CN112933052A

CN112933052A - Nano drug delivery system for improving tumor hypoxia microenvironment and enhancing immunotherapy

Info

Publication number: CN112933052A
Application number: CN202110095184.4A
Authority: CN
Inventors: 侯鹏; 王思蒙; 祭美菊
Original assignee: First Affiliated Hospital of Medical College of Xian Jiaotong University
Current assignee: First Affiliated Hospital of Medical College of Xian Jiaotong University
Priority date: 2021-01-25
Filing date: 2021-01-25
Publication date: 2021-06-11

Abstract

The invention belongs to the technical field of biomedicine and nano medicine, and particularly relates to a nano drug delivery system for improving a tumor hypoxia microenvironment and enhancing immunotherapy. The invention uses Human Serum Albumin (HSA) as a drug packaging carrier, and realizes the packaging of hydrophobic micromolecule drug Atovaquone (Atovaquone) under the action of chemical denaturation and renaturation, thereby obtaining the functionalized HSA-ATO NPs nano drug delivery system. The synthesis method is environment-friendly and convenient to operate. The functionalized nano drug delivery system can effectively improve the tumor hypoxia microenvironment and greatly enhance the treatment effect of immunotherapy, radiotherapy, chemotherapy drugs and targeted drugs on in-vivo tumors. Meanwhile, the system can remarkably improve the bioavailability and the tumor targeting property of the hydrophobic drug, and provides a new way for tumor delivery and tumor treatment of hydrophobic small molecules. The invention has good application prospect in the fields of cancer treatment research and drug transportation.

Description

Nano drug delivery system for improving tumor hypoxia microenvironment and enhancing immunotherapy

Technical Field

The invention belongs to the technical field of biomedicine and nano-medicine, relates to the technical field of biomedical materials for drug targeted tumor treatment, and particularly relates to a targeted hydrophobic drug delivery system and a construction technology thereof.

Background

Hypoxic microenvironments are one of the intrinsic features of most solid tumors, with the primary mechanisms being disorganized and structurally abnormal tumor vascularity. Hypoxia can cause a series of adaptive changes in cells, including enhanced anaerobic glycolysis, increased expression of protective stress proteins, and the like. Studies have shown that hypoxia not only causes radiation resistance, especially low energy linear density (LET) irradiation, but also is an important factor in causing drug resistance and tumor metastasis. Under normal conditions, oxygen in tissues can meet the requirement of cell metabolism; oxygen supply in tumor tissue is often lower than the growth metabolic needs of the cells, producing hypoxia (hypoxia). Hypoxia not only causes the resistance of tumor cells to radiation and chemotherapeutic drugs, but also promotes malignant transformation and metastasis of tumors, which is an important factor for poor prognosis of tumors.

The oxygen environment of the tumor is an equilibrium state determined by the supply and consumption of oxygen in the tumor. The tumor itself has a relatively high oxygen consumption rate, and thus, the interior thereof assumes a hypoxic state. Meanwhile, in this state, the blood vessels grow disorderly due to hypoxia inside the tumor, so that a high-pressure environment is formed inside the tumor, the delivery of oxygen is blocked, and the hypoxic state is further deteriorated. As shown by the oxygen consumption 3D model, approximately 30% inhibition of oxygen consumption may alleviate severe hypoxic conditions, restore vascular normalization, and also increase oxygen delivery within the tumor. Therefore, reducing the oxygen consumption capacity of the tumor's own height needs has become the most direct means to improve tumor hypoxia. The small molecular drug Atovaquone (Atovaquone) commonly used for treating malaria has the activity of inhibiting mitochondrial complex enzyme III, can greatly reduce the oxygen consumption of tumors and effectively improve severe anoxic regions. However, atovaquone, as a hydrophobic drug, is often low in bioavailability due to its physicochemical properties such as being insoluble in water, and is limited in market and clinical applications. To remedy the poor solubility of certain hydrophobic drugs, various carrier systems have been developed or patented to improve this deficiency. This is a problem of great concern in the markets of current medicine and the like, and further illustrates how to improve the bioavailability of hydrophobic small molecule drugs and avoid their side effects.

