CN110393803B

CN110393803B - Paclitaxel and polypeptide co-delivery system, preparation method and application

Info

Publication number: CN110393803B
Application number: CN201910726966.6A
Authority: CN
Inventors: 王庆杰; 黄艺; 林贵梅
Original assignee: Qilu Hospital of Shandong University
Current assignee: Qilu Hospital of Shandong University
Priority date: 2019-08-07
Filing date: 2019-08-07
Publication date: 2022-03-18
Anticipated expiration: 2039-08-07
Also published as: CN110393803A

Abstract

The disclosure belongs to the technical field of antitumor drug delivery, and particularly relates to a paclitaxel and polypeptide co-delivery system, a preparation method and application. In the prior art, the combination of immunotherapy and chemotherapy has good effect on tumor treatment. Alloferon-1, a basic peptide, has been studied in recent years to show that the polypeptide can promote immune system function by activating NK cells in the tumor microenvironment. The inventor considers that the combination of Alloferon-1 and paclitaxel is expected to have good treatment effect, in order to realize the co-delivery of the two drugs, the disclosure provides a PTX-DOTAP @ Alloferon-1-Heparin/Protamine nano co-delivery system, based on the degradation effect of heparanase-1 in a tumor microenvironment on Heparin, the disintegration of the whole system at a tumor part and the enrichment of drugs are realized, and the system has good effect particularly when being applied to the treatment of melanoma.

Description

Paclitaxel and polypeptide co-delivery system, preparation method and application

Technical Field

The invention belongs to the technical field of antitumor drug delivery, and particularly relates to a PTX-DOTAP @ Alloferon-1-Heparin/promamine co-delivery system, a preparation method thereof and application thereof in antitumor drug preparation.

Background

The information in this background section is only for enhancement of understanding of the general background of the disclosure and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Melanoma originates in melanocytes and is the most malignant tumor species among skin tumors. Metastatic melanoma reduces the five-year survival of patients from 98% to 17%. Typically, the stroma in the Tumor Microenvironment (TME) is composed primarily of structural proteins and glycosaminoglycans, the major component of the latter being Heparan Sulfate Proteoglycans (HSPGs). In TME, HSPGs store a large number of biological factors (e.g., b-FGF, VEGF and TGF- β), and these factors are highly strategic in this battle between the body and the tumor. However, the result is often that heparanase-1 in the tumor microenvironment degrades HSPGs, allowing for massive release of these biological factors, activating subsequent signaling pathways, leading to neovascularization, epithelial-to-mesenchymal transition (EMT), and tumor metastasis. In addition, stromal voids created in the tumor microenvironment also contribute to tumor cell invasion and metastasis. These studies indicate that malignant highly metastatic tumors often express high levels of heparanase-1, especially in melanoma.

Scientists have proposed various strategies and treatment regimens for such malignancies. Some researchers have shown that the causes of melanoma are not completely consistent across different ethnic groups, and therefore the corresponding measures taken for early prevention and examination vary. In addition, the method for staging melanoma is updated and improved by research, so that a more reasonable and effective treatment scheme can be formulated clinically, the curative effect of the medicament is improved, and the side effect is reduced. Still other investigators have summarized the changes in melanoma treatment regimens over the past five years and have proposed a rational first-line treatment regimen for patients with BRAF mutant melanoma. In summary, current methods of treating melanoma primarily include chemotherapy, molecular targeted therapy, immune checkpoint blockade therapy, and tumor vaccine therapy.

Paclitaxel, a classical broad-spectrum anticancer drug, can inhibit the proliferation of tumor cells and is commonly used for the clinical treatment of melanoma. Under normal conditions, there is a dynamic equilibrium between microtubules and tubulin dimers, but paclitaxel can disrupt this dynamic equilibrium, induce tubulin polymerization and prevent depolymerization, and stabilize microtubules, resulting in cells that cannot form spindles during mitosis, thereby inhibiting cell division and proliferation, and arresting tumor cells in the G2/M phase, thereby exerting an anti-tumor effect. However, the long-term use of paclitaxel may lead to paclitaxel resistance in patients. Fortunately, in recent years, the efficacy of immunotherapy has become increasingly satisfactory in the treatment of cancer. Researchers have attempted to combine immunotherapy with chemotherapeutic drugs and have found that this treatment greatly enhances the efficacy of the drug.

