CN120136830A - Nanoparticles targeting glioblastoma and their preparation and application - Google Patents
Nanoparticles targeting glioblastoma and their preparation and application Download PDFInfo
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Abstract
The invention belongs to the field of new auxiliary materials and new dosage forms of pharmaceutical preparations, relates to a nano preparation for targeting glioblastoma and preparation and application thereof, and in particular relates to a nano preparation for targeting glioblastoma, especially a chlorambucil-mycophenolic acid prodrug of temozolomide drug-resistant glioblastoma, and preparation and application thereof. The invention firstly constructs a chlorambucil-mycophenolic acid prodrug or pharmaceutically acceptable salt thereof, and then constructs a prodrug co-assembled nano preparation capable of realizing the synergistic delivery of three drugs of chlorambucil, mycophenolic acid and MHY1485 on the basis. Meanwhile, the prodrug co-assembled nano preparation is subjected to targeted modification, so that the ability of crossing the blood brain barrier is endowed. According to the invention, a targeting peptide modified nano drug delivery system is constructed by a prodrug co-assembly technology, so that the drug can be accurately delivered to a tumor part while crossing a blood brain barrier, and the synergistic anti-tumor effect of the drug combination is remarkably improved.
Description
Technical field:
The invention belongs to the field of new auxiliary materials and new dosage forms of pharmaceutical preparations, relates to a nano preparation for targeting glioblastoma and preparation and application thereof, and in particular relates to a nano preparation for targeting glioblastoma, especially a chlorambucil-mycophenolic acid prodrug of temozolomide drug-resistant glioblastoma, and preparation and application thereof.
The background technology is as follows:
The tumor is a serious disease which seriously threatens the life health of human beings, wherein glioblastoma (Glioblastoma) is recognized as one of the most difficult malignant tumors due to the characteristics of high treatment difficulty, high disability rate, high lethality rate and the like. At present, the standard treatment scheme of glioblastoma mainly adopts a comprehensive treatment mode of combining surgical excision with radiotherapy and chemotherapy. However, the biological nature of invasive growth of this tumor, lacking a clear margin from normal brain tissue, makes radical resection difficult to achieve with simple surgery. Meanwhile, when the tumor cells are killed by large-dose radiotherapy and chemotherapy, irreversible damage is often caused to the nerve function of a patient, and the life quality of the patient is seriously influenced.
In terms of drug treatment, temozolomide (Temozolomide) is the only first-line chemotherapeutic drug at present, and the clinical application of temozolomide is faced with serious drug resistance problem. Studies have shown that glioblastomas can effectively clear methylation damaged regions by O6-methylguanine-DNA methyltransferase (MGMT) and a base excision repair mechanism, thereby generating drug resistance to temozolomide. Therefore, the development of a novel therapeutic strategy for temozolomide drug-resistant glioblastoma has important clinical significance for improving the survival rate of patients and improving prognosis.
In the existing chemotherapeutics, chlorambucil is used as another important alkylating agent, and the action mechanism of chlorambucil is obviously different from that of temozolomide. The medicine forms irreversible cross-linking between DNA chains through SN 2 nucleophilic reaction, and the generated alkylation damage is not identified and cleared by MGMT, but is repaired by base excision repair mechanism.
Currently, chlorambucil is clinically used mainly for the maintenance treatment of chronic lymphocytic leukemia, hodgkin's lymphoma and ovarian cancer.
Notably, chlorambucil-induced DNA damage repair processes are highly dependent on base excision repair mechanisms, which require guanosine as a key raw material. The biosynthesis and metabolism of guanine are precisely regulated by hypoxanthine mononucleotide dehydrogenase 2 (IMPDH 2) and mammalian target protein complex 1 (mTorrC 1), respectively, which provides a new idea for developing novel combined therapeutic strategies.
MHY1485 is a small molecule mTOR agonist that activates the pathway by stabilizing the GTP-binding state of RAG GTPASE, a process that requires guanylate metabolism to provide GTP molecular support. Modulation of mTORC1 by MHY1485 affects the dynamic balance of the intracellular purine nucleotide pool, particularly by altering guanylate kinase activity to remodel the nucleotide metabolic network.
In the drug treatment of glioblastoma, the blood brain barrier constitutes a major obstacle to drug delivery. The barrier consists of brain microvascular endothelial cells, pericytes, basement membrane and astrocytes of the tail foot, and has high selective permeability. Furthermore, the non-specific distribution of chemotherapeutic agents within brain tissue may lead to severe neurotoxicity, which is also a major concern in glioblastoma treatment. Therefore, development of a delivery system capable of effectively penetrating the blood brain barrier and achieving lesion site-specific drug release has important clinical value for improving the therapeutic effect of glioblastoma.
The prior art has no report on the combined use of chlorambucil, mycophenolic acid and MHY1485, and has no report on the combined use of chlorambucil and mycophenolic acid after being prepared into prodrugs and MHY 1485.
The invention comprises the following steps:
aiming at the limitations of the prior art in the aspect of treating glioblastoma, in particular temozolomide drug-resistant glioblastoma, the invention creatively provides a multi-drug synergistic treatment strategy and constructs an intelligent drug delivery system with high-efficiency blood brain barrier penetration capability. The system is characterized in that chlorambucil is firstly utilized to generate specific DNA damage which is not regulated by MGMT, secondly, a ribosome biosynthesis pathway is activated by adopting an 'open source throttling' dual regulation mechanism, namely mTORC1 agonist MHY1485 is adopted to accelerate guanylate consumption, and meanwhile, an IMPDH2 inhibitor mycophenolic acid is utilized to selectively inhibit a de novo synthesis pathway of guanosine, so that effective exhaustion of intracellular guanylate level is realized, and finally, a base excision repair pathway is blocked.
Based on the above treatment strategy, the invention adopts the prodrug co-assembly technology, firstly constructs a chlorambucil-mycophenolic acid prodrug or pharmaceutically acceptable salt thereof, and then constructs a prodrug co-assembly nano-preparation capable of realizing three drug cooperative delivery on the basis. Meanwhile, the prodrug co-assembled nano preparation is subjected to targeted modification, so that the ability of crossing the blood brain barrier is endowed. The invention provides a brand-new treatment idea and technical scheme for targeting glioblastoma, in particular to temozolomide drug-resistant glioblastoma.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the invention provides chlorambucil as shown in formula I-a mycophenolic acid prodrug or a pharmaceutically acceptable salt thereof:
wherein X is a tumor microenvironment sensitive chemical bond.