Nanotechnology has the potential of revolutionarily changing cancer diagnosis and treatment modes, and with the progress of cancer drug engineering and material science and technology, a novel nano-targeted delivery system technology will bring new hopes for cancer patients. The nano-carrier can inhibit the degradation of the drug in vivo, improve the absorption of the drug at a target part and promote the interaction between the drug and cells; moreover, the drug can be controllably distributed and released by modifying and modifying the nano-carrier and regulating and controlling the composition of the carrier. Thus, a highly efficient nano-targeted drug delivery system would greatly improve the efficiency of cancer therapy, providing a potential support for clinical applications. The preparation of nano-carriers by utilizing natural macromolecules has unique advantages, so that the nano-carriers are more and more widely concerned in the field of biological medicines. First, natural macromolecules such as albumin. The polysaccharide and the like not only have wide sources and low price. No toxicity, good biocompatibility and biodegradability. Secondly, a plurality of natural macromolecules and drug molecules have strong interaction, and the high-efficiency embedding of the drugs can be realized. Therefore, it is very important to prepare natural polymer drug carriers with superior performance by a simple and green preparation method. At present, a nano drug-carrying system for embedding paclitaxel by using human serum albumin has a commercial product, which suggests that the preparation of functionalized nano-carriers by using human serum albumin has a very practical significance.

Human Serum Albumin (HSA) is often used as a targeting drug delivery vehicle to improve targeting of drugs in vivo. The targeting drug delivery carrier is mainly based on that the targeting drug delivery carrier is an endogenous substance and does not generate toxicity or immune response; meanwhile, the compound has good stability and can not be degraded due to the change of the internal environment of a human body or immune reaction, so that the stability of most exogenous drugs can be improved; the unique spatial structure of albumin is utilized, and the drugs are loaded in the albumin in a physical wrapping or chemical bond coupling mode, so that the solubility of the insoluble drugs in blood plasma can be increased, the drug toxicity is obviously reduced, and the albumin has a better protective effect on the easily oxidized drugs.

The research aims to construct a drug tumor delivery system with targeting capability and high biological safety and biocompatibility, realize the targeted specific delivery of hydrophobic micromolecule drugs, promote and improve the absorption and bioavailability of the micromolecule drugs, and further enhance the curative effect of related therapies on tumor treatment. The ultra-small nanometer size makes it have a good potential as a carrier for drug delivery systems. Therefore, in the present study, Human Serum Albumin (HSA) with high biocompatibility is used as a carrier of a drug delivery system, so that multiple pairs of disulfide bonds in the Human Serum Albumin (HSA) are opened under a reducing condition, and meanwhile, a molecular solution of atovaquone is added in the process, so as to realize the encapsulation and assembly of the hydrophobic drug through oxidative renaturation. Not only has the specificity of identifying cancer cells, but also obviously improves the bioavailability of the medicament and the curative effect of treating tumors, greatly improves the stability under the extracellular environment, and also improves the effective release amount under the intracellular environment.

Disclosure of Invention

Aiming at the defects of overhigh dosage, low bioavailability, no target identification and the like of the conventional hydrophobic small molecule medicament in the treatment process, the invention provides a nano drug delivery system for improving the tumor hypoxia microenvironment and enhancing the immunotherapy, and researches the application potential of the nano drug delivery system. The preparation method of the drug delivery system is simple, and the used micromolecule drugs and protein drugs are approved by FDA to be used as clinical treatment drugs, so that the curative effect of related drugs can be well enhanced. The experimental results prove that: the tumor targeted delivery system (HSA-ATO NPs) of the hydrophobic micromolecular drug has smaller nano size, excellent stability and excellent biological safety, obviously improves the tumor hypoxia microenvironment of the mouse colon cell carcinoma (MC38), and greatly improves the curative effect of tumor immunotherapy while reducing the drug dosage.

In order to achieve the purpose, the invention adopts the following technical scheme: a nano drug delivery system for improving tumor hypoxia microenvironment and enhancing immunotherapy is characterized by comprising the following preparation steps:

step (1) preparation of carrier solution: adding 100mg Human Serum Albumin (HSA) into 10ml reduced glutathione solution with the concentration of 50mM to obtain Human Serum Albumin (HSA) carrier solution;

preparing a medicine solution in the step (2): weighing 10mg of hydrophobic drug Atovaquone, and dissolving the hydrophobic drug Atovaquone in 1ml of DSMO to obtain a drug solution;

step (3) construction of a targeted hydrophobic drug delivery system: placing the carrier solution in the step (1) in a 50ml beaker, stirring at 37 ℃ and 200rpm/min for 5min, sucking 1ml of the medicinal solution in the step (2) and dropwise adding the medicinal solution into the carrier solution, fully mixing the medicinal solution with the carrier solution for 10min, then adding a protein renaturation reagent, blowing for 10 times and continuing to react for 15min, then placing the system in an ultrasonic environment for 3min to obtain a human serum albumin and atovaquone composite system, and standing for 30min and then carrying out dialysis purification;

step (4) purification of the nano-targeting hydrophobic drug delivery system: transferring the composite system in the step (3) into an MWCO 10kDa dialysis bag, dialyzing in 2L of PBS (pH 7.4) solution for 12h to remove DMSO and unencapsulated drugs and proteins, and then carrying out low-temperature freeze-drying; after freeze-drying, the product is dissolved according to the using concentration, and the HSA-ATO NPs nano delivery system is obtained by passing the product through a 0.2um filter membrane.

