A nanomedicine delivery system with tumor microenvironment regulation properties, its preparation method and application

Abstract

This invention belongs to the field of precision oncology diagnosis and treatment technology, specifically involving a nanomedicine delivery system with tumor microenvironment regulation capabilities, its preparation method, and its application. S1: An organic phase is prepared by dissolving ionizable lipids, helper phospholipids, cholesterol, and functionally modified lipids in a solvent at a molar ratio of 50:38.5:10:1.5. S2: Capsaicin is dissolved in the organic phase obtained in S1, and doxorubicin is dissolved in the solvent to prepare an aqueous phase. The organic and aqueous phases are rapidly mixed in a microfluidic device at a volume ratio of 3:1 to obtain a mixture. S3: The mixture obtained in S2 is placed in a dialysis bag and immersed in Tris buffer for magnetic stirring and dialysis, followed by concentration to obtain (C+D)@LNPPRT. This invention significantly enhances the precision detection of tumors, enables efficient early tumor detection and targeted chemotherapy, and improves treatment efficacy.

Description

Technical Field

[0001] This invention belongs to the field of precision diagnosis and treatment technology for clinical oncology, specifically relating to a nanomedicine delivery system with tumor microenvironment regulation properties, its preparation method, and its application. Background Technology

[0002] Because early symptoms of pancreatic cancer (PANC) are often subtle and traditional detection methods are often inaccurate, clinical diagnosis, effective treatment, and improved prognosis remain significant challenges. In recent years, neuro-cancer crosstalk therapy, based on the regulation of neurocancer crosstalk, has achieved remarkable results in tumor diagnosis and treatment, bringing new hope to patients. Neuro-cancer crosstalk treatment strategies utilize the nervous system's regulatory role in cancer to influence tumor progression, playing a crucial role in tumor diagnosis and treatment. Neuro-cancer crosstalk induces the release of cancer-related biomarkers and triggers cascade activities through neuromodulation, enabling precise tumor detection and diagnosis using specific detection methods. Simultaneously, it can reverse tumor drug resistance and enhance treatment efficacy by modulating the tumor microenvironment (TME) through neuro-vascular regulation. Currently, the main approach involves targeted intervention with neuromodulatory drugs to reduce neural innervation and inhibit tumor growth, invasion, and metastasis. Furthermore, blocking nerve growth factor-transmembrane tyrosine kinase receptor (NGF-Trk) signaling can regulate neural activity, remodel the TME, improve efficacy, and inhibit tumor metastasis. However, neurointerference therapies utilizing the aforementioned neuro-cancer crosstalk modulation strategies have shown poor efficacy, and numerous challenges remain in their practical application for early, accurate detection and enhanced therapeutic efficacy, severely limiting their clinical translation. Firstly, effective doses of existing neuromodulation drugs can induce side effects; secondly, existing Trk targets are prone to off-target effects, causing neurotoxicity; thirdly, there is currently a lack of effective diagnostic and therapeutic platforms for regulating neuro-cancer crosstalk; furthermore, the cascade signals and biomarkers inducing neuro-cancer crosstalk are difficult to detect accurately; and finally, the signaling pathways and specific molecular mechanisms of neuro-cancer crosstalk modulation remain unclear. Therefore, developing a diagnostic and therapeutic system with superior targeting and multifunctional response characteristics, capable of powerfully inducing novel neuro-cancer crosstalk targets, is crucial for achieving accurate tumor detection, improving treatment levels, monitoring treatment efficacy, and elucidating the molecular mechanisms of neuro-cancer crosstalk diagnostic and therapeutic strategies.

[0003] To address the shortcomings of existing neuro-cancer crosstalk modulation strategies, such as the side effects caused by effective doses of existing neuromodulation drugs and the tendency of existing Trk targets to go off-target and cause neurotoxicity, this invention proposes a multifunctional diagnostic and therapeutic platform (C+D)@LNPPRT (C represents capsaicin, D represents doxorubicin, and LNPPRT represents tumor- and TME-responsive lipid nanoparticles) that can specifically target and intervene in the novel neuro-cancer crosstalk target, transient receptor potential vanillin 1 (TRPV1). This platform achieves precise detection of PANC and enhances the targeted therapeutic effect through efficient modulation of neuro-cancer crosstalk. Summary of the Invention

[0004] To address the problems and shortcomings of existing technologies, this invention provides a method for preparing a nanomedicine delivery system with tumor microenvironment regulation properties, comprising the following steps:

[0005] S1: An organic phase was prepared by dissolving ionizable lipids, cofactor phospholipids, cholesterol, and functionally modified lipids in a solvent at a molar ratio of 50:38.5:10:1.5;

[0006] S2: Capsaicin is dissolved in the organic phase obtained in S1, and doxorubicin is dissolved in a solvent to prepare an aqueous phase. The organic phase and the aqueous phase are rapidly mixed in a microfluidic device at a volume ratio of 3:1 to obtain a mixture.