In the structural formula, the tumor microenvironment sensitive chemical bond is a pH sensitive bond, a redox environment sensitive bond, an enzyme sensitive bond and a hypoxia sensitive bond, wherein the pH sensitive bond comprises at least one of a hydrazone bond, an imine bond, an acetal bond and a beta-amino ester bond, the redox environment sensitive bond comprises at least one of a monosulfide bond, a disulfide bond, a trisulfide bond, a monoselenium bond, a diselenide bond and a boric acid ester bond, the enzyme sensitive bond comprises at least one of a metalloprotease sensitive bond, an esterase sensitive bond, a phosphatase sensitive bond, a transglutaminase sensitive bond and a thioredoxin reductase sensitive bond, and the hypoxia sensitive bond comprises at least one of an azo bond and a nitro aromatic bond.
Further, X is a redox context sensitive bond.
Specifically, the chlorambucil-mycophenolic acid prodrug or a pharmaceutically acceptable salt thereof has the following structure:
The invention further provides a synthesis method of the chlorambucil-mycophenolic acid prodrug, which specifically comprises the following steps:
step 1, reacting chlorambucil with oxalyl chloride to generate an acyl chloride intermediate, condensing with dithiodiethanol, extracting, preparing and separating liquid phase to obtain chlorambucil-dithiodiethanol intermediate.
Step 2, after reacting chlorambucil-dithiodiethanol intermediate with p-nitrophenyl chloroformate, adding mycophenolic acid and triethylamine, reacting overnight, extracting, preparing and separating liquid phase to obtain chlorambucil-mycophenolic acid prodrug.
The purity of the chlorambucil-mycophenolic acid prodrug prepared by the invention is more than 98%.
The chlorambucil-mycophenolic acid prodrug or the pharmaceutically acceptable salt thereof can be self-assembled into a chlorambucil-mycophenolic acid prodrug self-assembled nano-preparation (CMNP).
The chlorambucil-mycophenolic acid prodrug self-assembled nano-preparation comprises a chlorambucil-mycophenolic acid prodrug and a PEG modifier/active targeting modifier.
The mass ratio of the chlorambucil-mycophenolic acid prodrug to the PEG modifier/active targeting modifier is 1 (0.1-1).
The PEG modifier is an amphiphilic polymer such as DSPE-PEG, TPGS, PEG-PLGA or PEG-P or a targeting group, the active targeting modifier is a substance which can target specific tissues such as brain targeting peptide, antibody conjugate, ligand conjugate, cell penetrating peptide conjugate, receptor targeting conjugate, sugar residue, hormone and the like, and is selected from DSPE-PEG-SHp、DSPE-PEG-Angiopep、DSPE-PEG-T7、DSPE-PEG-RVG29、DSPE-PEG-cRGD、DSPE-PEG-Lactoferrin、DSPE-PEG-NGR、DSPE-PEG-TAT、DSPE-PEG-iRGD、DSPE-PEG-Mannose、DSPE-PEG-OTC、DSPE-PEG-GE11、DSPE-PEG-CREKA、DSPE-PEG-TH、DSPE-PEG-R8、DSPE-PEG-APOE,, preferably phospholipid-polyethylene glycol-apolipoprotein E (DSPE-PEG-APOE).
Further, the invention provides a preparation method of the chlorambucil-mycophenolic acid prodrug nano-preparation, which comprises the following steps:
and (3) dissolving a proper amount of PEG modifier/active targeting modifier and chlorambucil-mycophenolic acid prodrug together in an organic solvent, fully stirring and mixing, and slowly dripping the mixed solution into an aqueous phase. Under the self-assembly action of the prodrug molecules, the system spontaneously forms uniformly dispersed nanoparticles. And then, thoroughly removing the organic solvent by adopting a reduced pressure rotary evaporation method, and finally obtaining the pure PEG modified/active targeting modified chlorambucil-mycophenolic acid prodrug self-assembled nano preparation.
The chlorambucil-mycophenolic acid prodrug or the pharmaceutically acceptable salt thereof can also be co-assembled with other small molecular compounds as main drug components to form a chlorambucil-mycophenolic acid prodrug and small molecular compounds co-assembled nano preparation (tNP).
The small molecule compound is MHY1485.
The molar ratio of chlorambucil-mycophenolic acid prodrug or a pharmaceutically acceptable salt thereof to MHY1485 is 5:1-1:2, preferably 3:1-5:1, more preferably 3:1-4:1.
The chlorambucil-mycophenolic acid prodrug and small molecule compound co-assembled nanometer preparation comprises chlorambucil-mycophenolic acid prodrug, MHY1485 and PEG modifier/active targeting modifier.
The mass ratio of the total mass of the chlorambucil-mycophenolic acid prodrug and the MHY1485 to the PEG modifier/active targeting modifier is 1 (0.1-1).
The PEG modifier is an amphiphilic polymer such as DSPE-PEG, TPGS, PEG-PLGA or PEG-P or a targeting group, the active targeting modifier is a substance which can target specific tissues such as brain targeting peptide, antibody conjugate, ligand conjugate, cell penetrating peptide conjugate, receptor targeting conjugate, sugar residue, hormone and the like, and is selected from DSPE-PEG-SHp、DSPE-PEG-Angiopep、DSPE-PEG-T7、DSPE-PEG-RVG29、DSPE-PEG-cRGD、DSPE-PEG-Lactoferrin、DSPE-PEG-NGR、DSPE-PEG-TAT、DSPE-PEG-iRGD、DSPE-PEG-Mannose、DSPE-PEG-OTC、DSPE-PEG-GE11、DSPE-PEG-CREKA、DSPE-PEG-TH、DSPE-PEG-R8、DSPE-PEG-APOE,, preferably phospholipid-polyethylene glycol-apolipoprotein E (DSPE-PEG-APOE).
The invention also provides a preparation method of the chlorambucil-mycophenolic acid prodrug and MHY1485 co-assembled nano preparation, which comprises the following steps:
And (3) dissolving a proper amount of PEG modifier/active targeting modifier, chlorambucil-mycophenolic acid prodrug and MHY485 in an organic solvent together, fully stirring and mixing, and slowly dripping the mixed solution into an aqueous phase. Under the self-assembly action of the prodrug molecules, the system spontaneously forms uniformly dispersed nanoparticles. And then, thoroughly removing the organic solvent by adopting a reduced pressure rotary evaporation method, and finally obtaining the pure PEG modified/active targeting modified chlorambucil-mycophenolic acid prodrug and MHY1485 co-assembled nano preparation.
