CN118079019A - Mitochondrial thermosensitive release nano preparation, preparation method and application thereof in preparation of deep tumor therapeutic drugs - Google Patents
Mitochondrial thermosensitive release nano preparation, preparation method and application thereof in preparation of deep tumor therapeutic drugs Download PDFInfo
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Abstract
The invention belongs to the technical field of biological medicines, and in particular relates to a mitochondrial thermosensitive release nano preparation, a preparation method and application thereof in preparation of deep tumor therapeutic drugs. The mitochondrial thermosensitive release nano preparation is prepared by using a mitochondrial targeting group triphenylphosphine-tetradecyl alcohol (TPP-TCA) obtained by modifying Triphenylphosphine (TPP) as a thermosensitive carrier, simultaneously electrostatically adsorbing tumor specific hyaluronic acid (TPP-HA) connected with the mitochondrial targeting TPP on the surface of the thermosensitive carrier, taking a uncoupler and an initiator as delivered drug micromolecules, and synthesizing the mitochondrial thermosensitive release nano preparation TPP-HA-TDV by an emulsion solvent evaporation method. The mitochondria thermosensitive release nano preparation can spontaneously release drugs in mitochondria of tumor cells without external light stimulation, and can be used for drug delivery of deep tumors.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and in particular relates to a mitochondrial thermosensitive release nano preparation, a preparation method and application thereof in preparation of deep tumor therapeutic drugs.
Background
Hepatocellular carcinoma (hepatocellular carcinoma, HCC) was ranked fourth among the major causes of tumor-related death with a survival rate of 18% in 5 years. HCC is considered to be a typical inflammation-associated cancer, whose inflammatory response is mainly derived from hepatitis virus infection, nonalcoholic steatohepatitis and fatty liver. Because of the long-standing incurable inflammation in tumors, HCC has low therapeutic responsiveness to immune checkpoint inhibitors (immune checkpoint inhibitor, ICI), and only less than 20% -40% of patients can benefit from ICI treatment.
To solve the above problems, the prior studies have stimulated tumor cells to undergo immunogenic death (immunogenic CELL DEATH, ICD) mainly by photodynamic therapy and photothermal immunotherapy, releasing the relevant tumor antigen and activating antigen presenting cells, thereby eliciting tumor immune responses. However, HCC belongs to a tumor deeply distributed in the human body, and a light source which can be used in clinical means is insufficient to reach a liver cancer growth site, wherein the light source with the maximum tissue penetration depth is far infrared light with a wavelength of 780-1100nm, and the penetration depth is about 1-3 mm, and the HCC can be only used for treating superficial tumors, such as melanoma and the like. Thus, a therapeutic regimen for deep tumors remains highly desirable.
Disclosure of Invention
The invention aims to provide a mitochondrial heat-sensitive release nano preparation which realizes efficient administration of deep tumors by utilizing mitochondrial heat to trigger phase change.
The technical scheme adopted for solving the technical problems is as follows:
A mitochondrial thermosensitive release nano-preparation is provided, which is
The mitochondrial targeting group triphenylphosphine-tetradecanol (TPP-TCA) obtained by modifying Triphenylphosphine (TPP) with Tetradecanol (TCA) is used as a heat-sensitive carrier,
Meanwhile, the surface of the thermosensitive carrier is electrostatically adsorbed with mitochondria targeting TPP-connected tumor specific hyaluronic acid (TPP-HA),
The decoupling agent and the initiator act as small molecules of the drug being delivered,
The mitochondrial thermosensitive release nano preparation TPP-HA-TDV is synthesized by an emulsion solvent evaporation method.
The inventor finds that the cell mitochondria release a large amount of heat in the process of mitochondrial respiration, and the released heat enables the temperature of the mitochondria to be higher than the common temperature of cytoplasm by 37 ℃, so that the free radical initiator azo diiso Ding Mi-hydrochloride (VA-044) can be triggered to crack to generate free radicals. On the basis, the inventor designs a sensitive drug release nano preparation using mitochondria to trigger phase change, and the sensitive drug release nano preparation can spontaneously release drugs on mitochondria of tumor cells without external light stimulation, can be used for drug delivery of deep tumors, and provides a feasible scheme for clinical accurate administration. On the other hand, the mitochondria targeted administration of tumor cells can activate the immunogenic death of the tumor cells induced by mitochondrial damage and release liver cancer related antigens, thereby increasing the response of patients to ICI, providing new alternatives for liver cancer patients and having great practical significance.
