CN114246954A - Brain glioma whole-region targeted nano drug delivery system and preparation method and application thereof - Google Patents

Brain glioma whole-region targeted nano drug delivery system and preparation method and application thereof Download PDF

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CN114246954A
CN114246954A CN202011012212.3A CN202011012212A CN114246954A CN 114246954 A CN114246954 A CN 114246954A CN 202011012212 A CN202011012212 A CN 202011012212A CN 114246954 A CN114246954 A CN 114246954A
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dspe
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delivery system
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罗子淼
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Fudan University
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Abstract

The invention belongs to the field of pharmaceutical preparations, and relates to a brain glioma whole-region targeted nano drug delivery system, and a preparation method and application thereof. The drug delivery system comprises a double-layer targeting molecule, a drug and a foaming agent, wherein the targeting molecule is R9 polypeptide and hyaluronic acid HA, the foaming agent is 1-bromoheptafluorooctane, and the drug and the foaming agent are jointly encapsulated in a nano carrier; the targeting molecule R9 polypeptide is covalently connected on the surface of the nanoparticle; the targeting molecule HA is wrapped on the outer layer of the R9 polypeptide through electrostatic interaction. According to the invention, the HA of the outer-layer targeting molecule targets and accumulates in the marginal area of the brain glioma with high expression of hyaluronidase Hyal-2 and CD44, when the HA is hydrolyzed to expose R9 polypeptide, the drug is further delivered to the core area and the infiltration area of the glioma in a two-way manner by utilizing the high affinity of the R9 polypeptide with positive charges to the tumor cell membrane, the drug is irradiated by HIFU after accumulation, the drug is induced to be released instantaneously and sufficiently, and the drug concentration of the core area, the marginal area and the infiltration area of the brain glioma can be greatly improved.

Description

Brain glioma whole-region targeted nano drug delivery system and preparation method and application thereof
Technical Field
The invention belongs to the field of pharmaceutical preparations, and relates to a brain targeted drug delivery system, in particular to a nano drug delivery system which is modified layer by R9 polypeptide and Hyaluronic Acid (HA), controlled by High Intensity Focused Ultrasound (HIFU) and targeted to the whole area of brain glioma and a preparation method and application thereof.
Background
The prior art discloses that brain glioma is the most common primary malignant tumor with the highest mortality in the cranium, and the treatment of the brain glioma becomes one of the most troublesome tumors in clinical treatment of various tumors due to the particularity of the disease position and the unclear characteristics of the tumor boundary. The traditional surgical operation is difficult to completely remove tumor cells in an infiltration area, and relapse is easy to cause. The treatment effect of chemotherapy as adjuvant therapy or conservative therapy is limited by the delivery of Blood Brain Barrier (BBB), and the extremely low amount of drugs entering the brain makes the chemotherapy effect not ideal. Although the Enhanced Penetration and Retention (EPR) effect of tumors opens a window for passive targeted drug delivery, the EPR effect of brain tumors is relatively weak compared to peripheral tumors, for example, U87MG brain glioma, whose average pore size is only 7-100 nm, and therefore, the size of drugs for brain glioma treatment is very limited. Meanwhile, in the early stage, the middle stage or the late stage of development of the glioma, the EPR effect only exists in the tumor, namely the core region, and the BBB of the glioma margin and the infiltration region is always relatively complete, so that the treatment of the glioma margin region and the infiltration region also becomes a difficulty of chemotherapy, the relapse or metastasis of the tumor is often caused by incomplete treatment, and related reports aiming at the glioma margin region and the infiltration region drug delivery strategy are not seen at present.
Compared with the treatment of peripheral tumors, the active targeting drug delivery mode is more important for the treatment of brain tumors. On one hand, due to the particularity of the brain tumor position, the toxic and side effects of passive targeted drug delivery on normal brain tissues can seriously affect the life quality of patients; on the other hand, due to the obstruction of blood brain barrier, the amount of the drug entering the brain is extremely low, and finally, the drug amount passively targeted to the glioma part can not achieve the effect of effectively and thoroughly inhibiting the tumor. Hyaluronic Acid (HA) HAs been widely used in recent years for targeted modification of drug delivery systems due to its specific binding characteristics to tumor cells highly expressing CD44, and its advantages in terms of biocompatibility, biodegradability and low immunogenicity. Meanwhile, hyaluronidase Hyal-2 highly expressed in the tumor marginal zone can also be specifically bound with HA and hydrolyze HA. Thus, targeted delivery to the tumor margin can be achieved by modifying the surface of the delivery system with HA. The R9 polypeptide, a 9 cation-bearing polypeptide, is commonly used for modification of tissue penetrating drug delivery systems due to its very high affinity to negatively charged cell membranes. Therefore, the R9 polypeptide is modified in the inner layer of the drug delivery system, and HA is wrapped on the outer layer of R9 through electrostatic interaction, so that the drug can be actively targeted to the marginal area of the glioma, and is delivered to the core area and the infiltration area of the glioma in a two-way manner after the HA is hydrolyzed to expose R9, and the purpose of targeted drug delivery of the whole area of the glioma is achieved.
Doxorubicin (Dox) is one of the most effective broad-spectrum antineoplastic drugs for the treatment of circulating tumors and solid tumors, including brain glioma. Although the current clinically used liposome adriamycin greatly reduces the cardiac toxicity, the lack of targeting still remains a key problem limiting the clinical application of the liposome adriamycin.