The organic solvent is ethanol, acetone, tetrahydrofuran, dimethyl sulfoxide, N-dimethylformamide and other water-soluble organic solvents, preferably acetone.
The volume ratio of the organic solvent to the water is (0.1-1): 1, preferably 0.2-0.5:1.
The invention has the beneficial effects that:
(1) The invention provides an open source throttling strategy, and the guanosine raw material required by repairing DNA damage by depleting tumor cells forms a high-efficiency synergistic effect with chlorambucil, thereby effectively avoiding the drug-resistant mechanism of temozolomide. The strategy not only enriches the combined drug design thought of temozolomide drug-resistant glioblastoma, but also provides a new solution for clinical treatment.
(2) The invention designs and synthesizes the chlorambucil-mycophenolic acid prodrug, and successfully prepares the self-assembled nano preparation with regular shape and uniform particle size and the co-assembled nano preparation with MHY 1485. The particle size of the prepared nano preparation is less than 200nm, the optimal range is 50-150nm, the dispersion coefficient (PDI) is less than 0.15, and the preparation method is simple and easy to implement and has good repeatability and stability.
(3) According to the invention, the targeting peptide modified nano drug delivery system is constructed by a prodrug co-assembly technology, so that the drug can be accurately delivered to the tumor part across the blood brain barrier, the synergistic anti-tumor effect of the drug combination is obviously improved, and the key bottleneck of low drug delivery efficiency and poor treatment effect of brain diseases is hopefully broken through. In addition, the tumor redox responsive disulfide bond introduced in the prodrug design enables the nano preparation to activate the drug specifically in the tumor cells with reduced high expression, and keep inertia in normal tissues, so that the damage of the off-target effect to brain parenchyma is greatly reduced, and the treatment target of attenuation and synergy is realized.
Description of the drawings:
FIG. 1 is a mass spectrum of chlorambucil-mycophenolic acid prodrug of example 1 of the present invention.
FIG. 2 is a 1 HNMR spectrum of the chlorambucil-mycophenolic acid prodrug of example 1 of the present invention.
FIG. 3 is a graph showing particle size distribution of CMNP and tNP of example 3 of the present invention.
Fig. 4 is a transmission electron microscope image of CMNP and tNP of example 3 of the present invention.
Fig. 5 is a graph of the colloidal stability of CMNP and tNP of example 4 of the present invention.
Fig. 6 is a plot of long-term particle size and PDI change for CMNP and tNP of example 4 of the present invention.
Fig. 7 is a graph showing the change in the long-term standing potential of CMNP and tNP in example 4 of the present invention.
FIG. 8 is an in vitro drug release profile of tNP of example 5 of the present invention.
FIG. 9 is an in vitro drug release profile of CMNP of example 5 of the present invention.
Fig. 10 is a comet experimental plot of each prescription of example 7 of the present invention.
Fig. 11 is a graph showing the tail moment quantification of the comet assay for each prescription of example 7 of the present invention.
No significant differences, P <0.05 was considered significant differences (one-factor analysis of variance).
FIG. 12 is a graph showing the quantitative determination of the DNA content of the tail of the comet assay for each prescription in example 7 of the present invention.
No significant differences, P <0.05 was considered significant differences (one-factor analysis of variance).
FIG. 13 is a graph of intracellular GTP concentration for each of the formulations of example 7 of the present invention.
No significant differences, P <0.05 was considered significant differences (one-factor analysis of variance).
FIG. 14 is a quantitative detection chart of AP sites of each prescription of example 7 of the present invention.
No significant differences, P <0.05 was considered significant differences (one-factor analysis of variance).
FIG. 15 is a plot of in vivo tumor fluorescence signal for each of the prescriptions of example 8 of the present invention.
FIG. 16 is a graph of the in vivo tumor fluorescence signal quantification of each of the formulations of example 8 of the present invention.
FIG. 17 is a plot of the fluorescence signal of the major tissue and tumor ex vivo for each prescription of example 8 of the present invention.
FIG. 18 is a plot of fluorescence signal of isolated major tissues and tumors for each of the prescriptions of example 8 of the present invention.
Fig. 19 is an in vitro brain fluorescence signal plot for each prescription of example 8 of the present invention.
No significant differences, P <0.05 was considered significant differences (one-factor analysis of variance).
FIG. 20 is a graph of the bioluminescence of mouse temozolomide resistant glioblastoma in example 9 of the present invention.
FIG. 21 is a bioluminescence quantification of temozolomide resistant glioblastoma in mice of example 9 of the present invention.
No significant differences, P <0.05 was considered significant differences (one-factor analysis of variance).
FIG. 22 is a graph showing the weight change of mice in example 9 of the present invention.
FIG. 23 is a graph showing the survival rate of mice in example 9 of the present invention.
FIG. 24 is a median survival time plot for mice of example 9 of the present invention.
FIG. 25 is a H & E section of mouse brain tissue according to example 9 of the present invention.
The specific embodiment is as follows:
The present invention will be described in further detail with reference to examples.
EXAMPLE 1 Synthesis of chlorambucil-mycophenolic acid prodrug
Chlorambucil (5 mmol) and a catalytic amount of DMF are dissolved in anhydrous dichloromethane, ice bath is cooled to 0-5 ℃, oxalyl chloride (6 mmol) is rapidly added dropwise under stirring, the mixture is warmed to room temperature for reaction for 4 hours after the dripping is finished, and solvent is dried by spinning to obtain intermediate acyl chloride. Subsequently, dithiodiethanol (6 mmol) and triethylamine (10 mmol) were dissolved in anhydrous dichloromethane, cooled to 0-5 ℃ in an ice bath, and the acid chloride intermediate dissolved in the anhydrous dichloromethane was slowly added dropwise, and after the addition was completed, the reaction was carried out at room temperature overnight, the organic phase was extracted with a 10% citric acid solution, redissolved in acetonitrile after spin-drying, and the chlorambucil-dithiodiethanol intermediate was obtained by preparative liquid phase separation purification and spin-drying.
The chlorambucil-dithiodiethanol intermediate (3 mmol) and DIPEA (6 mmol) are dissolved in anhydrous dichloromethane, ice bath is cooled to 0-5 ℃, p-nitrophenyl chloroformate (4 mmol) dissolved in the anhydrous dichloromethane is slowly added dropwise, the mixture is cooled to room temperature for reaction for 2 hours after the dripping is finished, the solvent is dried by spinning, the mixture is redissolved in anhydrous DMF, mycophenolic acid (3 mmol) is added, the mixture is stirred at room temperature until the mycophenolic acid is completely dissolved, triethylamine (6 mmol) is rapidly added, the mixture is reacted at room temperature overnight, the organic phase is extracted by saturated sodium bicarbonate and 10% citric acid solution respectively, the mixture is redissolved in acetonitrile after spinning, and the chlorambucil-mycophenolic acid prodrug is obtained through preparation of liquid phase separation and purification and spinning.