Preferably, the decoupling agent is selected from one or more of 2, 4-Dinitrophenol (DNP), carbonyl cyanide-m-chlorophenyl (CCCP), carbonyl cyano-p-trifluoromethoxybenzohydrazone (FCCP) and valinomycin; the initiator is selected from one or more of 2.2' -azo [ N- (2-ethyl) -2-methylpropionate ] hydrate V057), azo-diisopropyl imidazoline hydrochloride (V044), azo-diisopropyl imidazoline (V061), azo-diisobutylamidine hydrochloride (V050) or azo-diisoheptonitrile.
Preferably, the uncoupler is selected from 2, 4-Dinitrophenol (DNP) and the initiator is selected from 2,2' -azo [ N- (2-carboxyethyl) -2-methylpropionamidine ] hydrate (V057).
The preparation method of the mitochondrial heat-sensitive release nano preparation comprises the following steps:
S1, preparation of TPP-TCA: the hydroxyl of TCA modifies the carboxyl of TPP-COOH through esterification reaction;
S2, preparation of TPP-HA: the hydroxyl of HA is modified into carboxyl of TPP-COOH through esterification reaction;
s3, preparation of TPP-HA-TDV:
S3.1, preparation of TDV solution:
Dissolving the mitochondrial targeting group triphenylphosphine-tetradecanol (TPP-TCA) prepared in the step S1 and a decoupling agent 2, 4-Dinitrophenol (DNP) in an organic solvent to obtain a mixed solution A;
Slowly adding the mixed solution A into 1-5% (w/V) polyvinyl alcohol (PVA) solution containing V057 to enable the solution A to be stably dispersed in water; carrying out ultrasonic treatment on the obtained mixture to form a water/oil emulsion; removing solvent DCM in the water/oil emulsion by distillation under reduced pressure;
centrifuging at 4 ℃ to obtain a nanocomposite TDV;
s3.2, mixing and incubating the prepared TPP-HA and TDV, and synthesizing the mitochondrion thermosensitive release nano preparation TPP-HA-TDV by utilizing electrostatic adsorption.
Preferably, in S3, the mass ratio of TPP-HA to TDV in the control system is 0.25-0.30:1, the incubation time is 30min to 40min.
Preferably, the organic solvent is selected from dichloromethane or chloroform.
Preferably, the conditions of centrifugation are: centrifugation was performed 3 times at 10000rpm for 5 minutes each.
Preferably, in S3.1, TPP-TCA, DNP, DCM and V057 are used in an amount of 10-20mg:1-2mg:1-2ml:2-6mg.
Preferably, S1, TPP-TCA is prepared:
Mixing 3.0-5.0g of TPP-COOH, 1.0-2.0g of EDC, 40-60 mg g of DMAP and 10-20ml of chloroform, degassing with nitrogen flow for 20min, and cooling to 0 ℃ to obtain a mixed solution B;
15ml of chloroform solution containing 1.0-3.0g of TCA is added into the mixed solution B to react with TPP-COOH in the system for 1h at 0 ℃; then the mixture is transferred to room temperature, stirred fully and washed by water to remove byproducts;
The organic phase was dried and the solvent was distilled off under reduced pressure to give the compound as a white solid, namely TPP-TCA. Preferably, S2, preparation of TPP-HA:
100-300mg of TPP-COOH was dissolved in 20-50ml DMSO/H 2 O v/v=1: 1, 200-400mg of DMAP and 500-1000mg of EDC are added to activate the carboxyl groups of TPP-COOH, stirred at 60℃for 1H, dissolved in 5-10ml of DMSO/H 2 O v/v=1: 1 and adding 100-500mg of excessive HA into the activated TPP-COOH, and reacting for 8 hours at room temperature;
After the reaction was completed, the resulting mixture was dialyzed against distilled water to remove unreacted impurities, and lyophilized to obtain purified TPP-HA.