The ultrasonic controlled release system is a drug controlled release system with exogenous stimulus response. The mechanism of the ultrasonic controlled release is that the control of the drug release is realized in a minimally invasive and remote way by the characteristic of high-frequency focusing through regulating and controlling the space and the temperature. High Intensity Focused Ultrasound (HIFU), which has been developed in recent years, is favored for tumor tissue ablation due to its ultra-high focusing power in the millimeter level and its characteristics of high attenuation and low damage outside the focus, and is also applied to controlled release of nano drug delivery systems.
Based on the current situation of the prior art, the inventor of the application intends to provide a brain glioma whole-region targeted nano drug delivery system and a preparation method and application thereof; further improving the chemotherapy effect of the whole area of the brain glioma.
Disclosure of Invention
The invention aims to provide a brain glioma whole-region targeted nano drug delivery system and a preparation method and application thereof based on the current situation of the prior art.
The invention provides a brain glioma whole-region targeted nano drug delivery system, which modifies R9 polypeptide and Hyaluronic Acid (HA) layer by layer on the surface of nanoparticles, delivers a drug to the marginal region of a brain glioma in a targeted manner through HA, delivers the drug to the core region and the infiltration region of the brain glioma in a two-way manner by utilizing the characteristic of high tissue penetrability of R9 polypeptide after the HA is hydrolyzed by hyaluronidase to expose R9 polypeptide, and induces the drug adriamycin (Dox) to be released sufficiently and instantaneously by HIFU irradiation, so that the comprehensive and effective inhibition effect on the core region, the marginal region and the infiltration region of the brain glioma is achieved, and the treatment effect of the brain glioma is improved remarkably.
The invention also provides a preparation method and application for constructing the brain glioma whole-region targeted nano drug delivery system.
More specifically, the invention provides a brain glioma whole-region targeted nano drug delivery system, which comprises double-layer targeted molecules, a drug, a foaming agent and a nano carrier, wherein the targeted molecules are respectively R9 polypeptide and Hyaluronic Acid (HA), the drug is adriamycin (Dox), the foaming agent is 1-bromoheptafluorooctane (PFOB), and the nano carrier is a mixture of polylactic acid-glycolic acid copolymer PLGA-COOH (molecular weight of 50000), distearoylphosphatidylethanolamine-polyethylene glycol DSPE-PEG (molecular weight of 2000) and distearoylphosphatidylethanolamine-polyethylene glycol-maleimide DSPE-PEG-MAL (molecular weight of 3400) modified by terminal carboxyl; the medicine and the foaming agent are jointly encapsulated in the nano-carrier; the targeting molecule R9 polypeptide is connected with polyethylene glycol on the surface of the nano carrier through a covalent bond; the targeting molecule hyaluronic acid is wrapped on the outer layer of the R9 polypeptide through electrostatic interaction; the foaming agent is instantly vaporized under HIFU irradiation, and the generated radial force enables the nano-carrier to be rapidly disintegrated, so that the drug is released at an accelerated speed; the sequence of the targeting molecule R9 polypeptide is 5 '-Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-3'; the mass ratio of PLGA-COOH to the sum of DSPE-PEG and DSPE-PEG-MAL in the nano carrier is 4: 1; the molar ratio of DSPE-PEG to DSPE-PEG-MAL in the nano-carrier is 5: 1; the molar ratio of the targeting molecule R9 polypeptide to the DSPE-PEG-MAL is 1: 10; the coating concentration of the targeting molecule HA is 0.4mg/mL PBS solution; the particle size of the drug delivery system HA-R9-NP-D/P is 40-50 nm, and the dispersion degree of the particle size is 0.1-0.2.
The invention provides a preparation method of the brain glioma whole-region targeted nano drug delivery system, which specifically comprises the following steps:
(1) taking a proper amount of PLGA-COOH, DSPE-PEG-MAL, a Dox methanol solution (6.66mg/mL) and PFOB (19.23mg/mL) and adding acetone to fully mix, wherein the mass ratio of PLGA-COOH to the sum of DSPE-PEG and DSPE-PEG-MAL is 4: 1; the molar ratio of the DSPE-PEG to the DSPE-PEG-MAL is 5: 1; the mass ratio of Dox to the sum of PLGA-COOH, DSPE-PEG and DSPE-PEG-MAL is 1: 50; the mass ratio of the PFOB to the sum of PLGA-COOH, DSPE-PEG and DSPE-PEG-MAL is 1: 100; then quickly injecting the acetone solution into Mili Q water, wherein the volume ratio of acetone to Mili Q water is 1:2, and removing the acetone by a vacuum pump at room temperature to obtain a Mili Q water-dispersed nano particle NP-D/P solution;
(2) dropwise adding a proper amount of R9 polypeptide aqueous solution into the nano NP-D/P solution obtained in the step 1, wherein the molar ratio of DSPE-PEG-MAL to R9 is 10:1, and magnetically stirring at 350rpm at room temperature for 2 hours to obtain an R9-NP-D/P aqueous solution;
(3) preparing an HA aqueous solution (0.4mg/mL), dropwise adding the R9-NP-D/P aqueous solution obtained in the step 2 into the HA aqueous solution, wherein the volume ratio of the R9-NP-D/P aqueous solution to the HA aqueous solution is 1:1, and magnetically stirring at 350rpm for 1h at room temperature to obtain the drug delivery system HA-R9-NP-D/P aqueous solution of claim 1.