The invention has the following advantages:

(1) the human serum albumin used in the invention is a protein existing in human plasma, is a nano material with proper size, has excellent biological stability and good biocompatibility, and has simple preparation process.

(2) The denaturing reagent reduced glutathione used in the invention replaces the traditional protein denaturing reagent with biotoxicity.

(3) All materials used in the invention are approved by FDA, the biological safety is excellent, and the carrier is an endogenous carrier, so that the carrier has the potential of realizing clinical application.

(4) The drug delivery system has good targeted delivery capability, and avoids off-target effect of the drug.

(5) The drug delivery system of the invention well solves the bottleneck problem of low bioavailability of the hydrophobic drug, greatly promotes the absorption of the hydrophobic drug, reduces the treatment concentration of the drug and enhances the treatment efficiency of the hydrophobic drug.

Drawings

FIG. 1 is a diagram of the synthesis and morphology of the HSA-ATO NPs delivery system.

FIG. 2 drug Loading and Release of HSA-ATO NPs delivery System.

FIG. 3 is tumor-targeted enrichment and safety assessment of HSA-ATO NPs.

FIG. 4 is the drug effect capacity of HSA-ATO NPs.

FIG. 5 shows the therapeutic effect of HSA-ATO NPs on tumors in combination with immunotherapy.

Detailed Description

For better understanding of the present invention, the present invention will be described in detail with reference to the following embodiments, wherein Human Serum Albumin (HSA) is taken as an example of the delivery system carrier, Atovaquone (Atovaquone) is taken as a representative of the hydrophobic small molecule drug, and the targeted hydrophobic delivery system is named HSA-ATO NPs, but the content of the present invention is not limited to the following embodiments.

Example 1: synthesis and characterization of HSA-ATO NPs

Adding 100mg of human serum albumin carrier into 10ml of 50mM GSH solution, incubating for 6h at 37 ℃, then placing the mixture into a 50ml beaker, stirring at 300rpm/min at 37 ℃, dropwise adding 10mg of Atovaquone dissolved in 1ml of DMSO solution, continuously stirring and uniformly mixing for 10min, then adding 30% hydrogen peroxide solution of a renaturation reagent to obtain a human serum albumin and Atovaquone composite system, and then dialyzing, purifying and freeze-drying.

FIG. 1 is a diagram of the synthesis and morphology of the HSA-ATO NPs delivery system. A schematic diagram of the overall synthesis scheme and subsequent mechanism of action of HSA-ATONPs is shown in FIG. 1 a. The shape and size of HSA-ATO NPs are detected by an electron microscope and a Malvern particle sizer respectively, and the results after sample loading detection are shown in fig. 1b and fig. 1c, wherein the particle size of the nanoparticles of the HSA-ATO NPs is about 164.5 nm.

The physical and chemical characteristics of the synthesized HSA-ATO NPs are obtained by detecting the synthesized HSA-ATO NPs through a Transmission Electron Microscope (TEM) and a laser particle sizer, and the results show that the diameters of the nanoparticles are about 164nm as shown in Table 1.

TABLE 1 physicochemical characterization of HSA-ATO NPs

Example 2: drug loading and drug release of HSA-ATO NPs

In order to verify whether atovaquone is successfully wrapped in an albumin system, an albumin carrier solution of a purified nanoparticle system and a control group is detected by using an ultraviolet spectrophotometry, and the result is shown in figure 2a, compared with an HSA solution, the HSA-ATO NPs system has a drug peak at the wavelength of 260nm, which indicates that the atovaquone is successfully wrapped by albumin; for the disaggregation properties of HSA-ATO NPs, we simulated the GSH concentration in tumor cells and studied the release behavior of atovaquone in PBS buffer at pH 7.4 (with or without 10mmol/L GSH). After dialysis for 48h, more than 80% of the drug-loaded atovaquone in the 10mmol/L GSH-containing group was released in PBS buffer, while only 30% of the drug-loaded without GSH group was released in PBS buffer as shown in FIG. 2b, which also demonstrates that the HSA-ATO NPs system will specifically disintegrate and release the drug in a high GSH environment.

