Novel nano-drug delivery system with tumor microenvironment regulation performance, preparation method and application

Abstract

The invention belongs to the technical field of clinical precise tumor diagnosis and treatment, and particularly relates to a novel nano-drug delivery system with tumor microenvironment regulation and control performance, a preparation method and application. The preparation method comprises the following steps: S1, dissolving ionizable lipid, auxiliary phospholipid, cholesterol and functional modified lipid in a solvent according to a molar ratio of 50: 38.5: 10: 1.5 to prepare an organic phase; s2, dissolving capsaicin in the organic phase obtained in S1, dissolving adriamycin in a solvent to prepare a water phase, and rapidly mixing the organic phase and the water phase in a microfluidic device according to a volume ratio of 3: 1 to obtain a mixed solution; and S3, putting the mixed solution obtained in the S2 into a dialysis bag, putting the dialysis bag into a Tris buffer solution, carrying out magnetic stirring dialysis, and concentrating to obtain the (C + D)-coated LNPPRT. According to the present invention, the precise tumor detection effect is significantly enhanced, the efficient early tumor detection and the targeted chemotherapy are achieved, and the treatment effect is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of clinical precision diagnosis and treatment of tumors, and specifically relates to a novel nano-drug delivery system with tumor microenvironment regulation performance, a preparation method and an application. Background Art

[0002] Because pancreatic cancer (PANC) has hidden early symptoms, traditional detection methods are usually unable to accurately detect it, making clinical diagnosis, efficient treatment and improved prognosis still facing major challenges. In recent years, neurointerference therapy based on regulating nerve-cancer crosstalk has achieved remarkable results in tumor diagnosis and treatment, bringing new hope to patients. The nerve-cancer crosstalk diagnosis and treatment program uses the regulatory effect of the nervous system on cancer to affect tumor progression and plays an important role in tumor diagnosis and treatment. The nerve-cancer crosstalk induces the release of cancer-related markers and triggers cascade activities through neural regulation, and uses specific detection methods to accurately detect and diagnose tumors. At the same time, it can also reverse tumor resistance and enhance treatment effects through neural-vascular regulation of the tumor microenvironment (TME). Currently, the main method of intervention in nerve-cancer crosstalk is to reduce neural innervation and inhibit tumor growth, invasion and metastasis through targeted intervention of nerve-cancer drugs. In addition, it can also regulate nerve activity and reshape the TME by blocking nerve growth factor-transmembrane tyrosine kinase receptor (NGF-Trk) signaling to improve efficacy and inhibit tumor metastasis. However, the neurointerference therapy using the above-mentioned neuro-cancer crosstalk regulation strategy is not very effective, and there are still many challenges in the actual application of early accurate detection and enhanced efficacy, which seriously limits the clinical translation application. First, the effective dose of existing neuromodulatory drugs can cause side effects; second, the existing Trk targets are prone to off-target and cause neurotoxicity in the body; third, there is currently a lack of effective diagnostic and therapeutic platforms for regulating neuro-cancer crosstalk; moreover, the cascade signals and biomarkers induced by neuro-cancer crosstalk are difficult to accurately detect; finally, the signaling pathways and specific molecular mechanisms regulated by neuro-cancer crosstalk are still unclear. Therefore, the development of a diagnostic and therapeutic system with excellent targeting and multifunctional response characteristics that can strongly induce new targets for neuro-cancer crosstalk is the key to achieving accurate tumor detection, improving treatment levels, monitoring treatment effects, and clarifying the molecular mechanisms of neuro-cancer crosstalk diagnostic and therapeutic strategies.