The invention provides an application of the brain glioma whole-region targeted nano drug delivery system, the nano drug delivery system is used as a chemotherapeutic drug for treating the brain glioma, and the application specifically comprises the following steps:
(1): injecting the delivery system intravenously;
(2): and 24h after administration, applying HIFU irradiation to the glioma part to induce the drug to be fully released at the glioma part.
The application of the brain glioma whole-region targeted nano drug delivery system provided by the invention can be used as a treatment scheme of the brain glioma, and can achieve ideal tumor inhibition effect on the targeted drug delivery of the core region, the marginal region and the whole infiltration region of the brain glioma.
The brain glioma whole-region targeted nano drug delivery system plays a role in the following aspects:
(1) the drug delivery system has the function of targeting the whole area of the brain glioma. Firstly, through the specific binding capacity of HA with Hyal-2 and CD44, the drug delivery system firstly targets and delivers the drug to the marginal zone of brain glioma with high expression of Hyal-2; secondly, after HA is hydrolyzed to expose R9 polypeptide, the drug delivery system bidirectionally delivers the high penetrability of the R9 polypeptide with positive charge to the core area and the infiltration area of the brain glioma, thereby achieving the effect of targeting the core area, the marginal area and the whole area of the infiltration area of the brain glioma.
(2) The drug delivery system provided by the invention induces the instantaneous and sufficient release of the drug through HIFU irradiation on the basis of targeted drug delivery to the whole area of the brain glioma, thereby further improving the chemotherapy effect to the whole area of the brain glioma.
(3) In the drug delivery system, the heat effect and the cavity effect generated by HIFU irradiation also play a certain role in ablating and inducing apoptosis on brain glioma cells.
(4) In the drug delivery system, the HIFU irradiation area is conservative, only the core area and the marginal area of glioma are irradiated, and the glioma cells in the infiltration area are treated mainly through the effect of drug chemotherapy, so that the drug delivery system has no mechanical damage to normal brain tissues.
Compared with the prior art, the invention has the following beneficial effects:
(1) due to the limitation of the blood brain barrier on the size of the drug delivery system, compared with a large-particle-size nano drug delivery system, the drug delivery system disclosed by the invention has the advantages of small particle size and good uniformity and is very suitable for drug delivery of brain tumors.
(2) Because the blood brain barrier of the infiltration area in each stage of brain glioma development is complete, the liposome adriamycin without target specificity in clinic and other existing drug delivery systems for target therapy of brain glioma cannot achieve better treatment effect on the infiltration area of glioma. The drug delivery system HAs the function of targeting the whole area of the brain glioma, and the drug is delivered to the marginal area, the core area and the infiltration area of the brain glioma through the layer-by-layer modification of R9 polypeptide and HA and two-step targeted delivery and accumulation, so that the purpose of targeted therapy of the whole area of the brain glioma is finally achieved.
(3) The drug delivery system of the invention has the obvious advantage of instantaneous full drug release. The drug delivery system can induce the drug adriamycin to be instantaneously and fully released at the brain glioma part by giving HIFU irradiation on the basis of fully accumulating the whole area of the brain glioma, thereby further improving the chemotherapy effect of the whole area of the brain glioma.
Drawings
FIG. 1 is a schematic view of the preparation process and release pattern of the drug delivery system of the present invention;
FIG. 2 is a graph comparing particle sizes of the delivery systems of the present invention;
FIG. 3 is a graph of the potential contrast of the delivery system of the present invention;
FIG. 4 is a stability assay for the delivery system of the present invention;
FIG. 5 is a graph of the H' -NMR identification of HA on the surface of the delivery system of the present invention;
FIG. 6 is a comparison of the surface morphology of the delivery system of the present invention with and without HIFU irradiation;
FIG. 7 is a graph showing the effect of the presence or absence of HIFU irradiation on drug release from a delivery system of the present invention;
FIG. 8 is a graph comparing the cumulative amount of drug released from different delivery systems with and without HIFU irradiation;
FIG. 9 is a confocal microscope fluorescence qualitative comparison graph showing the uptake of U87MG cells into the drug delivery system of the present invention;
FIG. 10 is a graph showing uptake of the drug delivery system of the present invention by U87MG cells (flow cytometric fluorescence vs. quantitation);
FIG. 11 is a pharmacokinetic profile of a delivery system of the present invention;
FIG. 12 is a comparison of in vivo fluorescence imaging of tumor-bearing mice for in vivo targeting verification of the drug delivery system of the present invention;
FIG. 13 is a comparison of in vivo targeting verification of the drug delivery system of the present invention- -in vitro fluorescence imaging of the brain of tumor-bearing mice;
FIG. 14 is a graph of in vivo targeting verification of the delivery system of the present invention- -distribution of the delivery system in brain tissue at various time points;
FIG. 15 is a photograph of fluorescent staining of hyaluronidase Hyal-2 in mouse brain glioma sections;
FIG. 16 is a TUNEL staining contrast of brain glioma sections from tumor-bearing mice of each treatment group;
FIG. 17 is a graph comparing survival curves of brain-loaded glioma mice in each treatment group;
FIG. 18 is a H & E section of the major organs of a mouse after a single administration, which is an in vivo safety test of the drug delivery system of the present invention.
