Application of sialic acid modified nano preparation in preparation of continuous low-dose drug delivery system

Abstract

The invention discloses application of a sialic acid modified nano preparation in preparation of a continuous low-dose drug delivery system, and belongs to the technical field of medicine, sialic acid is a sialic acid-cholesterol derivative, according to a continuous low-dose drug delivery scheme, the drug delivery concentration is 1 / 3-1 / 10 of the maximum tolerated dose, and the drug delivery concentration is 1 / 3-1 / 10 of the maximum tolerated dose. The sialic acid modified nano preparation is a sialic acid modified epirubicin liposome. Experiments prove that a continuous low-dose administration scheme of the sialic acid modified nano preparation can remarkably reduce liver and spleen accumulation, improve the targeting efficiency of a tumor site and maintain the blood concentration for a long time. The invention provides a solution for solving the problem that the curative effect of continuous low-dose administration of the PEGylated nano preparation is influenced by the anti-PEG antibody.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to the application of a sialic acid-modified nano preparation in the preparation of a continuous low-dose drug delivery system. Background Art

[0002] Currently, the main treatment options for cancer include surgery, chemotherapy, radiotherapy, targeted therapy, and immunotherapy. While newer therapies such as targeted therapy and immunotherapy have achieved remarkable clinical success and have profoundly impacted cancer treatment, these emerging therapies still require combination therapy with chemotherapy in many completed and ongoing clinical trials. Therefore, chemotherapy remains considered the most widely used and valuable treatment option for cancer.

[0003] Traditionally, the higher the dose, the more effective chemotherapy. However, a growing number of clinical results suggest that lowering the dose and decreasing the dosing interval may result in superior anti-tumor efficacy. Researchers have defined low-dose chemotherapy or maintenance dosing for certain cancers as metronomic chemotherapy (MCT), which involves continuous low-dose administration of chemotherapy drugs without extended rest periods. However, the concepts of "low dose" and "extended rest periods" are highly subjective, and MCT encompasses a variety of low-dose, frequent dosing regimens (e.g., daily, weekly, or every other day). Based on this, continuous low-dose metronomic chemotherapy (LDM) has been further defined as a specific MCT regimen: continuous or frequent administration of chemotherapy drugs at 1 / 3 to 1 / 10 of the maximum tolerated dose. LDM uses relatively low-dose, high-frequency, and continuous administration to maintain relatively low but effective blood concentrations for extended periods, thereby reducing toxic side effects and enhancing chemotherapy efficacy. Existing studies have shown that LDM is a multi-target treatment method that, in addition to inhibiting tumor angiogenesis, can also have direct or indirect effects on tumor cells and their microenvironment.

[0004] However, PEGylated liposomes can induce accelerated blood clearance (ABC) when administered in LDM, accelerating drug release and reducing the anti-tumor efficacy of the formulation. These adverse reactions are positively correlated with anti-PEG antibodies. Furthermore, numerous studies have shown that anti-PEG antibody levels in healthy individuals have increased annually, with the latest research showing that the positive rate of anti-PEG antibodies in healthy individuals has reached as high as 99.0%. Therefore, it is necessary to find new methods to reduce the adverse reactions of LDM administration of PEGylated nanoformulations and enhance their anti-tumor efficacy. Sialic acid (SA) is a type of nonacarbonyl amino sugar with a pyranose structure. Tumor proliferation and metastasis are closely related to the high expression of sialic acid in the tumor microenvironment (TME). These SAs can directly regulate the functions of various tumor-associated immune cells (TAICs), including macrophages, neutrophils, T cells, B cells, natural killer cells (NK), dendritic cells (DC) in the TME, and MDSCs in the peripheral blood, by interacting with SA-binding receptors. They can also "tame" the TAICs around the tumor and promote tumor development. Given the inseparable connection between tumors and TAICs, regulating TAICs may be an important strategy for tumor chemotherapy and immunotherapy. Therefore, SA can be modified in nanoformulations, indicating that by targeting TAICs, drugs can be delivered to the tumor site and the anti-tumor ability can be improved. Summary of the Invention

[0005] In response to the above problems, the main purpose of the present invention is to provide a sialic acid-modified nanoformulation for use in the preparation of a continuous low-dose drug delivery system, so as to reduce the accumulation of the preparation in the liver and spleen, improve the anti-tumor effect, and solve the problem of reduced anti-tumor effect of PEGylated nanoformulations due to the influence of anti-PEG antibodies.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The invention discloses an application of a sialic acid modified nano preparation in preparing a continuous low-dose drug delivery system. The sialic acid modified nano preparation is a sialic acid modified epirubicin liposome.