The structure of the chlorambucil-mycophenolic acid prodrug of example 1 was determined by mass spectrometry and nuclear magnetic resonance hydrogen spectrometry, and the results are shown in fig. 1 and 2. The results of the spectroscopic analysis are as follows:
1H NMR(400MHz,Chloroform-d)δ7.17–6.96(m,2H,d),6.64(d,J＝8.5Hz,2H,c),5.30(s,1H,p),5.23–5.03(m,2H,k),4.54(t,J＝6.8Hz,2H,j),4.34(t,J＝6.5Hz,2H,h),3.91–3.40(m,13H,a,b,n,o),3.07(t,J＝6.8Hz,2H,e),2.95-2.56(t,J＝6.4Hz,4H,i),2.47–2.14(m,9H,g,r,s),1.91(p,J＝7.5Hz,2H,f),1.79(d,J＝1.4Hz,3H,q).MS(ESI)m/z:808.1765[M+Na]+.
EXAMPLE 2 prescription screening of brain-targeted prodrug nanoformulations
The preparation method selects DSPE-PEG-APOE as a surface modification material, adopts a one-step nano precipitation method to prepare a brain targeting prodrug nano preparation, and comprises the following specific preparation processes:
Preparation of chlorambucil-mycophenolic acid prodrug self-assembled nano-formulations chlorambucil-mycophenolic acid prodrug and DSPE-PEG-APOE (mass ratio 20%, w/w) were dissolved in 0.5mL acetone to form a homogeneous solution. Under the condition of intense stirring, the solution is slowly and dropwise added into 5mL of deionized water, after full mixing, acetone is removed by reduced pressure rotary evaporation, and finally the chlorambucil-mycophenolic acid prodrug self-assembled nano preparation (CMNP) is obtained.
Preparation of the Navoside and MHY1485 Co-assembled nanoformulation the Navoside-mycophenolic acid prodrug and MHY1485 were mixed in different molar ratios of 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5 and dissolved in 0.5mL acetone with DSPE-PEG-APOE (mass ratio 20%, w/w) to form a homogeneous solution. Under the condition of intense stirring, slowly and dropwise adding the solution into 5mL of deionized water, fully mixing, removing acetone by reduced pressure rotary evaporation, and finally obtaining the chlorambucil-mycophenolic acid prodrug and MHY1485 co-assembled nano preparation.
Characterization of prodrug nanoformulations the prodrug nanoformulations were diluted 10-fold with deionized water and their particle size, aggregation index (PDI), and surface potential were measured using a malvern particle sizer.
Determination of prodrug nano-preparation encapsulation efficiency and drug loading, namely removing unencapsulated drug of the prodrug nano-preparation by using a centrifugation method, determining encapsulation efficiency by using a high performance liquid chromatography, and determining the detection wavelength of the prodrug of chlorambucil-mycophenolic acid by using a C18 reversed phase chromatographic column (4.6X250 mm,5 μm), wherein the mobile phase is methanol and water, the detection wavelength of the prodrug of chlorambucil-mycophenolic acid is 258nm, and the detection wavelength of MHY1485 is 340nm. The encapsulation and drug loading rates of chlorambucil-mycophenolic acid prodrug and MHY1485 were calculated according to the following formula:
Encapsulation efficiency = drug content in formulation/total drug dosed x 100%;
drug loading = theoretical drug loading x encapsulation efficiency x 100%.
Synergy index of prodrug nanoformulations the cytotoxicity of each ratio of co-assembled nanoformulations was first assessed using the MTT method, and the specific procedure was as follows, U87/TR cells (temozolomide resistant U87 cells) were seeded into 96-well plates (2 x 10 3 cells per well) and placed in an incubator for 12 hours of incubation to ensure stable adherence of the cells. Then, the original culture medium is replaced by the culture medium containing chlorambucil, mycophenolic acid, MHY1485 and CMNP with different concentration gradients and the co-assembled nano preparation culture medium with various proportions. Untreated cells served as control. After each group of cells was incubated in a 37 ℃ incubator for a further 48 hours, 20 μl of MTT solution was added to each well and incubation was continued for a further 4 hours to allow formazan crystals to form. Subsequently, the medium was discarded, formazan crystals were dissolved in DMSO, absorbance values were determined using a multifunctional microplate reader at 490nm wavelength, and the half maximal inhibitory concentration (IC 50) was calculated from the measured data using graphpad software. In addition, with the IC 50 of chlorambucil, mycophenolic acid, and MHY1485 as a control, the formula was calculated using the Chou-Talalay method as follows:
Wherein D 1、D1、D3 is the actual molar concentration of chlorambucil, mycophenolic acid and MHY1485 in the prodrug nano-preparation respectively, IC 50,1、IC50,2、IC50,3 is the IC 50.CI50 <1 of single drug of chlorambucil, mycophenolic acid and MHY1485 respectively, which shows synergistic effect, CI 50 =1 is additive effect, and CI 50 >1 shows antagonistic effect.
And (3) morphological observation of the prodrug nano preparation, namely diluting the prodrug nano preparation to 10 times of the sample volume by pure water, then taking a small amount of diluted liquid to be dripped on an electron microscope copper mesh, standing and naturally air-drying. Then, 0.25% phosphotungstic acid solution was added dropwise thereto for dyeing in a dark place, and the mixture was left to stand at room temperature for air drying. After sample preparation was completed, the morphological features of examine and accept rice grains were observed using a Transmission Electron Microscope (TEM).
The optimal assembly ratio is determined by examining the effect of the molar ratio of different chlorambucil-mycophenolic acid prodrugs to MHY1485 on the particle size, encapsulation efficiency and drug loading of the nanoformulation. The results are shown in Table 1, where the molar ratio of chlorambucil-mycophenolic acid prodrug to MHY1485 is 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, the preparation of the nanoformulation in colloidal state was successful. However, at 1:3, 1:4 and 1:5 ratios, the drug aggregates out. The blood brain barrier consists of a dense monolayer of cell membranes, tight junctions and matrices, strictly controlling the ingress and egress of substances. Even after surface modification, the nanoparticles with oversized particle size (> 200 nm) still have poor permeability and are difficult to penetrate the blood brain barrier. Thus, co-assembled nanofabricated formulations at ratios of 2:1, 1:1, and 1:2 cannot be a candidate prescription due to particle sizes of greater than 200 nm. When the molar ratio of chlorambucil-mycophenolic acid prodrug to MHY1485 is 5:1-3:1, the particle size of the nano preparation is less than 150nm, and the dispersion coefficient is less than 0.15. Thus, the molar ratio of chlorambucil-mycophenolic acid prodrug to MHY1485 is preferably 5:1-3:1.