Detailed Description
For a better understanding of the present invention, reference will now be made to the following examples, which are to be considered in all respects as illustrative and not restrictive.
Example 1:
preparation of nano drug delivery system HA-R9-NP-D/P:
(1) preparation of nanoparticle NP-D/P: 1mL of acetone and a mixed solution of PLGA-COOH (molecular weight 50000,16mg), distearoylphosphatidylethanolamine-polyethylene glycol DSPE-PEG (molecular weight 2000,3.35mg, 1.675. mu. mol), distearoylphosphatidylethanolamine-polyethylene glycol-maleimide DSPE-PEG-MAL (molecular weight 3400,0.65mg, 0.191. mu. mol), 60. mu.L of doxorubicin Dox methanol solution (6.66mg/mL) and 10. mu.L of perfluoroPFOA B (19.23mg/mL) were sufficiently dissolved. And quickly injecting the solution into 2mL of Mili Q water, and removing acetone by using a vacuum pump at room temperature to obtain the Mili Q water-dispersed nano particle NP-D/P solution.
(2) Preparation of R9 polypeptide modified nanoparticle R9-NP-D/P: and (2) dropwise adding 30 mu L of R9 polypeptide aqueous solution (0.655 mu mol/mL) into the nano-particle NP-D/P solution obtained in the step (1), and magnetically stirring at 350rpm at room temperature for 2 hours to obtain the R9-NP-D/P aqueous solution.
(3) Preparation of HA-modified nanoparticle HA-R9-NP-D/P: preparing an HA aqueous solution (0.4mg/mL), dropwise adding 100 mu L of the R9-NP-D/P aqueous solution obtained in the step 2 into 100 mu L of the HA aqueous solution, and magnetically stirring at 350rpm at room temperature for 1h to obtain the HA-R9-NP-D/P aqueous solution.
Preparation of fluorescein DiR or DiD labeled nanoparticle HA-R9-NP-D/P:
and (2) additionally adding 2 mu L of fluorescein DiR solution (1mg/mL) or 2 mu L of fluorescein DiD solution (1mg/mL) into the acetone solution in the step (1), and obtaining the DiR or DiD labeled HA-R9-NP-D/P aqueous solution without changing other steps.
FIG. 1 is a schematic diagram of the preparation process and drug release mode of HA-R9-NP-D/P.
Example 2:
the particle size and the potential of the nano-particle HA-R9-NP-D/P are measured:
1mg/mL of nano-particle NP-D/P, R9-NP-D/P and HA-R9-NP-D/P aqueous solutions are respectively prepared, and after the particle size, the particle size distribution and the potential of the nano-particle NP-D/P, R9-NP-D/P and HA-R9-NP-D/P are measured by a Malvern laser particle sizer, as shown in figure 2, the particle sizes of the NP-D/P, R9-NP-D/P and HA-R9-NP-D/P are respectively 34.05nm, 35.13nm and 45.73 nm; the particle size distributions PDI were found to be 0.149, 0.177 and 0.174, respectively. Therefore, the nanoparticle HA-R9-NP-D/P obtained by the preparation method HAs small particle size and good uniformity, can pass through a blood brain barrier more easily than a large-particle size drug delivery system, and is suitable for drug delivery of brain tumors. As shown in FIG. 3, the potentials of NP-D/P, R9-NP-D/P, and HA-R9-NP-D/P were found to be-11.4 mV, -9.4mV, and-34.63 mV, respectively. Therefore, the potential of the NP-D/P is increased after the NP-D/P is connected with the R9 polypeptide with positive charges, and the potential of the R9-NP-D/P is obviously reduced after the NP-D/P is wrapped with HA with negative charges, so that the success of layer-by-layer modification of the R9 and the HA on the surface of the nano-particle NP-D/P is proved.
Example 3:
determination of stability of the nanoparticle HA-R9-NP-D/P:
1mg/mL of nano-particle HA-R9-NP-D/P aqueous solution is prepared, and the change of the particle size and the potential of the nano-particle is monitored by a Malvern laser particle sizer within one week. As shown in FIG. 4, the HA-R9-NP-D/P showed little change in particle size and potential within one week, thus demonstrating that HA-R9-NP-D/P HAs good stability.
Example 4:
identification of the surface HA of the nanoparticle HA-R9-NP-D/P:
5mg of the freeze-dried nanoparticle HA-R9-NP-D/P was dissolved in 1mL of deuterated water and subjected to Varian 400MHz NMR detection. As shown in FIG. 5, the upper and lower panels show the nuclear magnetic results of NP-D/P and HA-R9-NP-D/P, respectively, and comparing the upper and lower panels can find that HA-R9-NP-D/P HAs more HA characteristic peaks than the nuclear magnetic results of NP-D/P, thereby further proving the success of HA modification.