[0008] Preferably, the sialic acid is a sialic acid-cholesterol derivative.

[0009] Preferably, the continuous low-dose administration regimen has an administration concentration of 1 / 3 to 1 / 10 of the maximum tolerated dose.

[0010] Preferably, the method for preparing the sialic acid-modified epirubicin liposomes comprises the following steps:

[0011] Blank liposomes were prepared by a modified ethanol injection method, and epirubicin was encapsulated using a pH gradient method.

[0012] Hydrogenated soy lecithin (HSPC), cholesterol (CH), and sialic acid-cholesterol derivative (SA-CH) were dissolved in anhydrous ethanol and stirred in a water bath at 50°C to 65°C to fully dissolve to prepare a membrane. After partial ethanol evaporation, a preheated citric acid-sodium citrate solution was injected into the membrane and stirred continuously for 15-20 minutes to obtain a preliminary liposome product. The preliminary liposome product was transferred to an extruder and extruded cyclically to obtain a blank liposome suspension. The blank liposome suspension was removed and sodium phosphate solution was added to adjust the pH of the external aqueous phase to 7.0 to obtain pH gradient liposomes. The pH gradient liposomes were mixed with an epirubicin solution, stirred for 10-20 minutes, and then placed in an ice-water bath for 2-5 minutes to terminate drug loading, resulting in sialic acid-modified epirubicin liposomes (EPI-SAL).

[0013] In the above preparation method, the sialic acid-cholesterol derivative (SA-CH) is prepared using the preparation method in CN113024400A.

[0014] In the above preparation method, the citric acid-sodium citrate solution is preheated to 50° C. to 65° C.

[0015] In the above preparation method, the preheated citric acid-sodium citrate solution is injected into the membrane material at a rate of 5 mL / min to 10 mL / min.

[0016] In the above preparation method, the liposomes were transferred to an extruder and sequentially passed through polycarbonate membranes of 400 nm, 200 nm and 100 nm under nitrogen pressure, with each pore size being extruded 8 times.

[0017] Beneficial effects of the present invention:

[0018] (1) The continuous low-dose administration regimen of sialic acid-modified nanoformulations can significantly reduce liver and spleen accumulation, improve tumor targeting efficiency, and maintain blood drug concentrations for a longer period of time. (2) The continuous low-dose administration regimen of sialic acid-modified nanoformulations can significantly improve the quality of life of patients and has the best anti-tumor effect. (3) The continuous low-dose administration regimen of sialic acid-modified nanoformulations has a better anti-tumor effect than PEGylated nanoformulations, and is the most effective in inhibiting tumor angiogenesis and stimulating immune responses. (4) The present invention provides a solution to the dilemma that the efficacy of continuous low-dose administration of PEGylated nanoformulations is affected by anti-PEG antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1The plasma clearance rates of EPI-SAL in S180 tumor-bearing SD rats under different dosing regimens of the present invention; A: drug-time curve after single injection; B: drug-time curve after multiple injections.

[0020] Figure 2 It is the plasma drug concentration before the tenth administration in the present invention.

[0021] Figure 3 The tissue drug concentrations after single and multiple injections of EPI-SAL at different dosing regimens in the present invention; A: tissue drug distribution after single injection; B: tissue drug distribution after multiple injections.

[0022] Figure 4 The drug accumulation per gram of tumor after single and multiple injections of EPI-SAL at different dosing regimens in the present invention; A: tissue drug distribution after single injection; B: tissue drug distribution after multiple injections.

[0023] Figure 5 The tumor volume and tumor inhibition index of each group of mice under different EPI-SAL administration regimens in the present invention; wherein A: mouse tumor volume; B: tumor inhibition index.

[0024] Figure 6 The weight changes of mice in each group during the pharmacodynamic period under different dosing regimens of EPI-SAL in the present invention; A: body weight; B: net body weight.

[0025] Figure 7 Figure 2 shows the weight changes of mice during the administration of EPI-SAL in the present invention under different dosing regimens; A: the weight change curve of mice during the administration period; B: the degree of change in weight of mice in each group after the end of administration compared with the weight before treatment.

