biocompatibility within the tested concentration range.

[0043] Example 3 This embodiment aims to evaluate the in vivo toxicity of tryptophan-sorbitol carbon quantum dots (WS-CQDs) in a C57 / 6J mouse model. The specific steps are as follows: Step (1): Male C57 / 6J mice, aged 7-8 weeks, were purchased from Hunan Borui Biotechnology Co., Ltd., and were allowed to acclimatize to the environment for 1 week before starting subsequent experiments. All animal experimental procedures were approved by the Medical Ethics Committee of Xiangya Hospital, Central South University (202112277).

[0044] Step (2): Twenty mice were divided into two groups: a PBS control group (n=10) and a WS-CQDs treatment group (n=10), with an injection dose of 50 mg / kg. Twenty-four hours after treatment, blood samples were collected from the eyes of the mice under anesthesia for complete blood count (WBC, RBC, PLT, and HGB) and liver and kidney function biochemistry (ALT, TBIL, LDH, UREA, and CK) analysis.

[0045] Step (3): The mice were then dissected to obtain and fix tissues of the heart, kidneys, liver, lungs and spleen, and H&E staining was performed to assess histopathological changes.

[0046] like Figure 3 As shown, in vivo toxicity assessment results indicated that WS-CQDs exhibited good biocompatibility in vivo, with no obvious toxic reactions observed, demonstrating its high safety potential as a medical nanomaterial. Specifically, no significant differences were observed between the WS-CQDs-treated group and the PBS control group in blood routine indicators (WBC, RBC, PLT, HGB) and liver and kidney function indicators (ALT, TBIL, LDH, UREA, CK). Further H&E staining histological analysis of major organs such as the heart, kidneys, liver, lungs, and spleen showed that WS-CQDs did not cause any obvious pathological damage.

[0047] Example 4 This embodiment aims to demonstrate, through in vitro experiments, that tryptophan-sorbitol carbon quantum dots (WS-CQDs) possess excellent anti-inflammatory effects. The specific steps are as follows: Step (1): To evaluate the anti-inflammatory effect of WS-CQDs, three different treatment modes were set up in the experiment: ① Pretreatment: WS-CQDs of different concentrations were pretreated for 1 hour, followed by co-culturing with 200 ng / mL LPS for 4 hours; ② Co-treatment: WS-CQDs and 200 ng / mL LPS were added to the cells simultaneously and co-cultured for 4 hours; ③ Posttreatment: WS-CQDs were stimulated with 200 ng / mL LPS for 1 hour, followed by co-culturing with different concentrations of WS-CQDs for another 4 hours. Under the three modes, the effect of WS-CQDs on the protein expression levels of inflammatory factors (TNF-α, IL-6, and IL-1β) in RAW264.7 cells was first detected by Western blotting.

[0048] Step (2): qPCR was used to detect changes in the mRNA levels of inflammatory factors (TNF-α, IL-6 and IL-1β) in RAW264.7 cells under the pretreatment mode.

[0049] like Figure 4 As shown, WS-CQDs effectively inhibited the expression of LPS-induced inflammatory factors TNF-α, IL-6 and IL-1β in RAW264.7 cells under all three treatment modes.

[0050] Example 5 This embodiment aims to demonstrate in vitro that tryptophan-sorbitol carbon quantum dots (WS-CQDs) can enhance the immune response of monocytes and macrophages. The specific steps are as follows: Step (1): Construction of the immunosuppression model. Mouse bone marrow-derived macrophages (BMDMs) were collected and first stimulated with 100 ng / mL LPS for 24 hours to induce immune tolerance. Then, the LPS was removed, and the cells were allowed to stand in a quiescent state for 4 hours without stimulation. Subsequently, they were stimulated again with 100 ng / mL LPS for 8 hours to establish an in vitro immunosuppression model.

[0051] Step (2): Detection of pro-inflammatory factor mRNA levels. The changes in the mRNA expression level of the pro-inflammatory factor TNF-α in the above model cells treated with WS-CQDs (40 μg / mL) after two LPS stimulations were detected by qPCR.