Example 5:
surface morphology identification of the nanoparticles HA-R9-NP-D/P under the condition of HIFU irradiation or not:
0.1mg/mL of aqueous solutions of the nanoparticles NP-D/P, R9-NP-D/P and HA-R9-NP-D/P were prepared. The HA-R9-NP-D/P aqueous solution was divided into 2 portions on average, one of which received HIFU irradiation at 5 steps (10.5W) for 1 min. All samples were stained with 2% phosphotungstic acid and the surface morphology of each group of nanoparticles was observed by transmission electron microscopy. As shown in FIG. 6, the surface of NP-D/P and R9-NP-D/P is round, and the outermost layer of HA-R9-NP-D/P HAs haze halo, namely HA modified by the outermost layer of nanoparticles, which further proves the success of HA modification. When HA-R9-NP-D/P receives HIFU irradiation, the HA-R9-NP-D/P HIFU + group nanoparticles are obviously expanded and disintegrated into two small nanoparticles, so that the structural collapse of the nanoparticle HA-R9-NP-D/P can be intuitively proved by the HIFU irradiation.
Example 6:
evaluation of the influence of HIFU irradiation on the release of the nanoparticle HA-R9-NP-D/P:
a5 mg/mL aqueous solution of nanoparticles HA-R9-NP-D/P was prepared and divided into 2 portions on average, wherein one sample received HIFU irradiation at 60min at 5 steps (10.5W) for 10 min. And respectively measuring the content of the adriamycin Dox in the sample solution by using an ultraviolet spectrophotometer for 0min, 60min and 1min, 5min, 10min and 60min after HIFU irradiation, wherein the ultraviolet absorption peak of the Dox is 485 nm. In FIG. 7, Control represents the group not receiving HIFU irradiation, and Dox In-put represents the amount of Dox loaded In the nanoparticles of the experimental group; it can be seen from fig. 7 that the longer the nanoparticle is subjected to HIFU irradiation, the greater the amount of Dox released, but the increase in release amount is slowed down 10min to 1h after irradiation. Comparing the cumulative release amount in fig. 8, the release of Dox in the nanoparticle HA-R9-NP-D/P is greatly accelerated by HIFU irradiation instantaneously, the cumulative release amounts at 1min and 10min are respectively as high as 53.68% and 70.37%, while the cumulative release amount of the drug in the group not receiving HIFU irradiation for 70min is only 2.1%.
Example 7:
the in vitro targeting examination of the nanoparticle HA-R9-NP-D/P-quantitative cell uptake result by a flow cytometry method:
glioma U87MG cell 2X 104The cell/mL density is inoculated on a 24-hole cell culture plate, after the cell/mL density is routinely cultured for 24 hours, 600 mu mL of NP-D/P, R9-NP-D/P, HA-R9-NP-D/P or HA-R9-NP-D/P + HAase is respectively added into each hole, wherein the Dox concentration is 50 mu g/mL, and the HAase is hyaluronidase with the concentration of 1 mg/mL. And continuously culturing for 0.5h, washing with PBS, digesting with trypsin, centrifuging for 5min at 140g, then resuspending the cells with PBS, and quantitatively detecting the uptake condition of the cells to the nanoparticles by a flow cytometer. As shown in FIG. 9, the fluorescence intensity of each group was R9-NP-D/P, HA-R9-NP-D/P + HAase, HA-R9-NP-D/P, and NP-D/P in this order from high to low, wherein the fluorescence intensity of HA-R9-NP-D/P group was 1.86 times that of NP-D/P group. This is because the R9 polypeptide has a positive charge, which is beneficial for the modified nanoparticle to bind to the negatively charged cell membrane and enter the cell; when the outer layer of R9-NP-D/P is wrapped by HA, the intake of cells is reduced, and after the HA at the outermost layer is hydrolyzed by HAase to expose R9 polypeptide, the potential of the nanoparticle is increased, and the uptake of the nanoparticle to the HA is increased, so that the success of layer-by-layer modification of the nanoparticle R9 and the HA and the U87MG cell targeting capability of the HA-R9-NP-D/P which is obviously improved compared with the NP-D/P can be further proved. In fig. 9, represents paired T test P < 0.01, and represents paired T test P < 0.001, indicating significant differences between the two groups.
Example 8:
the in vitro targeting examination of the nanoparticles HA-R9-NP-D/P-confocal microscope qualitative cell uptake result:
glioma U87MG cell 1 × 105The cell/mL density is inoculated on a confocal culture dish, 600 mu mL of NP-D/P, R9-NP-D/P, HA-R9-NP-D/P or HA-R9-NP-D/P + HAase is added into each hole after normal culture is carried out for 24 hours, wherein the concentration of Dox is 50 mu g/mL, and the concentration of HAase is 1mg/mL of hyaluronidase. Continuously culturing for 0.5h, removing the culture medium containing the medicine, washing with PBS for 2 times, fixing with 4% paraformaldehyde for 10min, dyeing with DAPI for 5min, and determining the intake condition of the cells to different medicines with laser confocal microscope. As shown in FIG. 10, the fluorescence intensities were alignedThe sequence is as follows: R9-NP-D/P > HA-R9-NP-D/P + HAase > HA-R9-NP-D/P > NP-D/P, which is consistent with the trend of the identification result of the flow cell method for cell uptake in example 7, and shows that the layer-by-layer modification success of the nanoparticles R9 and HA, and the uptake of the modified HA-R9-NP-D/P by the U87MG cells is obviously improved compared with that of the modified NP-D/P, namely, the HA-R9-NP-D/PANP HAs obviously enhanced in vitro targeting property on brain glioma U87MG cells. The scale in FIG. 10 is 100 μm.