Alloferon-1, (HGVSGHGQHGVHG), a basic peptide of 13 amino acids, which has a strong positive charge when dissolved in water. The peptide is used in the antiviral and antibacterial fields in the first place, but it has also been found to have a good effect on cancer treatment in recent years. Some reports also demonstrate that the basic peptide can reverse the immune system suppressed by tumor cells by activating Natural Killer (NK) cells in the tumor microenvironment, which release IFN- γ, TNF- β and perforin, thereby exhibiting an anti-tumor effect. Against the background of the above studies, the inventors considered: the treatment of melanoma with free drugs has obvious defects, including: systemic distribution and indiscriminate attack of the drug can reduce efficacy and cause damage to normal body tissues. Meanwhile, the drug without the protection of the carrier directly suffers from phagocytosis of macrophages and neutralization of opsonizing molecules, and the metabolic function of the liver and the excretory function of the kidney also reduce the half-life of the drug in the blood circulation. The adoption of the nanoparticle delivery form can obviously improve the defects, and increase the stability of the medicament and the enrichment effect of the medicament on tumor parts.

Disclosure of Invention

Against the background of the above studies, the inventors considered that the combination of paclitaxel and Alloferon-1 for the treatment of melanoma is expected to have a good therapeutic effect. However, since target cells of paclitaxel and Alloferon-1 are tumor cells and NK cells, respectively, there are technical difficulties in designing a nanosystem to achieve co-delivery of both drugs. As is well known, heparanase-1 can degrade heparan, and further research proves that the heparanase-1 also has the capability of degrading heparin, the discovery provides a research idea for providing a novel carrier material with enzyme reactivity, and the co-delivery of two medicaments is expected to be realized.

According to the research idea, research is carried out on a co-delivery system of paclitaxel and Alloferon-1, a nano system (PTX-DOTAP @ Alloferon-1-Heparin/Protamine) assembled by paclitaxel cationic micelles and Alloferon-1 polyelectrolyte complexes is obtained, and the nano system is a tumor microenvironment responsive nanoparticle based on Heparin and can finish the effects of co-loading and step-by-step delivery of paclitaxel and Alloferon-1. Combines chemotherapy and immunotherapy, and has the effects of good stability, simple preparation process, and increased enrichment of the medicine in tumor.

In order to achieve the technical effect, the present disclosure provides the following technical solutions:

in a first aspect of the present disclosure, a polyelectrolyte complex is provided, wherein the raw materials of the polyelectrolyte complex include polypeptide drugs, protamine sulfate and heparin.

Preferably, the polypeptide drug is an anti-tumor drug, and further is Alloferon-1.

Preferably, the heparin is heparin sodium.

Heparin sodium has a long-lasting antithrombotic effect, has been used as an anticoagulant for a long time, has various physiological activities such as smooth muscle cell proliferation resistance, anti-inflammation, anti-tumor, anti-virus and the like, and is loaded with a sugar chain structure with high-density negative charges on the surface. Protamine sulfate is a natural basic protein mainly composed of arginine, carries a large amount of positive charges, and is a pair of antagonistic substances which can make heparin lose anticoagulation effect by forming stable salt with heparin sodium. The two can form a stable compound for the delivery of the polypeptide drug, and can improve the instability of the free polypeptide drug to a certain extent. DOTAP is a positively charged phospholipid molecule commonly used in the preparation of cationic liposomes, and can be used to capture hydrophobic PTX by forming micelles. By optimizing the preparation process, the electrolyte complex with negative charge and proper particle size completely covers the DOTAP micelle with positive charge to form the final nano particle with negative charge, and a long-circulating drug carrier is expected to be formed.

In a second aspect of the present disclosure, there is provided a nanoparticle for co-delivery of paclitaxel with Alloferon-1; in the nanoparticle, the paclitaxel is in the form of paclitaxel cationic micelle, and the Alloferon-1 is in the form of polymer electrolyte in the first aspect.

Paclitaxel, as a cytotoxic antitumor drug, has a good therapeutic effect on solid tumors, but lacks targeting properties and has poor water solubility. The MediGene company in germany developed a paclitaxel cationic liposome, which is prepared from paclitaxel, 1, 2-dioleoyl-3-trimethylamine propane (DOTAP), and 1, 2-dioleoyl-sn-glycerol-3-phosphatidylcholine (DOPC) as raw materials, and is a drug targeting tumor new vessels. According to the paclitaxel and Alloferon-1 co-delivery system provided by the disclosure, the paclitaxel cation micelle and the polyelectrolyte complex are assembled, so that the whole nanoparticle surface presents strong negative electricity, and the nanoparticle is beneficial to realizing long circulation in blood.