[0052] Step (3): Immunophenotyping and activation status analysis. The expression of MHC-II, an activation marker on the surface of mouse peripheral blood mononuclear macrophages, was analyzed by flow cytometry. After anesthetizing mice, blood was collected from the inner canthus, treated with erythrocyte lysis buffer, and then subjected to live / dead staining, Fc receptor blocking, and antibody staining for surface markers such as CD45, CD11b, Ly6C, Gr-1, CD80, CD86, and MHC-II. Finally, the samples were analyzed by flow cytometry.

[0053] like Figure 5 As shown, compared with the model group, WS-CQDs treatment increased the secretion level of TNF-α in BMDM derived from CLP sepsis mice by approximately 5.7-fold under secondary LPS stimulation and significantly upregulated the expression of MHC-II molecules on the surface of BMDM. These results indicate that WS-CQDs can effectively enhance the immune response function of monocytes and macrophages under immunosuppressive conditions.

[0054] Example 6 This embodiment aims to verify that tryptophan-sorbitol carbon quantum dots (WS-CQDs) can effectively alleviate the "first hit" in CLP sepsis mice. The specific steps are as follows: Step (1): Constructing a CLP sepsis mouse model. Eight-week-old male C57BL / 6J mice were selected. After anesthesia and abdominal disinfection, the abdominal cavity was incised along the midline to expose the cecum. The distal end of the cecum was ligated with sterile silk sutures, and the cecum was punctured three times with a 22-G needle to squeeze out a small amount of intestinal contents into the abdominal cavity. The cecum was then returned to the abdominal cavity, and the incision was sutured layer by layer.

[0055] Step (2): Drug intervention and inflammatory factor detection. Two hours after CLP modeling, mice were randomly divided into two groups, and injected with PBS (n=4) or WS-CQDs at a concentration of 50 mg / kg (n=4) via the tail vein, respectively. Twelve hours after intervention, blood was collected from the eyeballs under anesthesia, serum was separated, and the expression levels of inflammatory factors TNF-α, IL-6, and IL-1β were detected by ELISA.

[0056] Step (3): Survival analysis. Another group of mice was injected with PBS (n=8) or WS-CQDs (n=8) via the tail vein 2 hours after CLP modeling. The survival status of each group of mice was continuously observed and recorded.

[0057] like Figure 6 As shown, compared with the PBS control group, WS-CQDs treatment significantly inhibited the expression levels of TNF-α, IL-6, and IL-1β in the serum of CLP-secreting mice and significantly improved their survival rate. This indicates that WS-CQDs can effectively alleviate the excessive inflammatory response (i.e., the "first hit") in the early stage of sepsis, demonstrating good therapeutic potential.

[0058] Example 7 This embodiment aims to verify that tryptophan-sorbitol carbon quantum dots (WS-CQDs) can effectively reduce mortality associated with subsequent "second-strike" (immunosuppression). The specific steps are as follows: Step (1): Constructing a sepsis "second hit" model. On the 3rd day after CLP surgery, surviving mice were anesthetized, fixed in a supine position, and their trachea exposed. An intravenous catheter was inserted through the tracheal ring gap, and 20-25 μL of Pseudomonas aeruginosa bacterial solution (1×10⁻⁶) was slowly injected using a microsyringe.7 CFU), after injection, keep the mouse in position for 1-2 minutes to prevent bacterial reflux and ensure effective bacterial invasion of the lungs, establish a secondary lung infection model, and simulate the "secondary attack" under immunosuppression.

[0059] Step (2): Drug intervention and survival analysis. Two hours after the “second hit” model was established, the mice were randomly divided into two groups and injected with PBS (n=8) or WS-CQDs at a concentration of 50 mg / kg (n=8) via the tail vein. Their survival was then continuously observed and recorded.