Preferably, in S3.2, the TDV is dispersed in an aqueous solution, after which it is added dropwise to the TPP-HA aqueous solution.
The invention relates to an application of a mitochondrial heat-sensitive release nano preparation in preparation of a deep tumor therapeutic drug. The application is specifically for thermosensitive drug release for deep tumors to increase drug accumulation in tumor cells, thereby increasing therapeutic effects.
The beneficial effects of the invention are as follows:
HCC is generally considered a rare "cold" tumor of immune response as a deep tumor. In recent years, immune Checkpoint Inhibitors (ICI), including cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1), have achieved significant clinical efficacy in a range of tumor types. However, tumor (including HCC) patients with intrinsic apoptosis resistance and immune evasion have poor response to ICI, and less than 20-40% of patients benefit from ICI treatment. To change this state of the art, researchers have employed specific therapies such as photothermal immunotherapy (PTI) and photodynamic therapy (PDT) to convert these "cold" tumors to "hot" tumors by inducing Immunogenic Cell Death (ICD) to stimulate an immune response. However, these treatments remain challenging in HCC treatment because HCC cells are deep tumors and have strong immune escape, making it difficult to reach the HCC deep layer.
The inventor aims to overcome the depth problem of HCC deep tumor, adopts mitochondrial inherent thermal therapy to trigger thermosensitive drug release, and further self-heating mitochondria through 2, 4-dinitrophenol uncoupling, thereby remarkably promoting free radical bomb and inducing tumor ICD. The synthesized mitochondrially targeted TPP-HA-TDV NPs produced specific free radicals in mitochondria without external stimulus and clearly elicited immune responses by increasing ICD marker release and CD4 + and CD 8+ T cell infiltration. The result shows that the mitochondria thermotherapy is probably an endogenous target for the release of the thermosensitive drug, so that the thermosensitive release nano-preparation (TPP-HA-TDV NPs) for mitochondria disclosed by the inventor can overcome the obstacle that the drug is difficult to penetrate in deep tumors, reduce the immune escape of HCC, and further enhance the anti-tumor immune response by combining with ICI.
Drawings
FIG. 1 is a morphological characterization of a mitochondrial thermosensitive release nano-formulation of the invention, wherein A is a TEM image of the mitochondrial thermosensitive release nano-formulation and B, C is the particle size and potential size of the nano-formulations TDV, TPP-HA-TDV;
FIG. 2 is a fluorescence micrograph of the thermosensitive release nano-preparation of the present invention, wherein A, B is the size distribution of TPP-HA-TDV NPs at 37℃and 45 ℃, C is the DNP content of TPP-HA-TDV NPs at 25℃and 37℃and 45 ℃, and D is the thermosensitive release of mitochondria;
FIG. 3 is an illustration of the induction of immunogenic death by the mitochondrial heat sensitive release nano-agent of the invention wherein A is SOSG the free radical generating capacity of TPP-HA-TDV, B, C the result of free radical induced immunogenic death by TPP-HA-TDV NPs;
fig. 4 is an illustration of in vivo efficacy of the mitochondrial heat sensitive release nano-formulation of the invention, wherein a is a photograph of a tumor of different groups, B is a statistic of the tumor weights of different groups, and C is the volume change of a mouse tumor during treatment.
Detailed Description
The technical scheme of the invention is further specifically described by the following specific examples. It should be understood that the practice of the invention is not limited to the following examples, but is intended to be within the scope of the invention in any form and/or modification thereof.
In the present invention, unless otherwise specified, all parts and percentages are by weight, and the equipment, materials, etc. used are commercially available or are conventional in the art. The methods in the following examples are conventional in the art unless otherwise specified.
The reagents used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.
In order to better illustrate the essence of the present invention, the effect thereof is further illustrated by the results of the pharmacological action experiments of the pharmaceutical combinations of the present invention.