[0003] To address the shortcomings of existing neuro-cancer crosstalk regulation strategies, such as side effects caused by the effective dose of existing neuromodulatory drugs and the off-target effects of existing Trk targets that can cause neurotoxicity, this paper proposes a novel multifunctional diagnostic and therapeutic platform (C+D)@LNP that can specifically target transient receptor potential vanilloid 1 (TRPV1), a new target for neuro-cancer crosstalk. PRT (C represents capsaicin, D represents doxorubicin, LNP PRTstands for tumor-targetable and TME-responsive lipid nanoparticles), a neural interference diagnosis and treatment strategy that achieves accurate detection of PANC and enhances targeted efficacy by efficiently regulating nerve-cancer crosstalk. Summary of the Invention

[0004] In view of the problems and shortcomings of the prior art, the present invention provides a method for preparing a novel nano drug delivery system with tumor microenvironment regulation performance, comprising the following steps:

[0005] S1: dissolving ionizable lipid, auxiliary phospholipid, cholesterol, and functional modified lipid in a molar ratio of 50:38.5:10:1.5 in a solvent to prepare an organic phase;

[0006] S2: dissolving capsaicin in the organic phase obtained in S1, dissolving doxorubicin in a solvent to prepare an aqueous phase, and rapidly mixing the organic phase and the aqueous phase in a microfluidic device at a volume ratio of 3:1 to obtain a mixed solution;

[0007] S3: The mixed solution obtained in S2 was placed in a dialysis bag and placed in Tris buffer for magnetic stirring dialysis and then concentrated to obtain (C+D)@LNP. PRT .

[0008] Furthermore, the molar ratio of capsaicin to doxorubicin in S2 is 6.5:3.5.

[0009] Furthermore, the flow rate during the mixing process described in S2 is 12 min / mL.

[0010] Furthermore, the pH of the Tris buffer described in S3 is 7.4.

[0011] Furthermore, the magnetic stirring speed in S3 is 200 rpm, the temperature is 4° C., and the time is 24 h.

[0012] Furthermore, the concentration described in S3 is ultrafiltration concentration, PEG 20k The ultrafiltration concentration was performed at a rate of 5000 rpm and a temperature of 4°C.

[0013] A novel nano drug delivery system with tumor microenvironment regulation performance comprises functionally modified lipids, ionizable lipids, cholesterol and auxiliary phospholipids, doxorubicin and capsaicin.

[0014] The invention relates to an application of a novel nano drug delivery system with tumor microenvironment regulation performance, the preparation of the novel targeted regulation nerve-cancer crosstalk nano drug delivery system, and its application in tumor targeting and enhanced tumor diagnosis and treatment.

[0015] Technical Effects

[0016] The present invention provides a method for preparing a novel nano-drug delivery system with tumor microenvironment regulation performance; the present invention utilizes functionalized lipids to synthesize a novel multifunctional diagnosis and treatment platform through a one-step microfluidic method, thereby realizing a neural interference diagnosis and treatment scheme for accurate tumor detection and targeted chemotherapy.

[0017] The effectiveness of neurointerference diagnosis and treatment is limited by the regulation strategy of nerve-cancer crosstalk. Existing neuromodulatory drugs and Trk targets lack specificity, and effective doses and off-target interventions can cause side effects. Therefore, the development of a diagnosis and treatment system with excellent targeting and multifunctional response characteristics and the ability to strongly induce new targets for nerve-cancer crosstalk is a key prerequisite for achieving precise diagnosis and treatment of tumors. The novel multifunctional diagnosis and treatment platform (C+D)@LNP synthesized in this invention PRT It can efficiently target tumors and trigger neural cascade activity and the expression and release of tumor-related markers by specifically activating TRPV1-mediated neural-cancer crosstalk, thereby enhancing the precise detection of tumors. At the same time, it regulates TME to enhance targeted efficacy to achieve a neural-cancer crosstalk regulation strategy for neural interference diagnosis and treatment.