In addition, heparanase-1 can be rapidly degraded by heparanase-1 in the outer layer of the nanoparticles due to high expression of heparanase-1 in the tumor microenvironment. Ion diffusion by the electrolyte in the gap dominates the disaggregation process of the nanoparticles, with protamine released by the concentration gradient, and degradation of the heparin molecules also results in a reduction of the electrostatic attraction between heparin and protamine. Eventually, this set of factors will form a positive feedback loop, accelerating the disintegration of the nanoparticles. Previous researches believe that electrolyte-induced ionic diffusion is an important factor for promoting the release of charged drug molecules in a polyelectrolyte complex, and the nanoparticles provided by the present disclosure promote nanoparticle depolymerization and alloferon-1 release through the synergistic effect of ionic diffusion and heparanase-1, so that the nanoparticles are more intelligent.

The nanoparticles will release Alloferon-1 to activate NK cells to reverse the tumor cell suppressed immune system. On the other hand, under the dual actions of enzymatic hydrolysis and ion diffusion, the DOTAP with the positive charge in the nanoparticle core is exposed, and the positively charged nanoparticles with reduced particle size are easier to permeate into the tumor and are easier to be endocytosed by the highly negatively charged tumor cells, so that the enrichment effect of the paclitaxel on the tumor cell part is improved, and the treatment effect of the chemical drug is improved.

In a third aspect of the present disclosure, a method for preparing nanoparticles described in the second aspect is provided, the method for preparing nanoparticles includes preparing paclitaxel cationic micelle, Alloferon-1 polyelectrolyte complex, and preparing nanoparticles.

In a fourth aspect of the present disclosure, an application of the polyelectrolyte complex of the first aspect and/or the nanoparticle of the second aspect in the preparation of an antitumor drug is provided.

Compared with the prior art, the beneficial effects of the present disclosure include:

1. the present disclosure provides a combination of paclitaxel and Alloferon-1, combining chemotherapy and immunotherapy, providing a therapy combining cytotoxic drugs and immunotherapeutic drugs, which can improve clinical therapeutic effects.

2. In order to realize the co-delivery of the two drugs, the disclosure provides a nano delivery system assembled by paclitaxel cation micelles and Alloferon-1 electrolyte complex, which utilizes the degradation effect of heparanase-1 on heparin to lead the nano system to be concentrated on a tumor part for disintegration. The disintegrated Alloferon-1 realizes the activation effect on NK cells in a tumor microenvironment, and the paclitaxel can easily enter the tumor cells under the action of charges, so that the treatment effect is enhanced. Especially has good therapeutic significance for melanoma, a tumor with high heparanase expression.

3. The preparation method disclosed by the invention has almost no chemical synthesis process, almost uses no organic solvent, and is simple and easy to implement. The nano particles prepared by the method have good size uniformity, and can be used for industrial production and clinical purposes in a pharmaceutical mode.

Drawings

The accompanying drawings, which are included to provide a further understanding of the disclosure, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure and are not to limit the disclosure.

FIG. 1 is a schematic representation of the action of PTX-DOTAP on tumor cells in the present disclosure;

FIG. 2 is a schematic representation of the preparation and characterization of PTX-DOTAP @ Alloferon-1-Heparin/Protamine in example 1;

wherein, FIG. 2a) is a schematic diagram of a preparation process of PTX-DOTAP @ Alloferon-1-heparin/Protamine;

FIG. 2b) is a schematic representation of the distribution of PTX and Alloferon-1 to different target cells during particle size reduction and charge inversion of PTX-DOTAP @ Alloferon-1-heparin/Protamine;

FIG. 2c) is a selection of the best ratio PTX-DOTAP @ Alloferon-1-heparin/Protamine;

FIG. 2d) is a size distribution of PTX-DOTAP @ Alloferon-1-heparin/Protamine;

FIG. 2e) TEM micrograph distribution of PTX-DOTAP @ Alloferon-1-heparin/Protamine;

FIG. 2f) is the zeta potential of PTX-DOTAP @ Alloferon-1-heparin/Protamine.

FIG. 3 is a graph showing the results of performance characterization of PTX-DOTAP and Alloferon-1-Heparin/Protamine in example 1;

wherein, FIG. 3a) is a graph of the ratio gradient of protamine and heparin in example 1;

FIG. 3b) is a TEM image of Alloferon-1-Heparin/Protamine nanoparticles of example 1;

FIG. 3c) is a graph showing the particle size distribution of Alloferon-1-Heparin/Protamine nanoparticles of example 1;

FIG. 3d) is a graph showing the potential distribution of Alloferon-1-Heparin/Protamine nanoparticles of example 1.

FIG. 3e) is a graph showing the particle size distribution of PTX-DOTAP nanoparticles of example 1;

FIG. 3f) is a graph showing the potential distribution of PTX-DOTAP nanoparticles of example 1.