TABLE 1 characterization of the co-assembled nano-formulations of chlorambucil-mycophenolic acid prodrugs with MHY1485 at different molar ratios
A) The different proportions are the molar ratio of chlorambucil-mycophenolic acid prodrug to MHY1485
The encapsulation efficiency and drug loading of the nano-formulation are two key indicators for evaluating the performance of the nano-formulation. The higher the encapsulation efficiency is, the stronger the self-assembly capability of the drug system of the nano preparation is, the better the reproducibility among different batches is, and the industrial production and the quality controllability are facilitated. The higher the drug loading rate is, the lower the proportion of non-drug components of the nano preparation is, and the potential toxic and side effects caused by the non-drug components can be effectively avoided. As shown in Table 2, when the molar ratio of chlorambucil-mycophenolic acid prodrug to MHY1485 is 5:1-1:2, the encapsulation efficiency of chlorambucil-mycophenolic acid prodrug can reach more than 75%, the encapsulation efficiency of MHY1485 can reach more than 60%, and the total drug loading is more than 50%. When the ratio of the two is 5:1-3:1, the encapsulation rate of the chlorambucil-mycophenolic acid prodrug can reach more than 98%, the encapsulation rate of MHY1485 can reach more than 90%, and the total drug loading rate can reach more than 60%. Especially when the chlorambucil-mycophenolic acid prodrug and the MHY1485 are assembled together in a ratio of 3:1, the encapsulation rate of the chlorambucil-mycophenolic acid prodrug and the MHY1485 reaches more than 99%, and the total drug loading rate reaches 65.43%.
TABLE 2 encapsulation efficiency and drug loading of Naphthol-mycophenolic acid prodrugs and MHY1485 Co-assembled nanoformulations at different molar ratios
A) The molar ratio of the chlorambucil-mycophenolic acid prodrug to the MHY1485 b) with different proportions is the sum of the drug loading of the chlorambucil, the mycophenolic acid and the MHY1485
IC 50 and CI 50 of each ratio of co-assembled nanofabricated formulations are shown in table 3. Co-assembled nanoformulations of chlorambucil-mycophenolic acid prodrug with MHY1485 constructed at molar ratios of 4:1, 3:1 and 1:1 showed significant synergistic effects (CI 50 < 1), whereas 5:1, 2:1 and 1:2 ratio combinations showed antagonistic effects (CI 50 > 1). Notably, the co-assembled nanoformulation constructed with chlorambucil-mycophenolic acid prodrug to MHY1485 at a molar ratio of 3:1 has the lowest IC 50 (15.7 μm) and the smallest CI 50 (0.39). The molar ratio of chlorambucil-mycophenolic acid prodrug to MHY1485 is preferably 3:1-4:1, as combined with the results of particle size, encapsulation efficiency and drug loading. And the co-assembled nano-preparation constructed when the molar ratio of the chlorambucil-mycophenolic acid prodrug to the MHY1485 is 3:1 has the optimal pharmaceutics and synergistic effect, so that the 3:1 is determined to be the optimal ratio to construct the chlorambucil-mycophenolic acid prodrug and the MHY1485 co-assembled nano-preparation.
TABLE 3 Co-assembly of chlorambucil-mycophenolic acid prodrugs with MHY1485 at different molar ratios IC 50 and CI of the nanoformulation 50
A) Molar ratio of chlorambucil to mycophenolic acid to MHY1485 after correction by encapsulation efficiency b) IC 50 units:. Mu.M
The following studies were conducted with chlorambucil-mycophenolic acid prodrug self-assembled nanoformulation (CMNP) and chlorambucil-mycophenolic acid prodrug co-assembled with MHY1485 (tNP) constructed with a molar ratio of chlorambucil-mycophenolic acid prodrug to MHY1485 of 3:1.
As shown in fig. 3 and 4, CMNP and tNP each had a particle size of about 90nm and a uniform particle size distribution (PDI < 0.15). The surface potential of the two nano-preparations is about-50 mV, so that the biocompatibility of intravenous injection administration is ensured. In addition, the observation result of the transmission electron microscope shows that the two are in uniform spherical structures and have good morphology.
EXAMPLE 3 preparation of brain-targeting prodrug nanoformulations
(1) Preparation of chlorambucil-mycophenolic acid prodrug self-assembled nanoformulation (CMNP):
The chlorambucil-mycophenolic acid prodrug and DSPE-PEG-APOE (mass ratio 20%, w/w) were dissolved in 0.5mL acetone to form a homogeneous solution. Under the condition of intense stirring, slowly and dropwise adding the solution into 5mL of deionized water, fully mixing, and removing acetone by reduced pressure rotary evaporation to obtain the aqueous solution.
(2) Preparation of chlorambucil-mycophenolic acid prodrug co-assembled with MHY1485 nanoformulation (tNP):
the chlorambucil-mycophenolic acid prodrug was mixed with MHY1485 in a 3:1 molar ratio and dissolved with DSPE-PEG-APOE (mass ratio 20%, w/w) in 0.5mL acetone to form a homogeneous solution. Under the condition of intense stirring, slowly and dropwise adding the solution into 5mL of deionized water, fully mixing, and removing acetone by reduced pressure rotary evaporation to obtain the aqueous solution.
EXAMPLE 4 colloidal stability of brain-targeted prodrug nanoformulations long-term shelf stability
Colloidal stability CMNP and tNP prepared in example 3 were diluted 1:10 in 10% Fetal Bovine Serum (FBS) in PBS and placed in a 37 ℃ shaker for incubation, samples were taken at 0, 2, 4, 8, 12, 24, 48 hours of incubation, and their particle size, aggregation index (PDI) was determined using a malvern particle sizer.
Long-term shelf stability CMNP and tNP prepared in example 3 were stored in a 4 ℃ refrigerator. Samples were taken on days 1, 7, 15, and 30, respectively, and the prodrug nano-formulations were diluted 10-fold with deionized water, and their particle size, aggregation index (PDI), and surface potential were measured using a malvern particle sizer.