[0018] This method also achieves precise regulation. It uses a new multifunctional diagnostic and therapeutic platform to specifically activate TRPV1 to mediate neural-cancer crosstalk for precise cancer detection. Compared to the traditional neural-cancer crosstalk target Trk, TRPV1 is simple, efficient, and multifunctional. Capsaicin is used to specifically activate TRPV1 to mediate neural-cancer crosstalk, enhancing its specific marker release and cascade activity. Combined with multimodal imaging detection technology, it improves the accuracy of early tumor detection, achieving precise tumor detection and diagnosis to enhance subsequent treatment effects. This method is expected to expand the practical application range of TRPV1, enhance the effectiveness of TRPV1-mediated neural-cancer crosstalk to enhance early tumor detection, and its translational development potential in clinical cancer precision testing medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the following is a brief introduction to the drawings required for use in the examples. It should be understood that the following drawings only illustrate certain embodiments of the present invention and should not be considered as limiting the scope. A person of ordinary skill in the art can also derive other relevant drawings based on these drawings without inventive effort. In the drawings:

[0020] Figure 1 (C+D)@LNP of the embodiment of the present invention PRT Characterization diagram of some characteristics of the diagnosis and treatment platform;

[0021] Figure 2 (C+D)@LNP of the embodiment of the present invention PRT Targeted tumors for precise detection of images;

[0022] Figure 3 (C+D)@LNP of the embodiment of the present invention PRT Activation of TRPV1 mediates neuro-cancer crosstalk, regulates neuro-vascularity, and improves TME hypoxia.

[0023] Figure 4 (C+D)@LNP of the embodiment of the present invention PRT Activation of TRPV1 mediates nerve-cancer crosstalk and enhances the efficacy of tumor chemotherapy;

[0024] Figure 5 (C+D)@LNP of the embodiment of the present invention PRT Biosafety assessment diagram;

[0025] Figure 6 (C+D)@LNP of the embodiment of the present invention PRT Schematic diagram of the construction of the diagnostic and treatment platform and its targeted intervention of nerve-cancer crosstalk for tumor diagnosis and treatment. DETAILED DESCRIPTION

[0026] The following is a diagram of the embodiment of the present invention. Figures 1 to 6 The technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0027] Example 1

[0028] S1: Ionizable lipid (SM-102): auxiliary phospholipid (DSPC): cholesterol: functional modified lipid (DSPE-SS-PEG 2K -FA) was dissolved in anhydrous ethanol at a molar ratio of 50:38.5:10:1.5 to prepare an organic phase;

[0029] S2: Dissolve 6.5 μmol capsaicin (CAP) in the organic phase. Dissolve 3.5 μmol doxorubicin (DOX) in deionized water to prepare the aqueous phase. Rapidly mix the organic and aqueous phases in a microfluidic device at a volume ratio of 3:1 at a flow rate of 12 min / mL to obtain a mixed solution.

[0030] S3: The mixture obtained in S2 was placed in a 14 kDa dialysis bag and placed in a Tris buffer solution with pH = 7.4. The mixture was dialyzed at 4°C and 200 rpm for 24 hours to remove ethanol and free drugs. The mixture was then concentrated by ultrafiltration at 4°C and 5000 rpm to obtain the final product (C+D)@LNP.PRT , and store at 4°C in the dark.

[0031] (C+D)@LNP obtained by Example 1 PRT , mainly including functional modified lipids, ionizable lipids, cholesterol and auxiliary phospholipids, DOX and Capsaicin. Functional modified lipids (DSPE-SS-PEG 2K -FA) is used for FA modification of the targeting ligand to improve tumor targeting ability; disulfide bond modification is used for rapid drug release in response to GSH; ionizable lipids (SM-102) are used to mediate cellular endocytosis and endosomal escape, promote drug release in response to pH, and enhance therapeutic effects; cholesterol and auxiliary phospholipids are used to enhance lipid tightness and stability, regulate membrane fluidity and rigidity, improve blood circulation time, enhance biocompatibility, promote drug encapsulation, and regulate LNP particle size and morphology; DOX is a chemotherapy drug used for efficient chemotherapy of tumors; Capsaicin is used to specifically activate TRPV1 to regulate nerve-cancer crosstalk and reshape TME to enhance tumor diagnosis and treatment effects.