[0007] S3: Place the mixture obtained in S2 into a dialysis bag and place it in Tris buffer. Perform magnetic stirring and dialysis, and then concentrate to obtain (C+D)@LNPPRT.

[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 Tris buffer described in S3 has a pH of 7.4.

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

[0012] Furthermore, the concentration mentioned in S3 is any one of ultrafiltration concentration, PEG20k concentration, or TFF concentration, wherein the ultrafiltration concentration rate is 5000 rpm and the temperature is 4°C.

[0013] A nanomedicine delivery system with tumor microenvironment regulation properties, the nanomedicine delivery system comprising functionally modified lipids, ionizable lipids, cholesterol and cofactor phospholipids, doxorubicin, and capsaicin.

[0014] Application of a nanomedicine delivery system with tumor microenvironment regulation properties, preparation of the targeted regulation neuro-cancer crosstalk nanomedicine delivery system, targeting of tumors, and application in enhancing tumor diagnosis and treatment.

[0015] Technical effect

[0016] This invention provides a method for preparing a nanomedicine delivery system with tumor microenvironment regulation properties; this invention utilizes functionalized lipids to synthesize a multifunctional diagnostic and therapeutic platform through a one-step microfluidic method, realizing a neuro-interference diagnostic and therapeutic solution for precise tumor detection and targeted chemotherapy.

[0017] The efficacy of neuro-interference therapy is limited by neuro-cancer crosstalk modulation strategies. Existing neuromodulation drugs and Trk targets lack specificity, and effective doses and off-target interventions can cause side effects. Therefore, developing a diagnostic and therapeutic system with excellent targeting and multifunctional response characteristics that can powerfully induce novel neuro-cancer crosstalk targets is a key prerequisite for achieving precision oncology. The multifunctional diagnostic and therapeutic platform (C+D)@LNPPRT synthesized in this invention can efficiently target tumors and induce neural cascade activity and tumor-related biomarker expression and release through specific activation of TRPV1-mediated neuro-cancer crosstalk, thereby enhancing the accuracy of tumor detection. Simultaneously, it modulates the TME to enhance targeted efficacy, thus realizing the neuro-cancer crosstalk modulation strategy for neuro-interference therapy.

[0018] Furthermore, this method achieves precise regulation by utilizing a multifunctional diagnostic and therapeutic platform to specifically activate TRPV1-mediated neuro-cancer crosstalk for precise cancer detection. Compared to the traditional neuro-cancer crosstalk target Trk, TRPV1 is simple, efficient, specific, and multifunctional. Capsaicin-specific activation of TRPV1 mediates neuro-cancer crosstalk, enhancing the release of its specific biomarkers and cascade activity. Combined with multimodal imaging technology, this improves the accuracy of early tumor detection, enabling precise tumor detection and diagnosis to enhance subsequent treatment outcomes. This method holds promise for expanding the practical application of TRPV1, improving the effectiveness of TRPV1-mediated neuro-cancer crosstalk in early tumor detection, and its translational potential in clinical precision oncology laboratory medicine. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort. In the drawings:

[0020] Figure 1 This is a partial characteristic representation diagram of the (C+D)@LNPPRT diagnostic and treatment platform according to an embodiment of the present invention;

[0021] Figure 2 This is an example of (C+D)@LNPPRT targeting tumors for precise detection in this invention.

[0022] Figure 3This is an image of (C+D)@LNPPRT activating TRPV1 to mediate neuro-cancer crosstalk regulation of neuro-vascular to improve TME hypoxia, according to an embodiment of the present invention.

[0023] Figure 4 This is a diagram illustrating the effect of (C+D)@LNPPRT activating TRPV1 to mediate neuro-cancer crosstalk and enhance tumor chemotherapy in an embodiment of the present invention.