FIG. 4 is a particle size and TEM image of PTX-DOTAP @ alloferon-1-heparin/Protamine when the PTX-DOTAP content is slightly higher or too high in example 1;

wherein, fig. 4a) is (protamine + heparin): TEM image of nanoparticles with DOTAP mass ratio of 4.2;

FIG. 4b) is (protamine + heparin): the distribution diagram of the particle size of the nanoparticles when the mass ratio of DOTAP is 4.2;

FIG. 4c) is (protamine + heparin): TEM image of 3.0 nanometer particle with DOTAP mass ratio;

FIG. 4d) is (protamine + heparin): the mass ratio of DOTAP is a distribution diagram of the particle size of 3.0 nanoparticles.

FIG. 5 is a graph showing the results of characterizing the release properties of PTX-DOTAP @ Alloferon-1-heparin/Protamine in example 2;

wherein, fig. 5a) is the amount of heparanase-1 expressed in B16F10 melanoma, liver and normal skin tissues;

FIG. 5b) in vitro release profiles of free DTX, free Alloferon-1 and PTX-DOTAP @ Alloferon-1-heparin/Protamine in the presence or absence of the heparin enzyme at 37 ℃ (n-3, results are shown as mean. + -. SD);

FIG. 5c) TEM photographs and schematic animations showing the distinct phenomena of PTX-DOTAP @ Alloferon-1-Heparin/Protamine at different times in four buffer environments without, enzyme, electrolyte and enzyme + electrolyte.

FIG. 6 is a graph of the results of in vitro cellular uptake of nanoparticles of example 3:

FIG. 7 is a graph showing the results of in vitro toxicity tests of the nanoparticles of example 4;

FIG. 8 is a graph of the results of in vivo profiles following administration in example 5;

wherein, fig. 8a) is a fluorescence image in vivo of a mouse after intravenous injection of a drug, and a dotted line indicates a tumor focus of the mouse.

FIG. 8b) is the drug profile in the isolated organ;

FIG. 8c) is the histogram of the drug content in the isolated organ.

FIG. 9 is a graph of immune system results from tumor cell suppression in the tumor microenvironment of example 5;

wherein, FIG. 9a) is a histogram of the change in IFN-. gamma.levels in peripheral blood of each group of mice;

FIG. 9b) is a histogram of the change in peripheral blood TNF- α levels in mice of each group;

FIG. 9c) is a histogram of the NKG2D content in tumor sections of various groups of mice;

FIG. 9d) is a histogram of CD94 content in tumor sections of each group of mice.

FIG. 10 is the results of the histopathological study of the mice in example 5;

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

As described in the background, the prior art has shown that immunotherapy in combination with chemotherapy has been shown to be effective in the treatment of tumors. Alloferon-1, a basic peptide, has been studied in recent years to show that the polypeptide can promote immune system function by activating NK cells in the tumor microenvironment. The inventor considers that the combination of Alloferon-1 and paclitaxel is expected to have good treatment effect, and in order to realize the co-delivery of the two drugs, the disclosure provides a PTX-DOTAP @ Alloferon-1-Heparin/Protamine nano co-delivery system, which realizes the disintegration of the whole system at the tumor part and the enrichment of the drugs based on the degradation of Heparin by heparanase-1 in the tumor microenvironment.

Preferably, the heparin is heparin sodium.

In some embodiments, the paclitaxel cationic micelle is made of paclitaxel, DOTAP.

In some embodiments, the nanoparticles are prepared from the following raw materials and in the following amounts: 1.200-1.600% of paclitaxel, 15.000-19.000% of DOTAP, 9.000-13.000% of protamine sulfate, 78-0.450% of Alloferon-10.150 and 60.000-80.000% of heparin sodium.

Furthermore, the medicine composition comprises 1.300-1.500% of paclitaxel, 16.000-18.000% of DOTAP, 10.000-12.000% of protamine sulfate, 0.400% of alloferon-10.200 and 65.000-75.000% of heparin sodium.

In specific examples, the amounts of nanoparticles are as follows: 1.401% of paclitaxel, 16.853% of DOTAP, 11.715% of protamine sulfate, alloferon-10.321% of heparin sodium 69.709%.

In some embodiments, the paclitaxel cationic micelle is prepared as follows: dissolving paclitaxel and DOTAP in chloroform, removing solvent by rotary evaporation at room temperature, and ultrasonic hydrating; furthermore, the hydration temperature of the ultrasonic is 20-40 ℃, and the ultrasonic time is 30-60 s.

In some embodiments, the Alloferon-1 polyelectrolyte complex is prepared as follows: mixing protamine sulfate and Alloferon-1, slowly dripping into the heparin sodium solution, and stirring to obtain the finished product.