[0032] (C+D)@LNP obtained in Example 1 PRT The basic characteristic parameters include particle size and potential: particle size ~140nm and electrical neutrality; stability: there is no obvious change in particle size and potential within 15 days; charge reversal characteristics: when pH = 7.5, the charge is -4.35±3.86mV, while when pH = 5.5, the charge reversal is 14.23±4.80mV; response drug release kinetics: within 48h, the normal release rate of DOX is 15.55±1.11% (pH = 7.5, GSH = 0mM), and the response release rate is 81.64±3.09% (pH = 5.5, GSH = 5mM); the normal release rate of CAP is 8.93±0.41% (pH = 7.5, GSH = 0mM), and the response release rate is 66.43±1.05% (pH = 5.5, GSH = 5mM).

[0033] A novel multifunctional targeted intervention of nerve-cancer crosstalk synthesized in Example 1 to enhance tumor precision detection and chemotherapy effect (C+D)@LNP PRT , and the diagnostic and therapeutic platform has good physical and chemical properties, making it suitable for research on neurointerference therapy;

[0034] Combine Figure 1 , Figure 1 a~b is (C+D)@LNP PRT TEM images; Figure 1 c is (C+D)@LNP PRT Particle size distribution diagram; Figure 1 d is (C+D)@LNP PRT Particle size stability image; Figure 1e is (C+D)@LNP PRT Charge reversal characteristics and stability images under different pH conditions; Figure 1 f is (C+D)@LNP PRT Image of environmentally responsive drug release characteristics.

[0035] (C+D)@LNP was synthesized by an improved microfluidic method. PRT Nano-diagnosis and treatment platform, and used TEM, DLS and Zeta potential instrument to detect its morphology, particle size and potential. The results showed that ( Figure 1 ), the diagnostic and therapeutic system is spherical, well-dispersed, with a uniform particle size of approximately 140nm, an electrically neutral potential, and good stability. In addition, it has a potential reversal effect under acidic conditions and has obvious responsive drug release characteristics under different pH and different GSH concentrations. The above results show that the synthetic route of this diagnostic and therapeutic platform is feasible, the process is stable, and it has good physical and chemical properties, laying the foundation for subsequent in vitro and in vivo experiments.

[0036] (C+D)@LNP obtained in Example 1 PRT It can efficiently target tumors in vivo and in vitro to enhance the precise detection of tumors; combined with Figure 2 , Figure 2 a is the effect diagram of cell binding, Figure 2 b is the in vitro imaging results of major organs and tumors. Figure 2 c is the distribution diagram in tumor tissue;

[0037] Investigation of (C+D)@LNP by confocal microscopy and small animal imaging equipment PRT The results showed that ( Figure 2 ), at the cellular level, via (C+D)@LNP PRT After treatment, the cells showed stronger fluorescence, indicating stronger cell binding ability and better tumor targeting effect. PRT It is mainly distributed in the liver and kidneys, which are the main detoxification and excretion organs, indicating that it is mainly metabolized through the liver and kidneys. Moreover, the blood shows obvious fluorescence and the fluorescence gradually decreases over time, while it accumulates in the tumor site ( Figure 2 b). The tumor was sliced and observed, and it was found that (C+D)@LNP PRT After treatment, LNPs in tumor tissue PRT and DOX fluorescence were evenly distributed, showing stronger penetration ability ( Figure 2 c) Explanation of (C+D)@LNP PRT Capsaicin can bind to TRPV1, activate nerve-cancer crosstalk and trigger TME vasodilation to enhance delivery and improve tumor targeting. The above results show that (C+D)@LNP PRTMediating nerve-cancer crosstalk can improve tumor detection and diagnostic effectiveness, and provide data support for subsequent studies on enhancing drug delivery and improving efficacy.

[0038] (C+D)@LNP obtained in Example 1 PRT Targeted regulation of nerve-cancer crosstalk can reshape the TME, and (C+D)@LNP was injected into orthotopic PANC mice. PRT Tumor tissue was then taken to investigate its ability to activate TRPV1 to mediate nerve-cancer crosstalk and regulate nerve-vascular remodeling TME. The results showed that ( Figure 3 ), capsaicin can activate TRPV1 to enhance neural activity, induce angiogenesis and expansion, effectively relieve tumor hypoxia, and provide a data basis for subsequent research on reversing tumor resistance.