[0024] Figure 5 This is a biosafety evaluation diagram of (C+D)@LNPPRT according to an embodiment of the present invention;

[0025] Figure 6 This diagram illustrates the construction of the (C+D)@LNPPRT diagnostic and treatment platform according to an embodiment of the present invention and its targeted intervention in neurocancer crosstalk for tumor diagnosis and treatment. Detailed Implementation

[0026] The following will be illustrated in conjunction with Embodiment 1 of the present invention. Figures 1-6 The technical solutions in the embodiments of the present invention are clearly and completely described herein. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0027] Example 1

[0028] S1: Prepare an organic phase by dissolving ionizable lipids (SM-102): cofactor phospholipids (DSPC): cholesterol: functionally modified lipids (DSPE-SS-PEG2K-FA) in anhydrous ethanol at a molar ratio of 50:38.5:10:1.5;

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

[0030] S3: The mixture obtained in S2 was placed in a 14 kDa dialysis bag and then in Tris buffer at pH 7.4. Dialysis was performed at 4°C and 200 rpm with magnetic stirring for 24 h to remove ethanol and free drug. The solution was then concentrated by ultrafiltration at 4°C and 5000 rpm to obtain the final product (C+D)@LNPPRT, and stored at 4°C protected from light.

[0031] The (C+D)@LNPPRT obtained in Example 1 mainly comprises functionally modified lipids, ionizable lipids, cholesterol and cofactor phospholipids, DOX, and Capsaicin. The functionally modified lipids (DSPE-SS-PEG2K-FA) enhance tumor targeting ability through FA modification; disulfide bond modification facilitates rapid drug release in response to GSH; the ionizable lipids (SM-102) mediate endocytosis and endosome escape, promote drug release in response to pH, and enhance therapeutic efficacy; cholesterol and cofactor phospholipids enhance lipid compactness 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 chemotherapeutic drug used for highly effective chemotherapy of tumors; and Capsaicin specifically activates TRPV1 to regulate neural-cancer crosstalk remodeling of the tumor microenvironment (TME), enhancing the efficacy of tumor diagnosis and treatment.

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

[0033] The (C+D)@LNPPRT, a multifunctional targeted intervention for neuro-cancer crosstalk that enhances the precision of tumor detection and chemotherapy efficacy, was synthesized in Example 1. This diagnostic and therapeutic platform has good physicochemical properties and is suitable for research on neuro-interference therapy.

[0034] Combination Figure 1 , Figure 1 a~b are TEM images of (C+D)@LNPPRT; Figure 1 c represents the particle size distribution of (C+D)@LNPPRT; Figure 1 d represents the particle size stability image of (C+D)@LNPPRT; Figure 1 e represents the charge reversal properties and stability of (C+D)@LNPPRT under different pH conditions; Figure 1 f is an image of the environmental response drug release characteristics of (C+D)@LNPPRT.

[0035] A (C+D)@LNPPRT nanotherapeutic platform was synthesized using an improved microfluidic method, and its morphology, particle size, and potential were detected using TEM, DLS, and Zeta potentiometry. The results showed that ( Figure 1 The proposed diagnostic system exhibits spherical, well-dispersed particles with a uniform size of approximately 140 nm, a neutral electrical potential, and good stability. Furthermore, it demonstrates potential reversal under acidic conditions and exhibits significant responsive drug release characteristics under different pH and GSH concentrations. These results indicate that the synthetic route of this diagnostic platform is feasible, the process is stable, and it possesses good physicochemical properties, laying the foundation for subsequent in vitro and in vivo experiments. The (C+D)@LNPPRT obtained in Example 1 can efficiently target tumors in vitro and in vivo, enhancing the accuracy of tumor detection; combined with… Figure 2 , Figure 2 a is an illustration of the effect of binding with cells. Figure 2 b shows the in vitro imaging results of the major organs and tumor. Figure 2 c shows the distribution of tumor tissue within the tumor.

[0036] The targeting ability of (C+D)@LNPPRT to tumors was investigated using confocal microscopy and small animal imaging equipment. The results showed that ( Figure 2 At the cellular level, cells treated with (C+D)@LNPPRT exhibited stronger fluorescence, indicating a stronger cell-binding ability and better tumor-targeting effect. Furthermore, at the in vivo level, (C+D)@LNPPRT was mainly distributed in major detoxification and excretion organs such as the liver and kidneys, indicating that it is primarily metabolized through the hepatic and renal pathway. Moreover, it showed significant fluorescence in blood, which gradually decreased over time, while accumulating at the tumor site. Figure 2 b). Observation of tumor sections revealed that after treatment with (C+D)@LNPPRT, LNPPRT and DOX fluorescence were uniformly distributed in the tumor tissue, exhibiting stronger penetration ability. Figure 2 c) This indicates that (C+D)@LNPPRT can activate neuro-cancer crosstalk by binding to TRPV1 through capsaicin, triggering TME vasodilation to enhance delivery and improve tumor targeting. These results suggest that (C+D)@LNPPRT-mediated neuro-cancer crosstalk can improve tumor detection and diagnostic efficacy, providing data support for subsequent studies on enhancing drug delivery and improving treatment efficacy.