In some embodiments, the nanoparticles are prepared as follows: at room temperature, the prepared paclitaxel cation is dripped into Alloferon-1 polyelectrolyte compound and stirred to obtain the paclitaxel cationic polymer; further, excess Alloferon-1-Heparin/protamine was removed by dialysis.

In some embodiments, the anti-neoplastic drug is an anti-melanoma drug.

In order to make the technical solutions of the present disclosure more clearly understood by those skilled in the art, the technical solutions of the present disclosure will be described in detail below with reference to specific examples and comparative examples.

In the following examples, the transmission electron micrograph was taken with a JEM-2100 transmission electron microscope (JEOL, Japan Electron Ltd.). Particle size analysis experiments a Zetasizer Nano ZS particle size potential analyzer (malmem britain).

EXAMPLE 1 preparation of PTX-DOTAP

1. Single factor test

In this example, conditions for PTX-DOTAP preparation were examined, and the examined factors included six factors of drug-lipid ratio, lipid concentration, hydration temperature, ultrasound time, ultrasound power, and rotary distilled water bath temperature.

1.1 drug to lipid ratio

For the determination of the mass ratio between PTX and DOTAP, this example was screened at five ratios of 1:5,1:10,1:15,1:20,1:25(PTX: DOTAP), and the encapsulation efficiency and drug loading were evaluated sequentially.

When the PTX: DOTAP is 1:10, the encapsulation efficiency is more than 90 percent, the increase is not large, but the drug loading capacity is greatly reduced, so that 1:12,1:15 and 1:18 are selected for further screening, and the drug loading capacity is mainly used in subsequent researches.

1.2 lipid concentration

For determination of DOTAP mass concentration, in this example, five amounts of 0.5,1,2,3,4mg/ml were screened, and drug loading was used as an evaluation index. The best drug loading was achieved when the DOTAP lipid concentration was around 2mg/ml, so 1.5,2.0,2.5mg/ml should be selected for further screening.

1.3 hydration temperature

For the determination of the hydration temperature during the ultrasonic treatment, five amounts of 20,30,40,50 and 60 ℃ are selected for screening in the embodiment, and the drug loading amount is used as a test index. The hydration temperature when ultrasonic treatment is about 30 ℃ has the best drug loading, so 25,30 and 35 ℃ should be selected for further screening.

1.4 ultrasound time

For the determination of the ultrasonic time, five quantities of 20s,40s,1min,5min,10min and 15min are selected for screening in the embodiment, and the drug loading quantity is used as a detection index. The best drug loading was achieved when the sonication time was around 40s, so 30s,40s,50s should be selected for further screening.

1.5 ultrasonic power

For the determination of the ultrasonic power, five amounts of 100,80,60,40 and 20% are selected for screening, the drug loading amount is taken as a test index, the drug loading amount of the liposome prepared by different ultrasonic powers is basically not changed, and the change of the ultrasonic power has no influence on the drug loading amount basically.

1.6 temperature of rotary steaming water bath

For the determination of the temperature of the rotary distilled water bath, five quantities of 20,30,40,50 and 60 ℃ are selected for screening, the drug-loading quantity is taken as a survey index, the temperature is changed from 20 ℃ to 60 ℃, but the drug-loading quantity is not changed basically, and the fact that the change of the temperature of the rotary distilled water bath does not have influence on the drug-loading quantity is proved.

2. Orthogonal design method optimization prescription

The results of single-factor experiments show that the factors which have larger influence on PTX-DOTAP @ Alloferon-1-Heparin/protamine nanoparticles are as follows: the drug-lipid ratio (A), the lipid concentration (B), the hydration temperature (C) and the ultrasonic time (D) are used as parameters for evaluating each prescription through an orthogonal design L9(3^4) table, and the following table is a factor level table and an orthogonal design investigation result respectively.

TABLE 1 orthogonal experiment factors and horizon

TABLE 2 results of orthogonal experiments

The final prescription was determined by combining the orthogonal test and the single factor experiment as follows: PTX: DOTAP is 1:12, the DOTAP lipid concentration is 1.5mg/ml, the hydration temperature is 35 ℃, and the ultrasonic time is 50 s. When the ratio of the drug to the lipid is 1:12, PTX-DOTAP has better drug loading (7.67% + -0.01) and encapsulation efficiency (99.70% + -0.15), the lipid concentration is 1.5mg/ml, the hydration temperature is 35 ℃, and the ultrasound time is 50 seconds. The particle diameter was 52.70. + -. 1.491nm (PDI: 0.163. + -. 0.012), and the zeta potential was 44.5. + -. 0.872 mV. Electron micrographs of PTX-DOTAP showed round and uniform particle size.