[0039] (C+D)@LNP obtained in Example 1 PRT Targeted regulation of nerve-cancer crosstalk can enhance the effect of tumor chemotherapy. (C+D)@LNP was injected into orthotopic PANC mice. PRT , and used small animal in vivo imaging to monitor the changes in mouse tumors and investigate the (C+D)@LNP PRT Enhance the effect of tumor chemotherapy. The results showed that ( Figure 4 ), (C+D)@LNP PRT After treatment, the growth of mouse tumors was significantly inhibited, indicating that (C+D)@LNP PRT The therapeutic effect of tumors can be enhanced by targeted regulation of nerve-cancer crosstalk, thus achieving an efficient neural interference treatment strategy.

[0040] (C+D)@LNP obtained in Example 1 PRT With good biosafety, combined Figure 5 , Figure 5 a is the weight change diagram of mice, Figure 5 b is the organ coefficient diagram, Figure 5 c is the morphological examination diagram of main organs; Figure 5 d is the histopathological test results of the corresponding organs after H&E staining, Figure 5 e is the result graph of hematological indicators, Figure 5 f is the results of liver, kidney, heart function and neurotoxicity indicators.

[0041] Injection of (C+D)@LNP into healthy mice PRT Afterwards, the biosafety was evaluated ( Figure 5 By tracking the weight changes of mice, taking the main organs of mice to calculate organ coefficients and observe organ pathological changes, and finally testing their hematological and biochemical indicators, the biosafety of the mice was comprehensively evaluated. The results showed that the weight of the mice increased steadily without abnormal changes ( Figure 5 a), there was no statistical difference among the coefficients of each organ ( Figure 5b), there is no obvious morphological change in the mouse organs ( Figure 5 c), there was no significant difference in the histopathological characteristics of the organs in each group, and all showed typical tissue structural characteristics ( Figure 5 d), there were no statistical differences in blood parameters, liver, kidney, heart function, and nerve function among the mice in each group, and all were within the normal range ( Figure 5 ef). The above results show that (C+D)@LNP PRT It has no obvious toxic side effects on mice and has good biosafety.

[0042] Combine Figure 6 You can get (C+D)@LNP PRT Schematic diagram of the construction and activation of TRPV1 to regulate nerve-cancer crosstalk and enhance tumor precision detection and targeted chemotherapy. Figure 6 a is (C+D)@LNP PRT Schematic diagram of the system construction process; Figure 6 b is (C+D)@LNP PRT Schematic diagram of capsaicin-activated TRPV1-mediated nerve-cancer crosstalk for precise tumor detection and enhanced chemotherapy.

[0043] The remarkable advancements of the present invention as well as the synergistic and unexpected effects are described in detail:

[0044] (C+D)@LNP PRT The functionally modified lipids are specially designed and synthesized according to the purpose of the present invention. The lipophilic drug CAP is used to trigger neural activity, regulate neural-cancer crosstalk for tumor detection and improve TME, and the hydrophilic drug DOX is used to improve the efficacy of multifunctional synergistic diagnosis and treatment research, which is the highlight of the present invention.

[0045] Moreover, currently LNP as a drug delivery carrier is focused on loading mRNA to increase the level of translation expression in cells, while there is no research on co-loading lipophilic and hydrophilic drugs. The aqueous phase of the present invention does not require sodium citrate buffer, only deionized water is required, and it can be directly dissolved and mixed using microfluidics. In addition, this method can simultaneously remove ethanol and free drugs through dialysis. During the concentration process, the appropriate method can be selected according to the different volumes of the solution. For small volumes of liquid (~0.1L), it can be concentrated by direct centrifugation through an ultrafiltration centrifuge tube with a molecular weight cutoff of 30kDa; for medium volumes of liquid (~0.5L), PEG can be used. 20K Cover the dialysis bag and perform "beach bath" concentration; for large volume liquid (>1L), tangential flow filtration system (TFF) can be used for concentration. In particular, the "beach bath" developed for medium volume concentration (for the solution after dialysis, the dialysis bag containing the solution is covered with a small amount of PEG 20KComplete thin layer coverage) not only solves the problem of high cost caused by the need for multiple ultrafiltration centrifuge tubes for small volumes of liquid, but also solves the problems of high cost of large-volume TFF equipment and waste caused by dead volume caused by inherent technology.