[0026] Figure 8 The figures show the trends of body weight changes of mice in each group 24 hours after each administration under different EPI-SAL administration regimens in the present invention; A: the degree of change in body weight of mice in the EPI-S group after each administration; B: the degree of change in body weight of mice in the EPI-S conventional administration group after each administration; C: the degree of change in body weight of mice in the EPI-SAL intensive dose group after each administration; D: the degree of change in body weight of mice in the EPI-SAL high-dose administration group after each administration; E: the degree of change in body weight of mice in the EPI-SAL continuous low-dose administration group after each administration; F: the average trend of body weight changes of mice after each administration.

[0027] Figure 9 The TSP-1 content in the serum of mice in each group under different EPI-SAL administration regimens in the present invention.

[0028] Figure 10The HMGB1 content in the tumor tissue of each group of mice under different EPI-SAL dosing regimens in the present invention; wherein A: HMGB1 fluorescence signal in the tumor tissue of each dosed mouse; B: HMGB1 positive cell ratio in the tumor of each dosed mouse; C: HMGB1 positive cell density in the tumor of each dosed mouse.

[0029] Figure 11 The MDR-1 content in tumor tissue of each group of mice under different EPI-SAL dosage regimens of the present invention; A: Western Blot graph; B: expression level of MDR-1 relative to ACTIN in different treatment groups; G1: Control; G2: EPI-S; G3: HD-EPI-SAL; G4: EPI-SAL; G5: DD-EPI-SAL; G6: LDM-EPI-SAL.

[0030] Figure 12 The tumor volume of each group of mice after continuous low-dose administration of different modified EPI liposomes of the present invention.

[0031] Figure 13 The tumor inhibition index of each group of mice after continuous low-dose administration of different modified EPI liposomes of the present invention.

[0032] Figure 14 These are the survival analysis results of mice in each group treated with different modified EPI liposomes in the present invention.

[0033] Figure 15 These are the HE staining results of the main organs of the different modified EPI liposomes in the present invention.

[0034] Figure 16 Figure 2 shows the tumor microvessel density after continuous low-dose administration of different modified EPI liposomes in the present invention; A: Fluorescence image of mouse tumor microvessels; B: Quantitative analysis results of tumor microvessel density; C: Concentration of the anti-angiogenic factor TSP-1 in mouse serum.

[0035] Figure 17 Figure 2 shows TAMs in tumor tissue after continuous low-dose administration of different modified EPI liposomes in the present invention; A: fluorescence image of mouse tumor microvessels; B: density of M1 tumor-associated macrophages; C: density of M2 tumor-associated macrophages; D: ratio of M1 TAMs to M2 TAMs in each group.

[0036] Figure 18 These are the results of serum enzyme-linked immunosorbent assay after continuous low-dose administration of different modified EPI liposomes in the present invention; wherein A: IL-10; B: TGF-β. DETAILED DESCRIPTION

[0037] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are merely illustrative and do not limit the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but such modifications or replacements fall within the scope of protection of the present invention.

[0038] Example 1: Preparation of sialic acid-modified epirubicin liposomes

[0039] Blank liposomes were prepared by a modified ethanol injection method, and epirubicin was encapsulated using a pH gradient method.

[0040] Dissolve HSPC, CH, and SA-CH in 500 μL of anhydrous ethanol. Stir in a 65°C water bath to fully dissolve. After most of the ethanol has evaporated, inject the preheated citric acid-sodium citrate solution (65°C) into the membrane material at a rate of 5 mL / min and continue stirring for 20 minutes to obtain the initial liposome product. Transfer the initial liposome product to an extruder and pass it through 400 nm, 200 nm, and 100 nm polycarbonate membranes in sequence under nitrogen pressure. Extrude each pore size 8 times to obtain a blank liposome suspension. Pipette 1.0 mL of the blank liposome suspension into a vial and add 0.4 mL of 500 mM sodium phosphate solution to adjust the pH value of the external aqueous phase to 7.0 to obtain pH gradient liposomes. The pH gradient liposomes were mixed with epirubicin solution at a drug-lipid ratio of 1:10 (w / w), stirred in a 60°C water bath for 20 min, and then placed in an ice water bath for 2 min to terminate drug loading, thereby obtaining sialic acid-modified epirubicin liposomes (EPI-SAL).