Proportional selection of Alloferon-1-Heparin/protamine

In the example, the Alloferon-1-Heparin/protamine ratio was screened, wherein the sequence of Alloferon-1 is shown in SEQ ID NO: 1. A series of protamine and heparin gradient curves were designed by slowly adding 0.3mg/ml protamine slowly dropwise to 0.3mg/ml heparin with stirring at room temperature. The results are shown in the following table, using the potential change of Alloferon-1-Heparin/protamine as an evaluation index:

TABLE 3 Heparin/protamine ratio screening

As shown in FIG. 2c), the ratio of the formulation components was saturated when the protamine to heparin mass ratio was 0.125.

Component ratio screening of PTX-DOTAP @ Alloferon-1-Heparin/protamine nanoparticles

In this example, PTX-DOTAP (0.5ml of 1.5mg/ml DOTAP) was slowly added dropwise to 0.3mg/ml Alloferon-1-Heparin/protamine with stirring at room temperature to design a series of (protamine + Heparin) to DOTAP ratio gradient curves. The potential change of PTX-DOTAP @ Alloferon-1-Heparin/protamine is taken as an evaluation index, and the results are as follows:

TABLE 4 screening Table for heparin globule-DOTAP ratio

As shown in fig. 3a), when (protamine + heparin): when the mass ratio of DOTAP is 4.85, the proportion of the formulation components is saturated. At this time, the electron micrograph of the nanoparticles showed uniform shape and particle size, with no excess heparin/Protamine in the background.

Characterization of formulations with higher DOTAP ratios

In this example, (protamine + heparin): the preparation of PTX-DOTAP @ Alloferon-1-Heparin/protamine nanoparticles was carried out at a mass ratio of 4.2 to 3.0, and no excess Alloferon-1-Heparin/protamine nanoparticles were removed using a 1000KD dialysis band.

As shown in FIG. 3, when the proportion of DOTAP in the formulation was increased, the size of the nanoparticles became very large, exceeding 200nm, even reaching 500nm, and the particle size was very non-uniform, as well as many Alloferon-1-Heparin/protamine nanoparticles that were not removed.

Investigation of physicochemical Properties of Alloferon-1-Heparin/protamine nanoparticles

6.1 form

Taking a proper amount of Alloferon-1-Heparin/protamine nanoparticle solution in a penicillin bottle, and observing that the appearance of the penicillin bottle presents light blue opalescence. And taking one to two drops of Alloferon-1-Heparin/protamine nanoparticle solution, diluting the solution with distilled water, dripping the diluted solution on a copper sheet, dyeing the copper sheet with 2 percent phosphotungstic acid solution, drying the copper sheet for 30min, and observing the microscopic form through a transmission electron microscope. Under TEM, the ethosome has round shape, smooth surface without adhesion and uniform particle size distribution.

6.2 particle size and potential

Taking a proper amount of the prepared Alloferon-1-Heparin/protamine nano particle solution, diluting by a proper time, and measuring by using a dynamic light scattering instrument, wherein the particle size is 26.19 +/-1.631 nm (PDI:0.146 +/-0.018) measured by using a Malvern laser particle size analyzer; the zeta potential is stabilized at-42.4. + -. 0.246-mV.

Preparation of PTX-DOTAP @ Alloferon-1-Heparin/Protamine

(1) Preparation of PTX-DOTAP

0.550ml of 1mg/ml PTX (dissolved in chloroform) and 6.616ml of 1mg/ml DOTAP (dissolved in chloroform) were mixed homogeneously and then rotary evaporated at room temperature for 15 minutes. Subsequently, 4.411ml of water were slowly added to the flask under ultrasound at a certain temperature and sonicated for a certain period of time.

According to the research result of single-factor investigation, in the step, the ultrasonic hydration temperature can be selected within 20-40 ℃, and the ultrasonic time is selected within 30-60 s, so that a good drug loading effect can be realized.

(2) Preparation of Alloferon-1-Heparin/protamine polyelectrolyte complex

After 15.33ml of 0.3mg/ml protamine sulfate and 0.42ml of 0.3mg/ml alloferon-1 were mixed, the mixture was slowly added dropwise to 91.215ml of 0.3mg/ml heparin sodium solution with stirring at room temperature and stirred for 30 minutes (600 rpm).

(3) Preparation of PTX-DOTAP @ Alloferon-1-heparin/protamine

4.411ml of PTX-DOTAP were slowly added dropwise to 106.97ml of Alloferon-1-heparin/protamine polyelectrolyte complex with stirring at room temperature for 10 minutes (200 rpm). The dialysis was then carried out in a 1000kD dialysis bag for 10 minutes to remove the slight excess Alloferon-1-Heparin/protamine.