EDC, 1-ethyl- (3-dimethylaminopropyl) carbodiimide (Shanghai national pharmaceutical group chemical reagent company);
DMAP, 4-dimethylaminopyridine (Shanghai pharmaceutical group chemical reagent company);
DNP,2, 4-dinitrophenol (ara-latin biochemical technologies limited);
V057, 2' -azo [ N- (2-carboxyethyl) -2-methylpropionamidine ] hydrate (Shanghai national pharmaceutical group chemical reagent company);
MitoTracker green FM mitochondrial green fluorescent probe (melengorgement);
HMGB1, high mobility group protein B1 (10829-1-AP, proteintech, wuhan three hawk biotechnology limited);
CRT, calreticulin (27298-1-AP, proteintech, wuhan three eagle biotechnology limited);
HSP 90, heat shock protein 90 (13171-1-AP, proteintech, WU Sanying biotechnology Co., ltd.)
TCA, tetradecanol (Shanghai milin Biochemical technologies Co., ltd.);
HA, hyaluronic acid (rohn, shanghai Yi En chemical technologies limited).
Example 1
A method for preparing a mitochondrial heat-sensitive release nano preparation, which comprises the following steps:
(1) TPP-TCA: the hydroxyl groups of TCA modify the carboxyl groups at TPP-COOH by esterification.
TPP-COOH (3.09 g), EDC (1.12 g), DMAP (48.8 mg) and chloroform (15 ml) were mixed in a flask, and then degassed with a nitrogen stream for 20min, and cooled to 0℃in an ice bath to obtain a mixed solution B.
Thereafter, to the mixed solution B, a chloroform (15 ml) solution containing TCA (1.5 g) was added, and reacted with TPP-COOH in the system at 0℃for 1 hour. The mixture was then transferred to room temperature and stirred for an additional 24h. The by-product was removed by extraction washing three times with the same volume of water.
The organic phase was then dried over anhydrous MgSO 4 and the solvent was removed by distillation under reduced pressure to give the compound as a white solid, namely TPP-TCA.
(2) TPP-HA: the hydroxyl group of HA modifies the carboxyl group at TPP-COOH through esterification.
TPP-COOH (200 mg) was dissolved in 20ml DMSO/H 2 O (v/v=1:1), and DMAP (267 mg) and EDC (678 mg) were added to activate the carboxyl group of TPP-COOH. Stirred at 60℃for 1h. Then, an excess of HA (147 mg) dissolved in 8ml DMSO/H 2 O (v/v=1:1) was added to the activated TPP-COOH and reacted for a further 8H at room temperature.
After the reaction was completed, the resulting mixture (MW 1000 Da) was dialyzed against distilled water for 24 hours to remove unreacted impurities. After lyophilization for 48h, a purified TPP-HA polymer was obtained.
(3) TPP-HA-TDV: the mitochondrial thermosensitive release nano preparation is synthesized by adopting an emulsion solvent evaporation method.
TPP-TCA (20 mg) synthesized in step (1) and DNP (2 mg) were dissolved in 1ml DCM to give a mixed solution A. The mixed solution A was slowly added to 2ml of a 1% PVA solution (m/V, deionized water) containing V057 (4 mg). The resulting mixture was sonicated with a probe for 5 minutes to form a water/oil emulsion. The solvent DCM in the water/oil emulsion was removed by distillation under reduced pressure at 28 ℃. Then, the mixture was centrifuged at 10000rpm for 3 times at 4℃for 5 minutes each. The centrifugal precipitate is TDV nanometer complex. And dispersing the centrifugal precipitate by ultrapure water to obtain a TDV solution.
And (3) dissolving the TPP-HA obtained in the step (2) in deionized water overnight to prepare a 1mg/ml TPP-HA solution, and preserving at 4 ℃. Adding TPP-HA into TDV solution, and incubating for 30min with the mass ratio of TPP-HA/TDV of 25% to obtain TPP-HA-TDV nanometer preparation.