[0041] Example 2: Differences in the concentration-time curves of EPI-SAL after single and multiple administrations at different dosing regimens

[0042] Experimental animals and cells: S180 mouse osteosarcoma cells (Shanghai Cell Bank, Chinese Academy of Sciences); SD rats (5-week-old, male, Shenyang Pharmaceutical University Experimental Animal Center).

[0043] Dosage regimen: S180 tumor-bearing SD rats were randomly divided into four groups: solution group (EPI-S), conventional dosing group (EPI-SAL), high-dose dosing group (HD-EPI-SAL), and continuous low-dose dosing group (LDM-EPI-SAL). These rats were designated as the single-dose group (LDM-1). Furthermore, S180 tumor-bearing SD rats were randomly divided into five groups: EPI-S, EPI-SAL, dose-dense group (DD-EPI-SAL), HD-EPI-SAL, and LDM-EPI-SAL. These rats were designated as the multiple-dose group (LDM-10). The EPI-S group received a dose of 5 mg / kg; the EPI-SAL group received a dose of 5 mg / kg; the DD-EPI-SAL group received a dose of 5 mg / kg; the HD-EPI-SAL group received a dose of 7.5 mg / kg; and the LDM-EPI-SAL group received a dose of 1.5 mg / kg. The EPI-SAL and DD-EPI-SAL groups were both administered a 5 mg / kg dose under the LDM-1 regimen, so the DD-EPI-SAL group was not included in the LDM-1 regimen study. Rats in the LDM-1 and LDM-10 groups were simultaneously tumor-bearing and began receiving injections at the same time.

[0044] Experimental Methods: Blood was collected from the orbital venous plexus of rats in each LDM-1 group at 0.083, 0.5, 1, 2, 4, 8, 12, and 24 hours after administration. Plasma was separated by centrifugation at 4500 rpm for 10 minutes, and the drug concentration in the plasma was measured using a microplate reader after processing.

[0045] Rats in each LDM-10 group were dosed 10 times consecutively. Blood was collected before the tenth dose to determine the residual drug concentration in plasma. Blood was collected from the orbital venous plexus at 0.083, 0.5, 1, 2, 4, 8, 12, and 24 hours after the tenth dose. Plasma was separated by centrifugation at 4500 rpm for 10 minutes, and the drug concentration was determined using a microplate reader after processing.

[0046] Results: As Figure 1 and Figure 2 As shown in Figure 2, the circulation time of the drug in the body is proportional to the injected dose. The higher the single dose, the longer the circulation time in the body, and vice versa. Continuous low-dose administration can maintain higher plasma drug concentrations.

[0047] Example 3: Differences in tissue distribution after single and multiple administrations of EPI-SAL at different dosing regimens

[0048] The experimental animals, cells, and administration regimen were the same as in Example 1.

[0049] Experimental Methods: Rats in each LDM-1 group were sacrificed by cervical dislocation 24 hours after drug administration. The liver, spleen, and tumor were removed, ground with saline, and drug concentrations were measured using a microplate reader. Rats in each LDM-10 group were dosed 10 times consecutively. The same procedure was repeated 24 hours after the last dose to measure drug concentrations.

[0050] Results: As Figure 3 As shown in the results, the liver and spleen accumulation in the LDM-EPI-SAL group was the smallest, regardless of single or continuous administration. After a single dose, the liver and spleen accumulation in the EPI-SAL and HD-EPI-SAL groups were significantly increased compared to the LDM-EPI-SAL group. Compared with the LDM-EPI-SAL group, the liver accumulation of EPI (epirubicin) in the EPI-SAL group increased by 1.43 times ( *** P < 0.001), spleen increased 13.33 times ( *** P<0.001); HD-EPI-SAL group liver increased by 1.19 times ( ** P < 0.01), spleen increased 26.34 times ( *** P<0.001).

[0051] After multiple administrations, the accumulation of EPI in the liver and spleen in the LDM-EPI-SAL group was significantly lower than that in the EPI-SAL, HD-EPI-SAL, and DD-EPI-SAL groups. The accumulation of EPI in the liver in the EPI-SAL group was 2.37 times that in the LDM-EPI-SAL group ( *** P<0.001), spleen was 7.05 times ( *** P<0.001); the EPI liver accumulation in the HD-EPI-SAL group was 1.61 times that in the LDM-EPI-SAL group ( *** P<0.001), spleen was 9.16 times ( *** P<0.001); the EPI liver accumulation in the DD-EPI-SAL group was 1.91 times that in the LDM-EPI-SAL group ( *** P<0.001), spleen was 3.33 times ( *** P<0.01). This result indicates that compared with other dosing regimens, the continuous low-dose administration regimen can significantly reduce the accumulation of the preparation in the liver and spleen.