PTX-DOTAP @ Alloferon-1-Heparin/protamine nanoparticle physical and chemical property investigation

8.1 form

A proper amount of PTX-DOTAP @ Alloferon-1-Heparin/protamine nanoparticle solution is put in a penicillin bottle, and the appearance of the penicillin bottle is observed to present light blue opalescence. And taking one to two drops of PTX-DOTAP @ Alloferon-1-Heparin/protamine nanoparticle solution, properly diluting with distilled water, dropwise adding the diluted solution on a copper sheet, dyeing with 2% phosphotungstic acid solution, drying for 30min, and observing the microscopic morphology through a transmission electron microscope. Under TEM, the ethosome has round shape, smooth surface without adhesion and uniform particle size distribution.

8.2 particle size and potential

Taking a proper amount of the prepared PTX-DOTAP @ Alloferon-1-Heparin/protamine nanoparticle solution, diluting by a proper time, and measuring by using a dynamic light scattering instrument and a Malvern laser particle size analyzer to obtain the particle size of 106.1 +/-1.113 nm (PDI:0.147 +/-0.005); the zeta potential is stabilized at-45.1. + -. 0.455 mV.

Example 2 in vitro Release behavior of PTX-DOTAP @ alloferon-1-heparin/Protamine

In this example, the in vitro release behavior of PTX-DOTAP @ alloferon-1-heparin/Protamine was studied. Tumor microenvironment was simulated with 0.01nM Tris-HCl (pH 6.5) using 20nM Ca²⁺Mimicking Ca in the tumor microenvironment²⁺Concentration and enhanced heparanase-1 activity. According to the graph in fig. 5b), the release of free alloferon-1 and free PTX was 85.22 ± 0.82% and 90.01 ± 1.41% respectively within 6 hours, and both groups rapidly completed the release. A PTX-DOTAP @ alloferon-1-heparin/Protamine set of 50. mu.l 0.0833IU/ml heparinase released alloferon-1 from 0 hours, the release rate of which was significantly reduced at 10 hours. The PTX group released rapidly from 2 hours later, since PTX in the nanoparticle core was protected by the alloperon-1-heparin/Protamine layer and was not released suddenly, the release rate decreased significantly at 16 hours. Finally, the released amounts of PTX and alloferon-1 were 83.82. + -. 1.12% and 81.67. + -. 0.76%, respectively. In the PTX-DOTAP @ alloferon-1-heparin/Protamine group without heparinase, only 12.77 +/-1.58% of PTX and 8.43 +/-1.79% of alloferon-1 are released in the whole release experiment, which indicates that the PTX-DOTAP @ alloferon-1-heparin/Protamine nano particles can provide excellent protection for internal drugs. The appearance of each group during release and electron micrographs are shown in fig. 5c), where the presence of nanoparticles causes the solution to exhibit a bluish opalescence. At 5min, the enzyme group and enzyme + electrolyte became cloudy, an insoluble white floc was produced, showing enzymatic hydrolysis of alloferon-1-heparin/Protamine (PTX-DOTAP @ the outermost layer of the alloferon-1-heparin/Protamine nanoparticles) and gradual release of heparin, exposing a portion of the positively charged Protamine. After 6 hours, the color of the nanoparticle aggregates became whiter, indicating that the alloperon-1-heparin/Protamine layer of the PTX-DOTAP @ alloperon-1-heparin/Protamine formulation was significantly degraded by heparinase and separated by ionic diffusion. At 6 hours, the electrolyte became very cloudy and the solution contained a large amount of white floc. This is due to ion diffusion by the electrolyte, so that the alloferon-1-heparin/Protamine layer of PTX-DOTAP @ alloferon-1-heparin/Protamine also dissociates slowly, and the nanoparticles expelled from the surface aggregate together. However, without the heparin enzymeThe participation, the progress of this physical phenomenon is markedly reduced. At 12 hours, the enzyme + electrolyte became very clear and the white flocs in solution almost completely disappeared. The electron micrograph also shows that the solution contains only a small amount of isolated nanoparticles with a particle size of about 50 nm. This is a result of the combination of heparin enzyme and ion diffusion, resulting in almost complete detachment of the allopron-1-heparin/Protamine layer of the PTX-DOTAP @ allopron-1-heparin/Protamine formulation, exposing the internal DOTAP core. The measurement results showed that the particle size and potential of the nanoparticles in the solution at this time were 59.30. + -. 0.783nm (PDI: 0.234. + -. 0.032) and 25.4. + -. 0.257mV, respectively. Depending on the amount of released PTX and alloferon-1 as measured by the release test, ionic diffusion of the electrolyte alone did not promote depolymerization of PTX-DOTAP @ alloferon-1-heparin/Protamine. Only the synergistic effect of heparinase and ion diffusion results in almost complete dissociation of the preparation, achieving particle size reduction and charge conversion of the nanoparticles. Thus, when the nanoparticles reach the tumor site, the particle size reduction process will change the particle size of the nanoparticles from about 100nm to 50nm to increase the amount of nanoparticles that enter the tumor.