Example 9:
in vivo pharmacokinetic evaluation of the nanoparticles HA-R9-NP-D/P:
normal BALB/c nude mice were 12, randomly divided into 3 groups, and 200. mu.L of DiD-labeled NP-D/P, R9-NP-D/P or HA-R9-NP-D/P, respectively, were injected into the tail vein. Blood samples of 40 μ L were collected by orbital bleeding at 0.033h, 0.25h, 0.5h, 1h, 2h, 4h, 8h, 12h, 24h and 48h, respectively, and the DiD in the blood samples was quantified by a microplate reader after 5-fold dilution with PBS. As shown in FIG. 11, the pharmacokinetic curve area AUC of HA-R9-NP-D/P was significantly greater than that of NP-D/P and R9-NP-D/P, indicating that HA-R9-NP-D/P had longer circulation time in vivo than the other two groups; the area under the curve of R9-NP-D/P is slightly smaller than that of NP-D/P, which is probably because the R9 polypeptide with the positively charged surface of R9-NP-D/P causes poor in vivo stability of the nanoparticles.
Example 10:
in vivo targeting investigation of the nanoparticle HA-R9-NP-D/P-mouse in vivo imaging experiment:
12 BALB/c nude mice were inoculated with U87MG cells in situ for 20 days, and randomized into 3 groups, and 200. mu.L of DIR-labeled NP-D/P, R9-NP-D/P or HA-R9-NP-D/P was administered into the tail vein, respectively. DiR fluorescence signals in the mice are detected by a small animal living body imager at 6h, 12h, 24h and 48h respectively. And after 48h, performing PBS heart perfusion on the mouse, taking out brain tissue and collecting the in vitro fluorescence imaging result of the brain. As can be seen from the fluorescence signals in the mice of each group at 4 time points in FIG. 12, the metabolism speed of the drug preparations of each group in vivo is R9-NP-D/P, NP-D/P and HA-R9-NP-D/P in sequence from fast to slow, which is consistent with the in vivo pharmacokinetic results of the drug preparations of each group in example 9. Meanwhile, as can be seen from the attached figure 12, the advantage of the fluorescence intensity of the brains of the HA-R9-NP-D/P group starts to appear from 24h and continues to 48h, and the in vitro fluorescence signals of the mouse brains in the figure 13 are combined, so that the HA-R9-NP-D/P group HAs the in vivo glioma targeting effect which is remarkably improved compared with the other two groups.
Example 11:
in vivo targeting investigation of the nanoparticle HA-R9-NP-D/P-mouse tissue distribution experiment:
after 24 BALB/c nude mice were inoculated with U87MG cells in situ for 20 days, they were randomly divided into 3 groups and injected into 200. mu.L of DID-labeled NP-D/P, R9-NP-D/P or HA-R9-NP-D/P, respectively, in the tail vein. Mice were sacrificed at 24h, 48h, PBS heart perfused, brain tissue removed and homogenized, and DiD in the samples quantified by a microplate reader. As shown in FIG. 14, the accumulation of HA-R9-NP-D/P in brain tissue is significantly higher than that of NP-D/P, while R9-NP-D/P is likely to be very small because the surface R9 is positively charged and is more easily cleared by the endothelial reticulum system, and the circulation time in vivo is shorter than that of NP-D/P, and this result is consistent with the results of in vivo and ex vivo imaging of mice in example 10, further demonstrating that HA-R9-NP-D/P HAs significantly improved glioma targeting and retention capacity compared with NP-D/P.
Example 12:
mouse brain glioma frozen section hyaluronidase Hyal-2 immunofluorescence staining analysis:
after the BALB/c nude mice are inoculated with U87MG cells in situ for 20 days, the nude mice are killed, PBS heart perfusion is carried out, the brain tissues of the mice are taken to be frozen sections, and hyaluronidase Hyal-2 immunofluorescence staining is carried out. As shown in fig. 15, the green fluorescence in the mouse brain glioma section is Hyal-2 positive signals, and it can be seen from the figure that the Hyal-2 positive signals are mainly distributed in the marginal area of the brain glioma, so that the binding area of hyaluronic acid, i.e. the area where the HA-R9-NP-D/P nanoparticles are distributed and accumulated first in the brain glioma tissue, can be illustrated.
Example 13:
in vivo pharmacodynamic evaluation of nanoparticles HA-R9-NP-D/P-TUNEL staining analysis of mouse brain glioma cryosection:
18 BALB/c nude mice were randomly divided into 6 groups (Saline, Dox, NP-D/P, R9-NP-D/P, HA-R9-NP-D/P and HA-R9-NP-D/P H +), and 3 mice per group were injected with 100. mu.L of Saline, Dox, NP-D/P, R9-NP-D/P, HA-R9-NP-D/P or HA-R9-NP-D/P (dose of Dox administered was 2MG Dox per kg of animal body weight) at 3, 6, 9, 12 days after in situ inoculation of U87MG cells, respectively. On the 13 th day, HIFU irradiation treatment is additionally carried out on HA-R9-NP-D/P H + group tumor-bearing mice under the irradiation condition of 5 grades (10.5W) for 60s (ultrasound 1s and interval 3 s).