[0052] Considering the effect of different dosing regimens on tumor volume, we calculated the EPI drug concentration per gram of tumor tissue. The results are shown in Figure 2. Figure 4 As shown. Figure 4As shown, after a single dose, the LDM-EPI-SAL group had lower tumor drug concentrations than the other dosing regimens due to the lower dose concentration (1.5 mg / kg vs. 5 mg / kg and 7.5 mg / kg). However, with increasing dosing frequency, tumor drug concentrations significantly increased compared to a single injection. After 10 consecutive doses, the LDM-EPI-SAL group had an 832.63% increase in tumor drug concentration compared to the initial dose (2028.46 ng / mL vs. 217.5 ng / mL). Furthermore, the LDM-EPI-SAL group also had significantly higher tumor drug concentrations than the other dosing regimens.

[0053] Example 4: Differences in anti-tumor effects of different EPI-SAL administration regimens

[0054] Construction of mouse S180 tumor model: Ascites of S180 cultured in Kunming mice (5-7 days) was carefully extracted, diluted with normal saline and the cell suspension density was adjusted to 1×10 7 After disinfecting the mouse skin, 0.2 mL of the cell suspension was extracted and inoculated into the subcutaneous tissue under the right anterior armpit of the mouse. 3 After 3 days (approximately 3 days after inoculation), the mouse S180 tumor model was established.

[0055] Dosing regimen: Thirty-six mice bearing S180 tumors were randomly divided into six groups: control group (Control), EPI solution group (EPI-S), high-dose EPI-SAL group (HD-EPI-SAL), conventional EPI-SAL group (EPI-SAL), dose-dense EPI-SAL group (DD-EPI-SAL), and continuous low-dose EPI-SAL group (LDM-EPI-SAL). The control group received an equal volume of saline. The solution group received five doses of 5 mg / kg each, separated by two days. The high-dose group received four doses of 6.25 mg / kg each, separated by three days. The conventional group received five doses of 5 mg / kg each, separated by two days. The dose-dense EPI-SAL group received five doses of 5 mg / kg each, separated by one day. The continuous low-dose group received 16 doses of 1.5 mg / kg each, separated by no interval. The control group received five doses of an equal volume of saline, separated by two days.

[0056] Throughout the pharmacodynamic period, data such as tumor volume, body weight, and mortality were recorded for each group of mice. The anti-tumor efficacy of EPI-SAL under different dosing regimens was then analyzed based on the following indicators.

[0057] Depend on Figure 5The LDM-EPI-SAL group showed the best anti-tumor effect. During the observation period after drug withdrawal, the tumor inhibition index of this group of mice showed a significant upward trend, indicating that this regimen minimized nonspecific damage.

[0058] Example 5: Body weight differences in different EPI-SAL dosing regimens

[0059] The construction of the mouse S180 tumor model and the administration regimen were the same as those in Example 4.

[0060] Depend on Figure 6 Throughout the pharmacodynamic period, the body weight and lean body mass of the LDM-EPI-SAL group were higher than those of the other dosing regimens. The results of body weight changes during the dosing period showed that the body weight of mice increased by 29.39% under the continuous low-dose dosing regimen, indicating a higher body weight gain.

[0061] To further explore the changes in body weight among different groups 24 hours after administration, the body weight change rate was calculated as (W 24h after administration ―W before administration ) / W before administration , the body weight changes of rats in each group after 24 hours of administration were as follows Figure 7 and Figure 8 As shown. The body weight of mice in the HD-EPI-SAL group showed a downward trend after the second dose, and the weight loss gradually increased with the increase in the number of doses (4.11% vs. -2.03% vs. -4.18% vs. -10.28%). The body weight of mice in the DD-EPI-SAL group also decreased after the second dose, but the weight loss did not increase after the subsequent three doses. The weight change of mice in the LDM-EPI-SAL group 24 hours after administration was "increase and decrease", with an overall upward trend. Comprehensive analysis of the changes in body weight after each dose showed that, except for the LDM-EPI-SAL group, the body weight of the other groups decreased 24 hours after each dose, indicating that each dose in the other groups will cause greater damage to the body while exerting anti-tumor effects, and the continuous low-dose administration regimen can minimize the damage to the body.