Comparative example 1:
Unlike example 1, the inventors first used TCA alone as a thermosensitive drug carrier, and carried out V057 and DNP encapsulation using the same protocol as in example 1, and found that TCA could not form a stable assembly, and could not be used for the construction of a thermosensitive carrier without precipitation by centrifugation.
To increase the ability of TCA to form an assembly, the inventors modified TCA with TPP-COOH having three benzene rings to synthesize TPP-TCA. TPP-TCA can achieve stable assemblies for entrapment of DNP and V057 by pi-pi stacking interactions.
Comparative example 2
In the case of preparing the TDV nanocomposite, V057 was dispersed in methylene chloride (DCM) solvent together with TPP-TCA and DNP, and V057 was found to be insoluble, so that V057 was not entrapped.
Thereafter, V057 was prepared by dissolving V057 in 1% pva solution in advance, dissolving TPP-TCA and DNP in DCM solvent, and synthesizing TDV by emulsion solvent evaporation method in example 1.
Comparative example 3:
The difference from example 1 is that TPP-HA: tdv=1: in the proportion of 1, the inventor finds that a large amount of aggregates precipitate after incubation, and the obtained TPP-HA-TDV nano particle size is larger than 1000nm.
Whereas example 1 uses TPP-HA: tdv=0.25: when the mass ratio of 1 is matched, the TPP-HA-TDV nano preparation which is uniformly dispersed in water and HAs the particle size of 300nm can be obtained.
Characterization and performance testing:
1. characterization of mitochondrial thermosensitive release nanoformulations
And taking the mitochondrial thermosensitive release nano preparation, and characterizing the morphology and particle size charge of the mitochondrial thermosensitive release nano preparation.
1.1 Characterization of morphology
Firstly, dispersing the prepared mitochondrial heat-sensitive release nano preparation into water, dripping the water onto a copper mesh and a conductive glass slide, drying the water by nitrogen, and then, characterizing the morphology by a Transmission Electron Microscope (TEM). The characterization result is shown in FIG. 1 (A).
The particle size of the mitochondrial thermosensitive release nano-preparation designed by the invention is about 300nm and takes the shape of a sphere as shown in fig. 1.
1.2 Charge size (zeta potential)
After diluting the prepared TDV and TPP-HA-TDV by a certain multiple, the particle diameter and potential of the nano particles are detected by a particle diameter instrument, as shown in FIG. 1 (B) and FIG. 1 (C).
The nano-formulation particle size and the potential size are shown in fig. 1 (B) and fig. 1 (C). (B) The zeta potential of TDV NPs is 35mV, a high positive charge leading to its rapid clearance from the blood and to poor hemolysis; (C) The zeta potential of NPs was varied from 35mV to-21 mV for TPP-HA-TDV, indicating successful modification of the TPP-HA chain.
2. Thermosensitive release of mitochondrial thermosensitive release nanoformulations
2.1 Particle size variation at different temperatures
After incubating TPP-HA-TDV NPs at 37℃and 45℃for 1 hour, the particle sizes were measured with a particle sizer, respectively. The detection results are shown in FIGS. 2 (A) and (B).
The size distribution of TPP-HA-TDV NPs at 37℃and 45℃is shown in FIGS. 2 (A) and (B). The TPP-HA-TDV size was kept at 300nm below 37℃with PDI as narrow as 0.208. However, after incubation for 1h at 45 ℃, the HA-TPP-TDV swells into a liquid form, increasing the particle size to above 1000. The results indicate that thermosensitive drug release is dependent on the solid-liquid transition of TCA (phase transition temperature of TCA is 38 ℃ -39 ℃).