On day 15 after in situ inoculation of U87MG cells, mice were sacrificed, heart perfused with PBS, brain tissue was removed, fixed in 4% paraformaldehyde for 48h, and cryo-sectioned TUNEL staining of brain tissue was performed. As shown in fig. 16, green fluorescence is a TUNEL positive signal, i.e. apoptotic cells in brain gliomas. As can be seen visually in fig. 16, the Dox group was essentially free of apoptotic cells due to the lack of targeting function of the clinically used liposomal doxorubicin; the number of apoptotic cells in the R9-NP-D/P group is much less than that in the NP-D/P group, because the in vivo circulation time of R9-NP-D/P is short and the target function is not available; the proportion of apoptotic cells in HA-R9-NP-D/P group tumor tissues is close to half, and tumor cells at the tumor margin and the infiltration area show positive signals; furthermore, after the HIFU irradiation treatment is superimposed, the brain glioma cells are basically completely ablated or induced to die, only a few die-cast tumor cell fragments are left in the original tumor region, and meanwhile, the edge region and the infiltration region of the tumor also have better treatment effect. Therefore, the HA-R9-NP-D/P H + treatment scheme of superposed HIFU irradiation can greatly improve the comprehensive treatment effect on the core area, marginal area and infiltration area of the brain glioma.
Example 14:
in vivo pharmacodynamic evaluation of nanoparticle HA-R9-NP-D/P-monitoring of mean survival of mice:
36 BALB/c nude mice were randomly divided into 6 groups (Saline, Dox, NP-D/P, R9-NP-D/P, HA-R9-NP-D/P and HA-R9-NP-D/P H +), and 6 mice in each group were injected with 100. mu.L of Saline, Dox, NP-D/P, R9-NP-D/P, HA-R9-NP-D/P or HA-R9-NP-D/P (dose of Dox administered was 2MG Dox per kg of animal body weight) at 3, 6, 9, 12 days after in situ inoculation of U87MG cells, respectively. On the 13 th day, HIFU irradiation treatment is additionally carried out on HA-R9-NP-D/P H + group tumor-bearing mice under the irradiation condition of 5 grades (10.5W) for 60s (ultrasound 1s and interval 3 s). Animal mortality was monitored daily and Kaplan-Meier survival curves were plotted. As can be seen from the analysis of the Kaplan-Meier survival curves in FIG. 17, the average survival periods of Saline, Dox, NP-D/P, R9-NP-D/P, HA-R9-NP-D/P and HA-R9-NP-D/P H + groups were 11, 16.5, 26.5, 24, 38 and 57 days, respectively; as can be seen from FIG. 17, compared with the NP-D/P, R9-NP-D/P treatment group, the HA-R9-NP-D/P treatment group significantly prolongs the life cycle of a tumor-bearing mouse, which indicates that the layer-by-layer modification of R9 and HA can significantly improve the targeting of the drug to the brain glioma and increase the accumulation in each region of the brain glioma, thereby improving the treatment effect on the brain glioma; due to the addition of HIFU irradiation treatment, HA-R9-NP-D/P H + group greatly accelerates the release of drug Dox by HIFU on the basis that HA-R9-NP-D/P is fully accumulated in each area of brain glioma, and further remarkably prolongs the survival period of tumor-bearing mice.
Example 15:
in vivo safety investigation of the nanoparticles HA-R9-NP-D/P-H & E staining analysis of paraffin sections of main organs of mice:
6 BALB/c nude mice were randomly divided into 2 groups (Saline, HA-R9-NP-D/P), and 3 mice per group were injected with 100. mu.L of Saline or HA-R9-NP-D/P, respectively, into the tail vein. After 48H, the mice were sacrificed, heart perfused with PBS, and the major organs of the mice were collected and stained with paraffin sections H & E. As shown in the attached figure 18, by comparing the H & E staining results of Saline and HA-R9-NP-D/P groups, it can be seen that the main organs of the mice of the HA-R9-NP-D/P group which are given with the same pharmacodynamics administration dose do not have obvious damage, and the staining results are similar to those of the Saline group, thereby indicating that HA-R9-NP-D/P HAs good in vivo safety.

Claims (6)

1. A brain glioma whole-region targeted nano drug delivery system is characterized by comprising a double-layer targeted molecule, a drug, a foaming agent and a nano carrier, wherein the targeted molecule is R9 polypeptide and Hyaluronic Acid (HA), the drug is adriamycin (Dox), the foaming agent is 1-bromoheptafluorooctane (PFOB), and the nano carrier is a mixture of polylactic acid-glycolic acid copolymer PLGA-COOH, distearoyl phosphatidyl ethanolamine-polyethylene glycol DSPE-PEG and distearoyl phosphatidyl ethanolamine-polyethylene glycol-maleimide DSPE-PEG-MAL modified by terminal carboxyl; the medicine and the foaming agent are jointly encapsulated in the nano-carrier; the targeting molecule R9 polypeptide is connected with polyethylene glycol on the surface of the nano carrier through a covalent bond; the targeting molecule HA is wrapped on the outer layer of the R9 polypeptide through electrostatic interaction; the foaming agent is instantly vaporized under HIFU irradiation, and the generated radial force enables the nano-carrier to be rapidly disintegrated, so that the medicine is rapidly released; the sequence of the targeting molecule R9 polypeptide is 5 '-Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-3'; the mass ratio of PLGA-COOH to the sum of DSPE-PEG and DSPE-PEG-MAL in the nano carrier is 4: 1; the molar ratio of DSPE-PEG to DSPE-PEG-MAL in the nano-carrier is 5: 1; the molar ratio of the targeting molecule R9 polypeptide to the DSPE-PEG-MAL is 1: 10; the coating concentration of the targeting molecule HA is 0.4mg/mL PBS solution; the particle size of the drug delivery system HA-R9-NP-D/P is 40-50 nm, and the dispersion degree of the particle size is 0.1-0.2.