[0062] Example 6: Differences in serum TSP-1 levels at different EPI-SAL administration regimens

[0063] The construction of the mouse S180 tumor model and the administration regimen were the same as those in Example 4.

[0064] After the efficacy experiment, blood was collected from the orbital venous plexus of mice and centrifuged at 3000 rpm for 10 min. The supernatant was collected to obtain serum and the TSP-1 concentration was detected using an ELISA kit.

[0065] Depend on Figure 9 It can be seen that the serum TSP-1 concentration in the LDM-EPI-SAL group was the highest after the administration, and there was a significant difference between it and the other groups.

[0066] Example 7: Differences in tumor HMGB1 content in different EPI-SAL dosing regimens

[0067] The construction of the mouse S180 tumor model and the administration regimen were the same as those in Example 4.

[0068] High mobility group box 1 (HMGB1), a key damage-associated molecular pattern (DAMP), can activate innate and adaptive immunity, thereby promoting the immunogenic effects of ICD. After the efficacy experiment, tumor tissues from each group of mice were dissected, fixed, dehydrated, and frozen. The sections were photographed under a fluorescence microscope, and the HMGB1 content in the tumor tissues was analyzed. The ratio and density of positive cells (red) were calculated.

[0069] Depend on Figure 10 It can be seen that compared with the Control group, the HMGB1 fluorescence signal (red) in the tumor tissue of each drug-treated group showed an increasing trend. The order of the HMGB1-positive cell ratio in each group of tumors is: LDM-EPI-SAL>EPI-SAL>DD-EPI-SAL>EPI-S>HD-EPI-SAL>Control, indicating that continuous low-dose rhythmic chemotherapy can induce a more powerful ICD response. Taking into account the differences in tumor area between different groups during the sectioning process, the HMGB1-positive cell density of the fluorescent sections was further analyzed. The results show that the HMGB1-positive cell density in the LMD-EPI-SAL group was the largest, and there was a very significant difference with the other groups ( *** p<0.001).

[0070] Example 8: Differences in tumor MDR-1 levels in different EPI-SAL dosing regimens

[0071] The construction of the mouse S180 tumor model and the administration regimen were the same as those in Example 4.

[0072] MDR-1, also known as P-glycoprotein (P-gp), belongs to the ATP-binding cassette (ABC) transporter family. Its overexpression in cancer is one of the main causes of chemotherapy resistance. By upregulating the expression of MDR-1, tumor cells can quickly pump chemotherapy drugs out of the cells, thereby reducing the killing effect of the drugs and leading to the occurrence of multidrug resistance (MDR). After the administration, the mice were killed by dislocation of the neck, and the tumor tissue was carefully dissected and washed 1 to 2 times with pre-cooled PBS solution. The tumor tissue was cut into small pieces and ground with tissue lysis buffer. The ground tumor tissue was then placed at 4°C for 30 minutes for lysis. After the lysis was completed, the mixture was centrifuged at 12,000 rpm for 10 minutes, and the supernatant was collected. The MDR-1 content in the tumor tissue of each group of mice was analyzed by protein immunoblotting.

[0073] Depend on Figure 11 It can be seen that the content of LDM-EPI-SAL group is the lowest, indicating that it can effectively inhibit the expression of MDR-1 and weaken the occurrence of MDR.

[0074] Example 9: Differences in antitumor pharmacodynamics of differently modified EPI liposomes

[0075] Tumor model construction: The method for constructing S180 tumor-bearing mice was the same as that in Example 4.

[0076] Dosage Regimen: Thirty-six S180 tumor-bearing Kunming mice were randomly divided into six groups: control (Control), EPI solution (EPI-S), EPI-CL continuous low-dose metronomic group (EPI-CL), EPI-PL continuous low-dose metronomic group (EPI-PL), EPI-SAL continuous low-dose metronomic group (EPI-SAL), and EPI-PL2 continuous low-dose metronomic group (EPI-PL2), with six mice in each group. Each group received a single dose of 0.75 mg / kg for 10 consecutive days. The control group received an equal volume of normal saline. Tumor volume, body weight, and mortality were recorded throughout the pharmacodynamic period.