PBS containing 10% FBS was used to simulate the physiological environment in vivo. The results are shown in fig. 5, where CMNP and tNP remained stable without significant particle size change over 12 hours of incubation. However, after 12 hours, CMNP particle size and PDI increased rapidly, indicating the breakdown of its nanostructures. In contrast tNP remained stable over 48 hours. This result suggests that tNP may have better in vivo stability.
The results of the long-term storage are shown in fig. 6 and 7, and no significant change in particle size and surface potential of CMNP and tNP occurred over a 30 day storage period, and PDI remained below 0.2, indicating that the prodrug nanoformulation was able to maintain its physical properties during long-term storage and was suitable for subsequent use.
Example 5 in vitro drug Release of brain-targeted prodrug nanoformulations
PBS containing 20% ethanol was used as a release medium, and Dithiothreitol (DTT) was added thereto at various concentrations, and final concentrations of 1 and 10mM, respectively, were set as a control. CMNP and tNP prepared in example 3 were placed in dialysis bags, respectively, and after sealing, the dialysis bags were placed in the corresponding release medium and subjected to shaking incubation at 37 ℃. To evaluate the drug release process, we sampled at different time points (1, 2,4, 8 and 12 hours) after incubation, and samples at each time point were repeated three times. The collected samples were quantitatively analyzed by High Performance Liquid Chromatography (HPLC) and chlorambucil, mycophenolic acid and MHY1485 were detected at wavelengths of 258nm, 250nm and 340nm, respectively.
As shown in fig. 8 and 9, tNP and CMNP exhibited excellent reductive response release characteristics, and both exhibited similar trends in the release behavior of chlorambucil and mycophenolic acid. In the absence of DTT, the prodrug nanoformulation released less than 10% of the drug within 12 hours. Whereas at a DTT concentration of 1mM almost all the drug was released completely within 12 hours. When the DTT concentration was increased to 10mM, chlorambucil and MHY1485 were released completely from tNP or CMNP in 4 hours, and mycophenolic acid was released completely in 8 hours. It is particularly noted that MHY1485 is not itself prodrug modified and therefore does not possess the property of a reductive response. However, when co-assembled with chlorambucil-mycophenolic acid prodrugs bridged by disulfide bonds, the reductive response of chlorambucil-mycophenolic acid prodrugs results in the cleavage of tNP nanostructures, thereby conferring simultaneous reductive response capacity to MHY 1485. This feature enables tNP to achieve smart responsive release of the triple drug, thereby providing unique advantages for reducing toxic side effects and improving therapeutic effects.
EXAMPLE 6 cytotoxicity and Selective assessment of brain-targeting prodrug nanoformulations
The cytotoxicity of the prodrug nanoformulations was assessed using the MTT method by seeding human glioblastoma U87 cells, U87/TR cells (temozolomide resistant U87 cells) and mouse hippocampal neuronal HT22 cells into 96-well plates (2 x 10 3 cells per well) and incubating them in an incubator for 12 hours to ensure stable adherence of the cells. Then, a Mixed solution (Mixed sol, molar ratio: chlorambucil: mycophenolic acid: MHY 1485=3:3:1, consistent with the ratio of three drugs in tNP) of temozolomide solution, chlorambucil, mycophenolic acid and MHY1485 with different concentration gradients, CMNP or tNP medium was used to replace the original medium. Untreated cells served as control. After each group of cells was incubated in a 37 ℃ incubator for a further 48 hours, 20 μl of MTT solution was added to each well and incubation was continued for a further 4 hours to allow formazan crystals to form. Subsequently, the medium was discarded, formazan crystals were dissolved in DMSO, absorbance values were determined using a multifunctional microplate reader at 490nm wavelength, and the half maximal inhibitory concentration (IC 50) was calculated from the measured data using graphpad software. The Selection Index (SI) was calculated from the resulting IC 50 as follows, selection index = IC 50 prescribed for tumor cells/IC 50 prescribed for HT22 cells. The results are shown in Table 4.
TABLE 4 IC 50 and selection index for each prescription
The results indicate that in both glioblastoma cells (U87 and U87/TR), the IC 50 values were higher than those of Mixed sol, since the prodrug nanoformulation was active only after drug release. As a first-line therapeutic for glioblastoma, temozolomide is significantly less cytotoxic than other combination therapies. In particular, in drug-resistant U87/TR cells, temozolomide has an 8.6-fold increase in IC 50 compared with wild-type U87, while other combination preparations have an increased IC 50 in drug-resistant cells, but the increase is not more than 2 times. Of these, IC 50 of the two prodrug nanoformulations was only 1.2-fold elevated. Notably, tNP exhibited greater cytotoxicity in prodrug nanoformulations due to the synergistic effect of the triple drugs. These results show that the combined drug strategy designed by the invention can target glioblastoma cells and further can effectively overcome the drug resistance of glioblastoma to temozolomide.
In addition, mouse hippocampal neuronal HT22 cells were used in place of normal brain tissue to assess off-target toxicity of each formulation. Experimental results show that Mixed sol has the strongest toxicity in HT22 cells, and the prodrug nano-preparation with stronger anti-tumor activity in glioma cells has significantly reduced toxicity in HT22 cells. To further analyze the selectivity of the drug, a selection index is calculated that measures the relative toxicity of the drug to tumor cells versus normal cells, with a larger number indicating a stronger drug toxicity to tumor cells and a lower drug toxicity to normal cells. The result shows that the selection index of temozolomide is even lower than 1, which shows that temozolomide has higher toxic and side effects on normal brain tissues. In contrast, tNP has the highest selection index, probably due to its responsive release mechanism to the tumor microenvironment and the lower proportion of chlorambucil (highly toxic drug) at the same molar concentration. The characteristic not only endows tNP with stronger glioblastoma resistance activity, but also reduces the nonspecific toxicity of the glioblastoma resistance to brain tissues, is not influenced by the drug resistance of temozolomide, and shows good therapeutic advantages.
Example 7 anti-tumor mechanism of brain-targeting prodrug nanoformulations
Comet assay (single cell gel electrophoresis) U87/TR cells were seeded in 6-well plates at a density of 2X 10 5 cells per well and incubated in an incubator for 12 hours to allow adherence. The medium was discarded, replaced with medium containing temozolomide (1000. Mu.M), mixed sol, CMNP and tNP (total dose 10. Mu.M), and incubation was continued for 24 hours with blank cell control. Cells were collected and washed, mixed with low melting agarose and spread on pretreatment slides. After solidification, placing the slide in a lysate to incubate the cell membrane in a dark place at 4 ℃ to release DNA, transferring the slide into an alkaline hydrolysis solution to cause the DNA to unwind and expose damage sites, and then electrophoresis in an alkaline electrophoresis buffer solution to cause the damaged DNA to form comet-like tailing. After electrophoresis, the tail DNA content was analyzed by Image J software by washing with neutralization buffer, EB staining, recording images under a fluorescence microscope.