Example 3: targeted enrichment and safety evaluation of HSA-ATO NPs

Detecting the enrichment condition of HSA-ATO NPs in main organs and tumors in a mouse body, and finding that the HSA-ATO NPs can be enriched in the tumors; and simultaneously has good biological safety.

As shown in FIG. 3, although all the raw materials involved in the preparation process were FDA approved, the biological safety of HSA-ATO NPs still needs to be further investigated. First, we examined the accumulation of drugs in major organs (heart, liver, spleen, lung, kidney) and tumors. Generally, such nanoparticles with a suitable particle size (. apprxeq.150 nm) have a better tumor targeting ability. As expected, the drug was mainly enriched in tumor and liver 6h after HSA-ATO NPs injection (FIG. 3 a). Meanwhile, 15 mice were treated with PBS (control group), HSA and HSA-ato NPs continuously for 7 days (days 1-7). On day 8, major organs and sera were excised and collected to assess the distribution of HSA-ATO NPs. As shown in FIGS. 3b and 3c, HSA-ato NPs had no effect on the liver and kidney weights of mice compared to mice administered with HSA or PBS. HE staining results are shown in figure 3 d. From the point of view of organ structure, there was no significant difference between the three groups. In addition, none of the relevant safety indices glutamic-oxaloacetic transaminase (AST), glutamic-pyruvic transaminase (ALT), serum Creatinine (CRE), Blood Urea Nitrogen (BUN) were significantly changed in the three groups of mice (fig. 3 e). In summary, our data indicate that HSA-ATO NPs retain a high degree of biocompatibility.

Example 4: validity testing of HSA-ATO NPs

As shown in FIG. 4, in order to better evaluate the drug efficacy of HSA-ATO NPs, we chose to validate the patient-derived xenograft (PDX) colon cancer model. During tumor progression we did not find any significant difference in tumor growth (fig. 4 a). By analyzing the hypoxic environment of each group of tumors, HSA-ATO NPs (atovaquone 6mg/kg) can obviously relieve the hypoxia of the tumors (FIG. 4 b).

Example 5: the curative effect of the synergistic immunotherapy of HSA-ATO NPs on tumors.

As shown in FIG. 5, HSA-ATO NPs significantly enhanced the immunotherapeutic effect of tumors against anti-PD-1 antibody.

To investigate whether HSA-ATO NPs act synergistically with anti-pd-1 antibodies and enhance immune responses, we established an MC38 allograft colon cancer model in the flank of normal C57 mice. When the tumor reaches 50-70 mm³Thereafter, all mice were randomly divided into 6 groups (5 per group) 1) PBS (control group); 2) anti-PD-1; 3) HSA-ATO NPs + anti-pd-1; 4) HSA-ATO NPs; 5) HSA; 6) atovaquone + anti-pd-1. All-grass of common swellingsTumor proliferation profile (FIG. 5a), tumor size (FIG. 5b) and tumor weight (FIG. 5c) results show that combination therapy with HSA-ATO NPs and anti-pd-1 synergistically inhibited tumor progression compared to anti-pd-1 alone.

While the foregoing is directed to embodiments of the present invention, it will be appreciated by those skilled in the art that various changes may be made in the embodiments without departing from the principles of the invention, and that such changes are encompassed within the scope of the invention.

Claims

1. A nano-drug delivery system for improving tumor hypoxia microenvironment and enhancing immunotherapy is characterized in that Human Serum Albumin (HSA) is used as a nano-particle carrier of a hydrophobic drug Atovaquone (Atovaquone), and the Atovaquone (Atovaquone) is wrapped by the Human Serum Albumin (HSA) and the Atovaquone through chemical denaturation and renaturation, and the nano-drug delivery system is named as HSA-ATO NPs.

2. The nano-delivery system for improving tumor hypoxia microenvironment and enhancing immunotherapy according to claim 1, wherein the HSA-ATO NPs are prepared by the following steps:

3. The nano-delivery system for improving tumor hypoxic microenvironment and enhancing immunotherapy according to claim 4, wherein the renaturation agent is 30% aqueous hydrogen peroxide.

4. The nano-delivery system for improving tumor hypoxia microenvironment and enhancing immunotherapy according to claim 1, wherein the HSA-ATO NPs have a particle size of 164.5 ± 10.3 nm.

5. The nano-delivery system for improving tumor hypoxia microenvironment and enhancing immunotherapy according to claim 1, wherein the HSA-ATO NPs drug loading rate is 8.70 ± 2.92%.

6. The nano-delivery system for improving tumor hypoxic microenvironment and enhancing immunotherapy prepared by the method of claim 1, and the application of the nano-delivery system in enhancing immunotherapy, radiotherapy, chemotherapy drugs and targeted drugs.