[0077] The preparation methods and processes of EPI-CL, EPI-PL, and EPI-PL2 are the same as those of EPI-SAL. Among them, EPI-CL: ordinary epirubicin liposomes (unmodified); EPI-PL: linear PEG-modified epirubicin liposomes with a molecular weight of 2000; EPI-PL2: PEG 2,n Modified epirubicin liposomes; PEG 2,nA compound obtained by covalently linking two linear methoxy-terminated PEG chains to lysine, wherein the total molecular weight of the two PEG chains is 2000. The compound was obtained according to the method described in patent CN113350512B.

[0078] Figure 12 The tumor volumes of mice in each group after continuous low-dose administration of different modified EPI liposomes;

[0079] Figure 13 The tumor inhibition index of each group of mice after continuous low-dose administration of different modified EPI liposomes;

[0080] Figure 14 Survival analysis of mice in each group after continuous low-dose administration of different modified EPI liposomes;

[0081] Depend on Figures 12 to 14 It can be seen that sialic acid-modified nanoformulations have the best anti-tumor effect under the continuous low-dose metronomic chemotherapy regimen.

[0082] Example 10: Histopathological analysis of different modified EPI liposomes

[0083] The tumor model was constructed as in Example 4; the administration regimen was the same as in Example 9.

[0084] After the pharmacodynamics experiments, mice in each group were killed and their hearts, livers, spleens, lungs, kidneys, and tumor tissues were removed. H&E staining was then used to analyze organ damage in each group.

[0085] The results show that ( Figure 15 ), while no obvious damage was observed in the major organs of mice in the EPI-SAL group. This indicates that sialic acid-modified EPI liposomes have good safety and tolerability.

[0086] Example 11: Analysis of tumor microvessel density with different modified EPI liposomes

[0087] The tumor model was constructed as in Example 4; the administration regimen was the same as in Example 9.

[0088] After the pharmacodynamics experiments, blood was collected from each group of mice and centrifuged to obtain serum. Serum TSP-1 concentrations were measured using an ELISA kit. Mice were then sacrificed by cervical dislocation, and tumor tissues were carefully dissected and fixed in 4% paraformaldehyde. Blood vessels were stained with CD31, and microvessel density (MVD) was calculated for each tumor group.

[0089] Depend on Figure 16It can be seen that although the continuous low-dose administration regimen has been shown to have the ability to inhibit tumor angiogenesis, our results show that not all EPI preparations can inhibit tumor angiogenesis. The order of MVD in each treatment group is: EPI-PL2>EPI-CL>EPI-PL>Control>EPI-S>EPI-SAL. Compared with the Control group, EPI-PL2 ( # P<0.05), EPI-CL( # P>0.05), EPI-PL( # P>0.05) after treatment, the MVD of the tumor did not decrease, but increased further, while the MVD of the tumor showed a decreasing trend after treatment with EPI-S and EPI-SAL. In addition, the MVD of the EPI-SAL group was significantly different from that of the EPI-PL2 ( ** P<0.01), EPI-CL( ** P<0.01), EPI-PL( * P<0.05) showed a significant difference, indicating that sialic acid cholesterol derivative modification can significantly inhibit tumor microangiogenesis, while EPI-PL and EPI-PL2 do not have this function. TSP-1 detection results showed that the EPI-SAL group of mice had the highest TSP-1 level ( * The TSP-1 concentrations in the EPI-CL, EPI-PL, and EPI-PL2 groups were significantly decreased. In conclusion, continuous low-dose administration of EPI-SAL can further reduce tumor MVD density by increasing serum TSP-1 concentrations.

[0090] Example 12: Immunofluorescence staining of differently modified EPI liposomes

[0091] The tumor model was constructed as in Example 4; the administration regimen was the same as in Example 9.

[0092] After the pharmacodynamics experiments, mice in each group were killed by cervical dislocation, and tumor tissues were carefully dissected and fixed in 4% paraformaldehyde. Immunofluorescence staining was performed using CD68 and D206 markers to evaluate the inhibitory effect of each treatment regimen on TAMs. Sections were observed and images were acquired using a fluorescence microscope. Image J was then used to quantitatively analyze the IOD values ​​of CD68 (red) and CD206 (green) in each image, calculating the positive density within the field of view.