2.2 Drug Release at different temperatures
0.5Ml of TPP-HA-TDV (1 mmol/L) solution was added to the MWCO:1000 dialysis bags, immersed in 3ml PBS containing 5% DMSO, incubated at 25℃at 37℃at 45℃with gentle shaking, simulating room temperature, normal body temperature and mitochondrial hyperthermia. During the experiment, 1ml of dialysis fluid outside the dialysis bag was collected periodically at different time points, and 1ml of fresh PBS (0.5% DMSO) was added. The DNP content of TPP-HA-TDVNPs was determined in parallel by High Performance Liquid Chromatography (HPLC). The detection result is shown in FIG. 2 (C)
In FIG. 2 (C) it is shown that TPP-HA-TDV NPs release only about 22% DNP after incubation at 25℃and 37℃due to the phase transition temperature of TPP-TCA being 38℃to 39 ℃. At 45 ℃, the drug release rate increases with the increase of time, and the release rate can reach about 60% after 50 min. These results indicate that body temperature (around 37 ℃) does not stimulate the phase transition of TPP-TCA and TPP-HA-TDVNPs releases small amounts of DNP. Meanwhile, the mitochondrial temperature (above 45 ℃) can effectively lead the dissolved TPP-HA-TDVNPs to release the drug, thereby ensuring the enrichment of the mitochondrial specific drug.
2.3 Mitochondrial thermosensitive Release
1X 10 5 Hepa1-6 cells were seeded in 6-well plates with slides and incubated for 24h, and serum-free medium containing methylene blue (MB, λex=664 nm) labeled HA-TD, TPP-HA-TCA and TPP-HA-TD was added instead of medium. Cells were incubated in the dark for 12 hours and then washed 3 times with PBS. To track mitochondria, cells were further visualized with a zeiss fluorescence microscope by staining with MitoTracker Green FM (λex/λem=490 nm/516 nm) in the dark for 0.5h, washing 3 times with pbs, DAPI anti-quench fluorescent dye-patch. MB and MitoTracker Green FM represent fluorescence of NPs and mitochondria, respectively. The detection result is shown in FIG. 2 (D)
The modification of TPP on HA polymer is shown in fig. 2 (D) to promote mitochondrial targeting of TPP-HA-TD-MB due to the positively charged effect of TPP. Due to uncoupling of DNP, TPP-HA-TD-MB is more fluorescent around the mitochondrial region than TPP-HA-TCA-MB.
3. Mitochondrial thermosensitive release nano preparation for inducing immunogenic death
3.1 Free radical Generation
The radical generating capacity of TPP-HA-TDV was measured using a Singlet Oxygen Sensor Green (SOSG). mu.LTPP-HA-TDV (V057: 0.67mM, DNP:3.4 mM) was added to 1ml H2O containing 1 mu M SOSG probe. Incubation was performed at 25℃and 37℃and 45℃and 60℃respectively. After incubation for 0.5h and 1h, SOSG fluorescence was detected with λex/λem=504 nm/525 nm. The detection results are shown in FIG. 3A.
FIG. 3 (A) shows that SOSG shows only a weak fluorescence enhancement at incubation below 45℃due to the lower rate of V057 decomposition. Whereas fluorescence of SOSG rapidly increased upon incubation at 60 ℃.
Detection of protein expression by WB
WB detects ICD related signal proteins HSP90, CRT, HMGB. Briefly, cells were seeded in six well plates and treated for 24h with control, HA-TDV, TPP-HA-TD, TPP-HA-TV and TPP-HA-TDV. In animal tumors, the collected tumor tissues are homogenized, and whole protein precipitates are extracted. Then cleaved with RIPA containing 1% PMSF at 4℃for 30min, respectively. Protein concentration was determined using BCA protein assay kit. After electrophoresis, proteins of different molecular weights were separated and blotted onto PVDF membranes. Next, blocking with 5% skim milk was followed by incubation with different antibodies in a primary anti-dilution (P0023A, beyotime), overnight at 4 ℃. Then, PVDF membranes were incubated with enzyme-labeled anti-rabbit IgG or anti-mouse IgG secondary antibodies for 1 hour at room temperature. All blots were soaked with ECL and scanned with ChemiDoc Touch image system. The detection results are shown in FIGS. 3 (B) and (C).
The results of free radical induced immunogenic death by TPP-HA-TDVNPs are shown in FIGS. 3 (B) and (C). In vitro and in vivo, the expression of HSP90, CRT and HMGB1 was most significantly increased in the TPP-HA-TDVNPs group compared to the control group, and the production of surface radicals was critical for ICD activation.