Intracellular GTP concentration measurement U87/TR and U87 cells were seeded in 6-well plates at 2X 10 5 cells per well and incubated in an incubator for 12 hours to promote adherence. The medium in the U87/TR wells was removed, replaced with medium containing temozolomide (1000. Mu.M), mixed sol, CMNP and tNP (total dose 10. Mu.M), and incubation was continued for 24 hours with blank U87/TR and U87 cells as controls. Cell samples were collected, lysed with cell lysates and centrifuged to obtain supernatant. According to the operation of the GTP concentration detection kit, adding a GTP standard substance with known concentration and cell lysis supernatant into an ELISA plate coated with a specific antibody, incubating again after washing the plate, adding an ELISA secondary antibody, washing the plate, adding a substrate solution for light-shielding color development, adding a stop solution when color development is proper, finally measuring the absorbance at 450nm by using an ELISA plate, and comparing with a standard curve to obtain the intracellular GTP concentration.
AP site quantitative detection, namely inoculating U87/TR cells into a 6-well plate according to 2X 10 5 cells per well, and incubating the cells in an incubator for 12 hours to enable the cells to adhere to the wall. The medium was discarded and replaced with medium containing temozolomide (1000. Mu.M), mixed sol, CMNP and tNP (total dose 10. Mu.M), and incubation was continued for 24 hours with blank cell control. After the incubation, cells were collected, and the AP site detection kit was used to equilibrate the materials and reagents to room temperature, purify genomic DNA and dilute to 100. Mu.g/mL, perform ARP reaction, and dilute ARP-derived DNA sample to 1. Mu.g/mL after the reaction product was treated. Adding a standard substance diluent and a sample into a DNA high-binding plate, sequentially adding a DNA binding solution, a streptavidin-enzyme conjugate and a substrate solution, incubating and washing after each addition, measuring absorbance at 450nm of a microplate reader after adding a termination solution, and comparing the absorbance with a standard curve to calculate the intracellular AP site content.
Comet assay (single cell gel electrophoresis) is a highly sensitive method of detecting DNA damage by which damaged DNA fragments migrate by electrophoresis to form a typical "comet-like" structure. In general, the tail length, tail moment and tail DNA content of comets can quantitatively reflect the degree of DNA damage, and higher values indicate more severe damage. As shown in fig. 10, 11 and 12, temozolomide-treated U87/TR cells showed only a very small amount of tailing, indicating weak DNA damage to drug-resistant cells. The tail of the comet of the Mixed sol group has the longest tail moment and the highest tail DNA content, which indicates that the DNA damage is most obvious and tNP times. This trend is consistent with the results of cytotoxicity experiments, further demonstrating that temozolomide alone is difficult to effectively induce DNA damage in temozolomide resistant U87/TR cells, while the combination, especially Mixed sol and tNP, still significantly destroyed DNA integrity of drug resistant glioblastoma cells.
Temozolomide resistance mechanisms of glioblastomas typically involve upregulation of guanylate metabolism to enhance DNA repair capacity and to address chemotherapy-induced DNA damage. As shown in FIG. 13, the intracellular GTP concentration of U87/TR cells was significantly increased compared to wild-type U87 cells, indicating that the guanylate metabolic pathway was more active in drug resistant cells. Temozolomide increases GTP levels even further under different drug treatments, whereas Mixed sol and tNP are based on an "open source throttled" guanylate regulation strategy, i.e. MHY1485 activates the mTOR pathway to promote guanylate consumption (open source) while IMPDH2 inhibits guanylate de novo synthesis (throttling), eventually leading to a significant decrease in intracellular GTP concentration, leading to guanylate depletion. In contrast, CMNP, while also reducing GTP levels, only a single mechanism of guanylate synthesis inhibition by IMPDH2 works far less well than the "open source throttling" strategy of Mixed sol and tNP.
The AP site (apurinic site ) refers to an abasic site formed by the deletion of a purine base due to hydrolysis, oxidation or chemical modification in a DNA strand. In tumor cells, when chlorambucil induces crosslinking of DNA chains, the cells need to cleave off damaged bases and activate a base cleavage repair pathway for DNA repair, and guanylate is an indispensable raw material in this repair process. When intracellular guanylate is depleted, DNA repair is blocked and unrepaired AP sites accumulate, exacerbating DNA damage. As shown in FIG. 14, the trend of the change in the number of AP sites was consistent with the trend of DNA damage and was inversely related to the intracellular GTP concentration. The result shows that the combined medication scheme based on the open source throttling strategy can obviously reduce the intracellular guanylate level, aggravate the DNA damage induced by chlorambucil and effectively inhibit the viability of temozolomide-resistant glioblastoma cells, thereby prompting the treatment advantage of overcoming the drug-resistant glioblastoma.
Example 8 biodistribution of brain-targeted prodrug nanoformulations
Preparation of DiR markers CMNP and tNP DiR markers CMNP and tNP were prepared in the same manner as in example 3, the only difference being that 10% DiR (mass ratio, w/w) was additionally added as fluorescent marker during the preparation to facilitate fluorescent tracer analysis.
Preparation of DiR-labeled non-brain targeting tNP (tNP/nT) tNP/nT the preparation method was identical to the tNP preparation method described in example 3, except that DSPE-PEG-APOE was replaced with equal mass DSPE-PEG during preparation, while 10% DiR (mass ratio, w/w) was added additionally as fluorescent marker to achieve fluorescent tracing. Finally, the prepared nanoparticle was designated tNP/nT.
And (3) constructing a temozolomide drug-resistant glioblastoma model, namely constructing a glioblastoma in-situ model by using an immunodeficiency Balb/c nude mouse. After isoflurane anesthesia, nude mice were fixed to a stereotactic instrument, and after skull drilling, digested U87/TR-luc cells (5X 10 5, PBS diluted) were slowly injected into the right caudate nucleus region. After injection is completed, the wound is sealed with tissue glue. After 10 days of tumor inoculation, the bioluminescence signal of the tumor was recorded and analyzed by intraperitoneal injection of potassium fluorescein (150 mg/kg) using a biopsy system. Subsequently, the successfully modeled nude mice were randomly grouped for subsequent experiments.