[0093] Depend on Figure 17 It can be seen that the infiltration of M2 TAMs in the EPI-SAL group was the lowest; compared with the EPI-PL group, the M2 infiltration in the EPI-PL2 group was significantly reduced ( ++P<0.01). Compared with the control group, the infiltration of M1 TAM in EPI-S and EPI-CL groups was reduced, while that in EPI-PL and EPI-SAL groups was increased. *** P<0.001), EPI-PL2( *** These results suggest that EPI-PL, EPI-SAL, and EPI-PL2 can significantly reduce M2 TAM infiltration in tumors, polarizing TAMs toward the M1 type, thereby reversing TME immunosuppression and restoring antitumor activity.

[0094] Generally speaking, high-density TAMs infiltration is often observed in the late stage of tumors. However, a single analysis of TAMs (the number of M1+M2 cells) infiltration in the tumor site cannot accurately predict the occurrence and development of cancer. Further analysis of the ratio of M1 and M2 TAMs is considered to be a more biologically meaningful indicator. A lower M1 / M2 ratio usually indicates a poor prognosis for cancer patients, while a higher M1 / M2 ratio indicates a better prognosis for patients. Therefore, we further analyzed the M1 / M2 ratio in the tumor site, and the results showed that the order of the ratios of each drug administration group was: EPI-SAL>EPI-PL2>EPI-PL>EPI-CL>Control>EPI-S group, indicating that mice in the EPI-SAL administration group had the best prognostic effect.

[0095] Example 13: Anti-tumor immune effects of continuous low-dose administration of differently modified EPI liposomes

[0096] The tumor model was constructed as in Example 4; the administration regimen was the same as in Example 9.

[0097] After the pharmacodynamic experiment, the blood of each group of mice was collected and centrifuged to obtain serum, and the IL-10 and TGF-β concentrations in the serum were detected using ELISA kits.

[0098] Depend on Figure 18 It can be seen that after EPI-SAL treatment, the levels of IL-10 and TGF-β in mice were the lowest, indicating that it can significantly improve the anti-tumor immune effect. It is worth noting that after continuous low-dose administration of EPI-PL, the levels of IL-10 and TGF-β in mice were the highest, indicating that the anti-tumor immune effect of continuous low-dose administration of EPI-PL was relatively low. Although EPI-PL2 further reduced the concentrations of IL-10 and TGF-β in mice, the levels were still higher than those in the EPI-S group ( * P<0.05). This result indicates that continuous low-dose administration of PEG-modified nanoformulations can produce immunosuppressive effects.

Claims

1. Application of a sialic acid-modified nanoformulation in the preparation of a continuous low-dose drug delivery system.

2. The use according to claim 1, characterized in that The sialic acid is a sialic acid-cholesterol derivative.

3. The use according to claim 1, characterized in that For the continuous low-dose administration regimen, the dosage concentration is selected to be 1 / 3 to 1 / 10 of the maximum tolerated dose.

4. The use according to claim 1, characterized in that The sialic acid modified nano preparation is sialic acid modified epirubicin liposome.

5. A method for preparing the sialic acid-modified epirubicin liposomes according to claim 4, characterized in that: The following steps are involved: Hydrogenated soybean lecithin, cholesterol, and sialic acid-cholesterol derivatives are dissolved in anhydrous ethanol and stirred in a water bath at 50°C to 65°C to fully dissolve to prepare a membrane material. After some ethanol is evaporated, a preheated citric acid-sodium citrate solution is injected into the membrane material and continuously stirred to obtain a preliminary liposome product. The preliminary liposome product is transferred to an extruder for cyclic extrusion to obtain a blank liposome suspension. The blank liposome suspension is removed, and sodium phosphate solution is added. The pH value of the external aqueous phase is adjusted to 7.0 to obtain a pH gradient liposome. The pH gradient liposome is mixed with an epirubicin solution, stirred, and placed in an ice-water bath to terminate drug loading to obtain sialic acid-modified epirubicin liposomes.

6. The preparation method according to claim 5, characterized in that In the preparation method of the sialic acid-modified epirubicin liposomes, a preheated citric acid-sodium citrate solution is injected into the membrane material at a speed of 5 mL / min to 10 mL / min.

7. The preparation method according to claim 5, characterized in that In the preparation method of the sialic acid-modified epirubicin liposomes, the primary liposomes are transferred to an extruder and sequentially passed through 400 nm, 200 nm and 100 nm polycarbonate membranes under nitrogen pressure, with each pore size being extruded 8 times in a cycle.

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