4. Antitumor effect
The in vivo anticancer effect of TPP-HA-TDV was evaluated using H22 tumor-bearing model. These tumor-bearing mice were randomly divided into 6 groups (n=5): (1) control group, (2) TPP-HA-TD NPs, (3) anti-CTLA-4, (4) TPP-HA-TV NPs, (5) TPP-HA-TDV, (6) TPP-HA-TDV+anti-CTLA-4. NPs and anti-CTLA-4 were dosed at 3mg/kgV057 and 10. Mu.g/mouse anti-CTLA-4, respectively, when the tumor volume reached around 200mm 3. Equal amounts of PBS were injected as controls. Body weight and tumor volume of each group of mice were measured and recorded daily. Mice were sacrificed after 12d, tumor sizes were photographed and weighed, and each group of tumor tissue was collected. The detection results are shown in FIG. 4.
The in vivo anti-tumor effect is shown in fig. 4. Compared with the control group, the TPP-HA-TDV+ anti-CTLA-4 tumor is obviously smaller, and the weight of each tumor is as follows: control group: 0.95.+ -. 0.23g, TPP-HA-TD group: 0.51+ -0.17 g; CTLA-4 antibody panel: 0.33+ -0.06 g; TTP-HA-TV group; 0.23+ -0.07 g; TPP-HA-TDV group: 0.13+/-0.04 g; TPP-HA-TDV+anti-CTLA-4 group: 0.05 plus or minus 0.01g.
The mitochondrial heat-sensitive release nano preparation has the therapeutic effect on tumors: the mitochondria thermosensitive release nano preparation is utilized to reach tumor cells through endocytosis, and the tumor cells reach mitochondria through accurate targeting of TPP. Due to the high temperature of the mitochondria itself (above the thermal phase transition temperature of TCA), TCA changes from solid to liquid phase, releasing DNP and V057. Since DNP uncouples to release a large amount of heat, the mitochondrial temperature is further raised, causing excessive mitochondrial heating. Too high thermal cleavage of V057 generates a large number of free radicals, which cause immunogenic death. Immunogenic death (ICD) can cause release of related injury-associated molecular patterns (DAMPs), including Adenosine Triphosphate (ATP), calreticulin (CRT), heat Shock Proteins (HSPs) and high mobility group box 1 (HMGB 1), ultimately resulting in a T cell mediated tumor neoantigen immune response.
In summary, the mitochondrial heat sensitive release nano-preparation (TPP-HA-TDVNPs) of the invention can be an effective treatment method for deep tumors through mitochondrial induced self-heating free radical explosion.
In this specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, so that the same or similar parts between the embodiments are referred to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant points refer to the description of the method section.
The mitochondrial heat-sensitive release nano preparation, the preparation method and the application thereof in preparing deep tumor therapeutic drugs are described in detail. The principles and embodiments of the present invention have been described herein with reference to specific examples, the description of which is intended only to facilitate an understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that various modifications and adaptations of the invention can be made without departing from the principles of the invention and these modifications and adaptations are intended to be within the scope of the invention as defined in the following claims.
Claims (11)
1. A mitochondrial heat sensitive release nano-formulation characterized by: the mitochondrion thermosensitive release nano preparation is
The mitochondrial targeting group triphenylphosphine-tetradecanol (TPP-TCA) obtained by modifying Triphenylphosphine (TPP) with Tetradecanol (TCA) is used as a heat-sensitive carrier,
Meanwhile, the surface of the thermosensitive carrier is electrostatically adsorbed with mitochondria targeting TPP-connected tumor specific hyaluronic acid (TPP-HA),
The decoupling agent and the initiator act as small molecules of the drug being delivered,
The mitochondrial thermosensitive release nano preparation TPP-HA-TDV is synthesized by an emulsion solvent evaporation method.