Biodistribution experiments DiR solution, diR-labeled CMNP, tNP, and tNP/nT were administered to the molded mice by tail vein injection at a dose of 2mg/kg calculated as DiR equivalents. At 4, 8, 12 and 24 hours after dosing, mice were anesthetized separately and DiR fluorescence images of tumor sites were acquired using a live imaging system while quantitative analysis was performed. At the 24 hour endpoint, mice were sacrificed, the major viscera (heart, liver, spleen, lung, and kidney) and brain tissues were isolated, and DiR fluorescence signals of each tissue were imaged and quantitatively analyzed using a biopsy imager to assess the tissue distribution of the drug.
The experimental results are shown in fig. 15 and 16, the DiR solution and the DiR marker tNP/nT without brain targeting modification detected only very low fluorescence signal in brain tissue, while both DiR markers CMNP and tNP showed significant fluorescence accumulation in brain. Over time, the fluorescence intensity of both brain-targeted prodrug nanoformulations gradually increased in the brain and peaked at 12 hours. This phenomenon suggests that CMNP and tNP both successfully cross the blood brain barrier and accumulate in brain tumor areas, verifying their good brain targeting delivery capability, emphasizing the importance of performing brain targeting modifications.
The fluorescence distribution of the main organs and brain tissues at 24 hours was as shown in fig. 17 and 18, and the results were consistent with the observation results of in vivo fluorescence imaging. DiR solutions and non-brain-targeted modified DiR markers tNP/nT have only very low fluorescence signals in the brain, but are distributed primarily in the lungs and liver. In contrast, both CMNP and tNP of the DiR markers successfully cross the blood brain barrier and are significantly enriched in brain tumor areas. As shown in fig. 19, the fluorescence intensity of the DiR-labeled tNP/nT was only 1.5-fold higher than that of the DiR solution, while the DiR-labeled CMNP and tNP were 6.1-fold and 6.5-fold higher, respectively, further demonstrating their effective accumulation in the brain. The accumulation effect of the two prodrug nano-formulations on brain tumors is not obviously different, and the two prodrug nano-formulations can be related to the same targeting modification and similar surface physicochemical properties, so that similar in-vivo distribution characteristics are caused. In conclusion, the brain targeting prodrug nano-preparation constructed by the research can realize accurate brain glioma targeting delivery, and lays an important foundation for further playing an anti-tumor role.
Example 9 in vivo antitumor investigation of brain-targeting prodrug nanoformulations
An immunodeficient Balb/c nude mouse was used to construct glioblastoma in situ model. After isoflurane anesthesia, nude mice were fixed to a stereotactic instrument, and after skull drilling, digested U87/TR-luc cells (5X 10 5, PBS diluted) were slowly injected into the right caudate nucleus region. After injection is completed, the wound is sealed with tissue glue. After 10 days of tumor inoculation, the bioluminescence signal of the tumor was recorded and analyzed by intraperitoneal injection of potassium fluorescein (150 mg/kg) using a biopsy system. Subsequently, the successfully modeled nude mice were randomly grouped for subsequent experiments.
In the in vivo anti-tumor experiment, the anti-tumor curative effect of different medicines is evaluated by adopting a glioblastoma nude mouse in-situ model. Temozolomide is administered by intraperitoneal injection at a dose of 60mg/kg (309. Mu.M/kg), and Mixed sol, CMNP, and tNP are all administered by intravenous injection, with total dose set at 21.87. Mu.M/kg, and specific doses are shown in Table 5, with PBS as a control. Dosing was started on day 0, once every 3 days for a total of 5 times. On days 0, 4, 8, 12, 16, respectively, the tumor growth was monitored by intraperitoneal injection of potassium fluorescein (150 mg/kg) and recording and analyzing the bioluminescence signal of the tumor using a biopsy imaging system. When the mice body weight decreased to 20% of the initial body weight, the experiment was terminated and euthanasia was performed. On day 16, mouse brain tissue was collected for H & E staining, the remaining mice continued to receive survival experimental observations, and body weight changes and survival were monitored every 2 days until the experiment was terminated.
TABLE 5 specific dosage for each prescription
A) Mu represents mu M/kg and M represents mg/kg
Tumor growth is shown in fig. 20, 21 and 22, and survival of mice is shown in fig. 23 and 24. In the PBS control group, the tumor grew rapidly, the mice body weight continued to drop, and the median survival was only 18 days. In a glioblastoma model with temozolomide resistance, the clinical first-line drug temozolomide hardly inhibits tumor growth, mice begin to die on day 14, and median survival time cannot be prolonged. In addition, although Mixed sol showed better anti-tumor activity in cell experiments, the anti-tumor effect of the Mixed sol is similar to that of temozolomide, mice also begin to die on the 14 th day, and the median survival time is even shortened to 16 days, which is lower than that of a PBS control group. This result suggests that the Mixed sol in free drug form cannot cross the blood brain barrier and is difficult to reach the brain tumor area to act, thus underscores the key role of drug delivery strategies in brain glioma treatment.
In contrast CMNP and tNP exhibit more excellent anti-tumor effects in drug-resistant glioblastoma models due to their brain targeting ability. Wherein tNP shows stronger inhibition, not only obviously slows down the tumor growth, but also effectively delays the weight reduction of mice, and prolongs the median survival time to 30 days. Since CMNP was demonstrated to be comparable to the brain-targeted delivery capacity of tNP in example 8, the stronger antitumor activity of tNP was attributed to the synergistic effect of the triple drug based on the "open source throttling" strategy. As shown in fig. 25, the H & E staining results of the mouse brain tissue further demonstrated that tNP significantly inhibited the growth of drug-resistant glioblastoma, CMNP times, whereas the tumor sizes of temozolomide and Mixed sol treated groups were similar to PBS control groups, consistent with the drug effect results.
In conclusion, the brain-targeting prodrug nano-preparation tNP constructed by the invention exacerbates guanylate depletion in tumor cells by an open source throttling strategy, thereby enhancing the sensitivity of cells to the alkylating agent chlorambucil. Meanwhile, the brain targeting modification enables tNP to efficiently cross the blood brain barrier, achieves targeting delivery to brain tumor areas, and finally remarkably inhibits the growth of temozolomide-resistant glioblastoma. The research provides an innovative treatment strategy for overcoming the drug resistance of glioblastoma, and provides a new theoretical basis and potential application value for the accurate treatment of clinical temozolomide drug-resistant patients.
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