[0037] The (C+D)@LNPPRT obtained in Example 1 can target and regulate the neuro-cancer crosstalk remodeling TME. Tumor tissue was harvested from orthotopic PANC mice after injection of (C+D)@LNPPRT to investigate its ability to activate TRPV1-mediated neuro-cancer crosstalk-regulated neuro-vascular remodeling TME. The results showed that ( Figure 3Capsaicin can activate TRPV1 to enhance neural activity, induce angiogenesis and dilation, and effectively alleviate tumor hypoxia, providing a data basis for subsequent research on reversing tumor drug resistance.

[0038] The (C+D)@LNPPRT obtained in Example 1 can target and regulate neuro-cancer crosstalk to enhance the effect of tumor chemotherapy. (C+D)@LNPPRT was injected into orthotopic PANC mice, and tumor changes were monitored using a small animal in vivo imaging system to investigate the effect of (C+D)@LNPPRT on tumor chemotherapy. The results showed that ( Figure 4 After treatment with (C+D)@LNPPRT, tumor growth in mice was significantly inhibited, indicating that (C+D)@LNPPRT can enhance the therapeutic effect of tumors by targeting and regulating neuro-cancer crosstalk, thus achieving a highly efficient neuro-interference therapy strategy.

[0039] The (C+D)@LNPPRT obtained in Example 1 exhibits good biocompatibility, combined with Figure 5 , Figure 5 a is a graph showing the changes in mouse body weight. Figure 5 b is the organ coefficient diagram. Figure 5 c is a morphological examination image of the main organs; Figure 5 Image d shows the histopathological examination results of the corresponding organ after H&E staining. Figure 5 e is a graph showing the results of hematological indicators. Figure 5 f represents the results of liver, kidney, and cardiac function and neurotoxicity indicators.

[0040] The biosafety of (C+D)@LNPPRT was evaluated in healthy mice. Figure 5 The study assessed the biosafety of mice by tracking their body weight changes, calculating organ coefficients from major organs, observing pathological changes in those organs, and finally detecting hematological and biochemical indicators. Results showed that the mice experienced stable weight gain without any abnormal changes. Figure 5 a) There were no statistically significant differences in the coefficients among the various organs. Figure 5 b), no obvious morphological changes were observed in the mouse organs ( Figure 5 c) No significant differences were found in the pathological characteristics of the organs and tissues in each group; all groups exhibited typical histological features. Figure 5 d), there were no statistically significant differences in blood parameters, liver, kidney, heart function, and neurological function among the groups of mice, all of which were within the normal range. Figure 5 The above results indicate that (C+D)@LNPPRT has no significant toxic side effects on mice and exhibits good biocompatibility.

[0041] Combination Figure 6 This yields a schematic diagram illustrating the construction of (C+D)@LNPPRT and the activation of TRPV1 to regulate neuro-cancer crosstalk, enhancing precise tumor detection and targeted chemotherapy through neural interference diagnosis. Figure 6 a is a schematic diagram of the (C+D)@LNPPRT system construction process; Figure 6 Schematic diagram of (C+D)@LNPPRT activating TRPV1 via capsaicin to mediate neuro-cancer crosstalk for precise tumor detection and enhanced chemotherapy.

[0042] The significant advancements, synergistic effects, and unexpected benefits of this invention are described in detail below:

[0043] The functionally modified lipids in (C+D)@LNPPRT are specially designed and synthesized according to the purpose of this invention. The highlight of this invention is its multifunctional synergistic diagnostic and therapeutic research, which utilizes the lipophilic drug CAP to induce neural activity, regulates neuro-cancer crosstalk for tumor detection and improves TME, and utilizes the hydrophilic drug DOX to enhance efficacy.

[0044] Currently, LNPs used as drug delivery carriers are mainly focused on loading mRNA to improve intracellular translational expression levels, while research on co-loading lipophilic and hydrophilic drugs is lacking. This invention eliminates the need for sodium citrate buffer in the aqueous phase, requiring only deionized water. Direct dissolution and microfluidic mixing are sufficient. Furthermore, this method allows for the simultaneous removal of ethanol and free drug via dialysis. During concentration, an appropriate method can be selected based on the solution volume. For small volumes (~0.1 L), concentration can be achieved directly by centrifugation using ultrafiltration centrifuge tubes with a molecular weight cutoff of 30 kDa. For medium volumes (~0.5 L), PEG20K can be coated onto the dialysis bag for "beach bath" concentration. For large volumes (>1 L), tangential flow filtration (TFF) systems can be used for concentration. In particular, the "beach bath" method (completely covering the dialysis bag with a thin layer of PEG20K after dialysis) developed for medium-volume concentration not only solves the cost problem of requiring multiple ultrafiltration centrifuge tubes for small volumes but also addresses the high cost of large-volume TFF equipment and the waste caused by dead volume due to inherent technology.