2. The mitochondrial heat sensitive release nano-formulation according to claim 1, wherein: the decoupling agent is one or more selected from 2, 4-Dinitrophenol (DNP), carbonyl cyanide-m-chlorophenyl (CCCP), carbonyl cyano-p-trifluoromethoxybenzohydrazone (FCCP) and valinomycin; the initiator is selected from one or more of 2.2' -azo [ N- (2-ethyl) -2-methylpropionate ] hydrate V057), azo-diisopropyl imidazoline hydrochloride (V044), azo-diisopropyl imidazoline (V061), azo-diisobutylamidine hydrochloride (V050) or azo-diisoheptonitrile.
3. A method of preparing a mitochondrial heat sensitive release nano-formulation according to claim 1, characterized in that the method comprises the steps of:
S1, preparation of TPP-TCA: the hydroxyl of TCA modifies the carboxyl of TPP-COOH through esterification reaction;
S2, preparation of TPP-HA: the hydroxyl of HA is modified into carboxyl of TPP-COOH through esterification reaction;
s3, preparation of TPP-HA-TDV:
S3.1, preparation of TDV solution:
Dissolving the mitochondrial targeting group triphenylphosphine-tetradecanol (TPP-TCA) prepared in the step S1 and a decoupling agent 2, 4-Dinitrophenol (DNP) in an organic solvent to obtain a mixed solution A;
slowly adding the mixed solution A into 1-5% (w/V) polyvinyl alcohol (PVA) solution containing V057 to enable the solution A to be stably dispersed in water; carrying out ultrasonic treatment on the obtained mixture to form a water/oil emulsion; removing solvent DCM in the water/oil emulsion by distillation under reduced pressure;
centrifuging at 4 ℃ to obtain a nanocomposite TDV;
s3.2, mixing and incubating the prepared TPP-HA and TDV, and synthesizing the mitochondrion thermosensitive release nano preparation TPP-HA-TDV by utilizing electrostatic adsorption.
4. A method of preparation according to claim 3, characterized in that: in S3, controlling the mass ratio of TPP-HA to TDV in the system to be 0.25-0.30:1, the incubation time is 30min to 40 min.
5. A method of preparation according to claim 3, characterized in that: the organic solvent is selected from dichloromethane or chloroform.
6. A method of preparation according to claim 3, characterized in that: the conditions for centrifugation are: 10000 Centrifugation was performed 3 times at 5 minutes each at rpm.
7. A method of preparation according to claim 3, characterized in that: in S3.1, the dosages of TPP-TCA, DNP, DCM and V057 are 10-20 mg:1-2 mg:1-2 ml:2-6 mg.
8. A process according to claim 3, characterized in that S1, TPP-TCA is prepared:
Mixing TPP-COOH 3.0-5.0 g, EDC 1.0-2.0 g, DMAP 40-60 mg and chloroform 10-20 ml, degassing with nitrogen flow 20 min, cooling to 0deg.C to obtain mixed solution B;
Adding trichloromethane 15 ml solution containing TCA 1.0-3.0 g into the mixed solution B to react with TPP-COOH in the system at 0 ℃ to obtain 1 h; then the mixture is transferred to room temperature, stirred fully and washed by water to remove byproducts;
the organic phase was dried and the solvent was distilled off under reduced pressure to give the compound as a white solid, namely TPP-TCA.
9. A process according to claim 3, characterized in that S2, TPP-HA is prepared:
TPP-COOH 100-300 mg was dissolved in 20-50 ml DMSO/H 2 O v/v=1: 1, adding DMAP 200-400 mg and EDC 500-1000 mg to activate the carboxyl groups of TPP-COOH, stirring 1H at 60 ℃ to dissolve in 5-10 ml DMSO/H 2 O v/v=1: 1 and adding the excessive HA 100-500 mg in the step 1 into the activated TPP-COOH, and reacting 8-h at room temperature;
After the reaction was completed, the resulting mixture was dialyzed against distilled water to remove unreacted impurities, and lyophilized to obtain purified TPP-HA.
10. A method of preparation according to claim 3, characterized in that: in S3.2, TDV is dispersed in an aqueous solution, followed by dropwise addition to an aqueous TPP-HA solution.
11. Use of the mitochondrial heat sensitive release nano-formulation of claim 1 in the preparation of a medicament for the treatment of deep tumors.
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