[0060] Step (3): Bacterial load detection. Another group of mice was injected with PBS (n=4) or WS-CQDs (n=4) via the tail vein 2 hours after the “second hit” model was established. Peripheral blood, lung tissue homogenate and bronchoalveolar lavage fluid (BALF) were collected 24 hours after intervention, diluted and plated, and colony forming units (CFU / mL) were counted.

[0061] Step (4): Histopathological analysis. Two hours after the “secondary blow” model was established, PBS (n=4) or WS-CQDs (n=4) were injected via the tail vein and treated for 24 hours. Lung, liver and kidney tissues were then collected for H&E staining to observe the pathological damage.

[0062] like Figure 7 As shown, WS-CQDs significantly improved the survival rate of mice in the immunosuppressed phase (“second-hit”), reduced bacterial load in lung tissue and blood, and alleviated pathological damage to the liver, lungs, and kidneys. These results strongly suggest that WS-CQDs have a significant therapeutic effect on the immunosuppressed phase of sepsis.

[0063] Example 8 This embodiment aims to verify that tryptophan-sorbitol carbon quantum dots (WS-CQDs) can remodel the immune microenvironment homeostasis in mice with sepsis-induced secondary infection. The specific steps are as follows: Step (1): Establish a lung secondary infection model according to step (1) in Example 7 to simulate the "secondary attack" under immunosuppression.

[0064] Step (2): Drug intervention. Two hours after the “second hit” model was established, the mice were randomly divided into two groups and injected with PBS (n=4) or WS-CQDs at a concentration of 50 mg / kg (n=4) via the tail vein.

[0065] Step (3): Detection of inflammatory factors in lung tissue. Lung tissues of mice were collected at 4 h, 12 h and 24 h after intervention. The expression level of TNF-α in the supernatant of lung tissue homogenate was detected by ELISA and the mRNA expression level of key cytokines (TNF-α, iNOS, IL-18 and IL-10) in lung tissue was detected by qPCR.

[0066] Step (4): Serum inflammatory factor detection. Serum cytokine levels were detected 12 h after intervention using the BD Cytometric Bead Array (CBA) Mouse Inflammatory Cytokine Kit.

[0067] like Figure 8 As shown, WS-CQDs not only upregulated cytokine expression in the lung tissue of mice with sepsis-secondary infection, but also maintained serum cytokine levels similar to those in the sham-operated group. These results strongly suggest that WS-CQDs can reshape the immune microenvironment homeostasis in mice with sepsis-secondary infection.

[0068] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of a tryptophan-sorbitol carbon quantum dots (WS-CQDs) in the preparation of a drug for treating sepsis.

2. Use according to claim 1, characterized in that, The WS-CQDs are synthesized using tryptophan and sorbitol as carbon source precursors via a one-step hydrothermal method.

3. Use according to claim 2, characterized in that, The preparation method of the WS-CQDs includes the following steps: (1) Dissolve tryptophan and sorbitol in water and mix well to obtain a mixed solution; (2) The mixed solution is subjected to a hydrothermal reaction to obtain crude WS-CQDs; (3) The crude WS-CQDs were purified to obtain purified WS-CQDs.

4. Use according to claim 3, characterized in that, The mass ratio of tryptophan to sorbitol in step (1) is 1:(4~6).

5. Use according to claim 3, characterized in that, The hydrothermal reaction conditions described in step (2) are: temperature 150~170 ℃, time 8~12 hours.

6. Use according to claim 3, characterized in that, The purification in step (3) includes centrifugation and dialysis.

7. Use according to claim 6, characterized in that, The centrifugation conditions are 11000~13000 g, 14~16 minutes.

8. Use according to claim 6, characterized in that, The dialysis uses dialysis bags with a molecular weight cutoff of 8-12 kDa.

9. Use according to claim 3, characterized in that, The medicament for treating sepsis contains a therapeutically effective amount of WS-CQDs and a pharmaceutically acceptable carrier or excipient.

10. Use according to claim 9, characterized in that, The drug for treating sepsis is available in injectable form.

Images