[0046] In summary, the innovation of the present invention lies in (C+D)@LNP PRT Design and development of two-phase microfluidic chip pipelines and two-phase drug loading, "beach bath" concentration method, capsaicin activation of TRPV1 to regulate nerve-cancer crosstalk, neural interference diagnosis and treatment strategies that use nerve-cancer crosstalk to reshape TME to enhance diagnosis and treatment effects, and application expansion of LNP and TRPV1.

[0047] The present invention co-loads hydrophilic and lipophilic drugs through LNP, and uses the lipophilic drug capsaicin to activate TRPV1 to regulate nerve-cancer crosstalk to enhance the delivery efficiency of the hydrophilic drug doxorubicin, while reshaping the TME to improve the diagnosis and treatment effect, and realize an efficient nerve interference diagnosis and treatment strategy. The present invention also expands the application field of LNP and the research scope of TRPV1. The results obtained in Example 1 show that the (C+D)@LNP obtained by the present invention PRT The delivery effect is better than that of the LNP delivery system in the existing technology, and can effectively activate TRPV1 to specifically regulate nerve-cancer crosstalk, efficiently reshape TME, and significantly enhance the diagnosis and treatment effect. The present invention also has good clinical transformation prospects and commercial production value.

Claims

1. A method for preparing a novel nano drug delivery system with tumor microenvironment regulation performance, characterized in that: The following steps are involved: S1: dissolving ionizable lipid, auxiliary phospholipid, cholesterol, and functional modified lipid in a molar ratio of 50:38.5:10:1.5 in a solvent to prepare an organic phase; S2: dissolving capsaicin in the organic phase obtained in S1, dissolving doxorubicin in a solvent to prepare an aqueous phase, and rapidly mixing the organic phase and the aqueous phase in a microfluidic device at a volume ratio of 3:1 to obtain a mixed solution; S3: The mixed solution obtained in S2 was placed in a dialysis bag and placed in Tris buffer for magnetic stirring dialysis and then concentrated to obtain (C+D)@LNP. PRT .

2. The method for preparing a novel nano drug delivery system with tumor microenvironment regulation performance according to claim 1, characterized in that: The molar ratio of capsaicin to doxorubicin in S2 was 6.5:3.

5.

3. The method for preparing a novel nano drug delivery system with tumor microenvironment regulation performance according to claim 1, characterized in that: The flow rate during the mixing process described in S2 was 12 min / mL.

4. The method for preparing a novel nano drug delivery system with tumor microenvironment regulation performance according to claim 1, characterized in that: The pH of the Tris buffer described in S3 is 7.

4.

5. The method for preparing a novel nano drug delivery system with tumor microenvironment regulation performance according to claim 1, characterized in that: The magnetic stirring described in S3 was performed at a rate of 200 rpm, a temperature of 4° C., and a time of 24 h.

6. The method for preparing a novel nano drug delivery system with tumor microenvironment regulation performance according to claim 1, characterized in that: The concentration described in S3 is ultrafiltration concentration, PEG 20k The ultrafiltration concentration was performed at a rate of 5000 rpm and a temperature of 4°C.

7. A novel nano drug delivery system with tumor microenvironment regulation performance obtained by the preparation method according to any one of claims 1 to 6, characterized in that: The nano drug delivery system comprises functionally modified lipids, ionizable lipids, cholesterol, auxiliary phospholipids, doxorubicin and capsaicin.

8. The use of a novel nano drug delivery system with tumor microenvironment regulation performance according to claim 7, characterized in that: The novel targeted regulation of nerve-cancer crosstalk nanodrug delivery system is prepared and used in tumor targeting and in enhancing tumor diagnosis and treatment.

Images