[0045] In summary, the innovations of this invention lie in the design and development of (C+D)@LNPPRT, two-phase microfluidic chip pipelines and two-phase drug loading, the "beach bath" concentration method, capsaicin-activated TRPV1 regulation of neuro-cancer crosstalk, the neuro-interference diagnostic and therapeutic strategy that utilizes neuro-cancer crosstalk to reshape the TME and enhance the diagnostic and therapeutic effect, and the application expansion of LNP and TRPV1.

[0046] This invention utilizes a liquid nitrogen (LNP) to co-load hydrophilic and lipophilic drugs, and leverages the lipophilic drug capsaicin to activate TRPV1, regulating neuro-cancer crosstalk and enhancing the delivery efficiency of the hydrophilic drug doxorubicin. Simultaneously, it reshapes the tumor microenvironment (TME) to improve therapeutic efficacy, achieving a highly efficient neuro-interference treatment strategy. This invention also expands the application areas of LNPs and the research scope of TRPV1. The results of Example 1 demonstrate that the (C+D)@LNPPRT delivery system obtained by this invention outperforms existing LNP delivery systems and can effectively activate TRPV1 to specifically regulate neuro-cancer crosstalk, efficiently reshape the TME, and significantly enhance therapeutic efficacy. This invention also possesses promising clinical translation prospects and commercial production value.

Claims

1. A method for preparing a nanomedicine delivery system with tumor microenvironment regulation properties, characterized in that, Includes the following steps: S1: An organic phase was prepared by dissolving ionizable lipids, cofactor phospholipids, cholesterol, and functionally modified lipids in a solvent at a molar ratio of 50:38.5:10:1.5; the ionizable lipid was SM-102, the cofactor phospholipid was DSPC, and the functionally modified lipid was DSPE-SS-PEG. 2K -FA; S2: Capsaicin is dissolved in the organic phase obtained in S1, and doxorubicin is dissolved in a solvent to prepare an aqueous phase. The organic phase and the aqueous phase are rapidly mixed in a microfluidic device at a volume ratio of 3:1 to obtain a mixture. S3: Place the mixture obtained in S2 into a dialysis bag and place it in Tris buffer. Perform magnetic stirring and dialysis, and then concentrate to obtain (C+D)@LNPPRT.

2. The method for preparing a nanomedicine delivery system with tumor microenvironment regulation properties according to claim 1, characterized in that, The molar ratio of capsaicin to doxorubicin in S2 is 6.5:3.

5.

3. The method for preparing a nanomedicine delivery system with tumor microenvironment regulation properties according to claim 1, characterized in that, The flow rate during the mixing process described in S2 is 12 min / mL.

4. The method for preparing a nanomedicine delivery system with tumor microenvironment regulation properties according to claim 1, characterized in that, The pH of the Tris buffer solution described in S3 is 7.

4.

5. The method for preparing a nanomedicine delivery system with tumor microenvironment regulation properties according to claim 1, characterized in that, The magnetic stirring speed described in S3 is 200 rpm, the temperature is 4 ℃, and the time is 24 h.

6. A method for preparing a nanomedicine delivery system with tumor microenvironment regulation properties according to claim 1, characterized in that, The concentration described in S3 is any one of ultrafiltration concentration, PEG20k concentration, or TFF concentration, wherein the ultrafiltration concentration rate is 5000 rpm and the temperature is 4 ℃.

7. A nanomedicine delivery system with tumor microenvironment regulation properties obtained by the preparation method according to any one of claims 1 to 6, characterized in that, The nanomedicine delivery system comprises functionally modified lipids, ionizable lipids, cholesterol and cofactor phospholipids, doxorubicin, and capsaicin; the ionizable lipid is SM-102, the cofactor phospholipid is DSPC, and the functionally modified lipid is DSPE-SS-PEG. 2K -FA.

8. The application of the nanomedicine delivery system with tumor microenvironment regulation properties according to claim 7 in the preparation of drugs for enhancing tumor diagnosis and treatment, characterized in that, The tumor is pancreatic cancer.

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