2. The brain glioma whole region targeted nano drug delivery system of claim 1, wherein the molecular weight of polylactic acid-glycolic acid copolymer PLGA-COOH modified by terminal carboxyl is 50000, the molecular weight of distearoylphosphatidylethanolamine-polyethylene glycol DSPE-PEG is 2000, and the molecular weight of distearoylphosphatidylethanolamine-polyethylene glycol-maleimide DSPE-PEG-MAL is 3400.
3. The method for preparing the brain glioma whole-area targeted nano drug delivery system according to claim 1, which is characterized by comprising the following steps:
(1) taking a proper amount of PLGA-COOH, DSPE-PEG-MAL, Dox methanol solution and PFOB, adding acetone, and fully mixing, wherein: the concentration of the Dox methanol solution is 6.66mg/mL, and the concentration of the PFOB is 19.23 mg/mL; the mass ratio of PLGA-COOH to the sum of DSPE-PEG and DSPE-PEG-MAL is 4: 1; the molar ratio of the DSPE-PEG to the DSPE-PEG-MAL is 5: 1; the mass ratio of Dox to the sum of PLGA-COOH, DSPE-PEG and DSPE-PEG-MAL is 1: 50; the mass ratio of the PFOB to the sum of PLGA-COOH, DSPE-PEG and DSPE-PEG-MAL is 1: 100; the acetone solution was then rapidly injected into MiliQ water, where: the volume ratio of acetone to MiliQ water is 1:2, and the acetone is removed by a vacuum pump at room temperature to obtain a MiliQ water-dispersed nano particle NP-D/P solution;
(2) dropwise adding a proper amount of R9 polypeptide aqueous solution into the nano NP-D/P solution obtained in the step (1), wherein the molar ratio of the DSPE-PEG-MAL to the R9 polypeptide aqueous solution is 10:1, and magnetically stirring at 350rpm at room temperature for 2 hours to obtain an R9-NP-D/P aqueous solution;
(3) preparing an HA aqueous solution with the concentration of 0.4mg/mL, dropwise adding the R9-NP-D/P aqueous solution obtained in the step (2) into the HA aqueous solution, wherein the volume ratio of the R9-NP-D/P aqueous solution to the HA aqueous solution is 1:1, and magnetically stirring at 350rpm at room temperature for 1h to obtain the HA-R9-NP-D/P aqueous solution of the drug delivery system.
4. Use of the brain glioma whole-region targeted nano drug delivery system of claim 1 in the preparation of chemotherapeutic drugs for treating brain gliomas.
5. Use according to claim 4, characterized in that: the nano drug delivery system is used as a chemotherapeutic drug for treating brain glioma by the following steps:
(1): injecting the delivery system intravenously;
(2): and 24h after administration, HIFU irradiation is given to the brain glioma part to induce the drug to be fully released at the brain glioma part.
6. Use according to claim 4, characterized in that: the nano drug delivery system is used for targeted drug delivery to the core region, the marginal region and the whole region of the infiltration region of the brain glioma so as to inhibit the tumor.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116869968A (en) * 2023-09-07 2023-10-13 四川大学 Nanoparticulate targeting brain and brain glioma, and synthesis method and application thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20110076836A (en) * 2009-12-29 2011-07-06 경북대학교 산학협력단 Targeted drug delivery system for diagnosis or therapy of cancer comprising liposome marked peptide specifically targeting interlukin-4 receptor, and method for preparing the same
CN107050040A (en) * 2017-03-27 2017-08-18 华东师范大学 Glioma targeted nano delivery system of HIFU controlled releases and its production and use
CN109893662A (en) * 2019-03-13 2019-06-18 苏州大学 Inhibit the preparation method and application of the load prodrug metastatic encephaloma targeting drug delivery system of Mfsd2a
CN110548152A (en) * 2018-05-30 2019-12-10 复旦大学 Tumor-targeted multi-drug co-loaded liposome modified by transmembrane anti-inflammatory peptide and preparation method thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20110076836A (en) * 2009-12-29 2011-07-06 경북대학교 산학협력단 Targeted drug delivery system for diagnosis or therapy of cancer comprising liposome marked peptide specifically targeting interlukin-4 receptor, and method for preparing the same
CN107050040A (en) * 2017-03-27 2017-08-18 华东师范大学 Glioma targeted nano delivery system of HIFU controlled releases and its production and use
CN110548152A (en) * 2018-05-30 2019-12-10 复旦大学 Tumor-targeted multi-drug co-loaded liposome modified by transmembrane anti-inflammatory peptide and preparation method thereof
CN109893662A (en) * 2019-03-13 2019-06-18 苏州大学 Inhibit the preparation method and application of the load prodrug metastatic encephaloma targeting drug delivery system of Mfsd2a

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116869968A (en) * 2023-09-07 2023-10-13 四川大学 Nanoparticulate targeting brain and brain glioma, and synthesis method and application thereof
CN116869968B (en) * 2023-09-07 2023-11-24 四川大学 Nanoparticulate targeting brain and brain glioma, and synthesis method and application thereof

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