Example 3 cellular uptake assessment of PTX-DOTAP @ alloferon-1-heparin/Protamine

In the embodiment, the B16F10 cell is used as a model to evaluate the uptake effect of the tumor cell on the nanoparticles, and the cell uptake condition is indicated by coumarin 6. The fluorescence intensity was measured by fluorescence microscopy. As shown in FIG. 6, the fluorescence intensity was lowest in the free C6 group, i.e., the blank group. The fluorescence intensity of the C6-DOTAP group is strongest, which indicates that PTX-DOTAP @ alloferon-1-heparin/Protamine can expose positively charged DOTAP under the action of enzymatic degradation in a tumor microenvironment, and the cellular uptake efficiency of the nanoparticles is improved.

In addition, the uptake of nanoparticles by coumarin 6-HEP NP and heparin preincubation + coumarin 6-HEP NP was much higher than that by free coumarin 6. Pre-incubation of cells with heparin did not affect cellular uptake of coumarin 6-HEP NP, indicating that heparin molecules are not tumor targeted. Finally, coumarin 6-CS NP and coumarin 6-HA NP were taken up. Compared with coumarin 6-HEP NP, the result shows that hyaluronic acid and chondroitin sulfate have a certain targeting effect on tumor, and the uptake of nanoparticles by tumor cells is enhanced.

Example 4 in vitro cytotoxicity assay of PTX-DOTAP @ alloferon-1-heparin/Protamine

In this example, the cytotoxicity of different nanoparticles in vitro was measured using a Cell Counting Kit (CCK) -8(Cell Counting Kit-8, BestBio, Shanghai, China).

B16-F10 cells were seeded in 96-well plates at 37 ℃ and 6000 cells/well and cultured overnight in 100. mu.L of complete medium. Blank NPs (DOTAP @ heparin/Protamine), free PTX, free alloferon-1, free combinations, PTX NPs (PTX-DOTAP @ heparin/Protamine), alloferon-1 NP (DOTAP @ alloferon-1-heparin/Protamine), PTX/alloferon-1 NP (PTX-DOTAP @ alloferon-1-heparin/Protamine) and controls (with an equal volume of blank complete medium added) were added to each well at paclitaxel concentrations of 0.003,0.03,0.3, 3,30 and 300. mu.g/mL (the concentration of alloferon-1 being one eighth of paclitaxel) and 0.14,1.4,14,140 and 1400. mu.g/mL and blank nanoparticles were incubated at 3 ℃ with 5% CO at 3 ℃ and 5% CO₂For 24 hours or 48 hours. Prior to analysis, CCK-8 solution (10. mu.L/well) was added and then incubated for a further 1 hour. The maximum absorbance was set at 450nm and the Optical Density (OD) of each well was scanned by a microplate reader (BioTek Synergy H1, BioTek Instruments, inc., Winooski, VT, USA).

Relative cell survival (RCV) (%) was calculated as RCV (%) ═ OD test/OD control x 100%, where OD test and OD control represent the OD of cells treated with test and control groups, respectively. The experiments were independently repeated six times and half maximal inhibitory concentrations (IC50) were calculated for the test groups using GraphPad Prism 7.0 software.

To explore the apoptosis-inducing properties in vitro, B16-F10 cells were plated at 2X 10⁵The density of individual cells/well was seeded in 12-well plates. After co-incubation with different groups of free drug or nanoparticles, detection was performed by flow cytometry (CytExpert 3.0 software) within 30 minutes.

B16-F10 was seeded in 6-well plates at a density of 5X 10⁵Individual cells/well, treated with different sets of free drugs or nanoparticles. After 24 hours of incubation, cells were collected by flow cytometryThe intensity and distribution of propidium iodide was analyzed by surgery (FACSCalibur, BD Biosciences, Franklin Lakes, NJ, USA). Data were analyzed and processed using ModFit 3.1 software. The research result shows that the PTX/alloferon-1 NP group has the strongest cytotoxicity with the same concentration of the administration dose, can obviously reduce the activity of tumor cells, and the inhibition effect is enhanced along with the increase of the administration concentration. The effect of each group of drugs on the cell cycle is shown in FIG. 7, in which PTX blocks the cell proliferation cycle of G2/M phase, and PTX/alloferon-1 NP further increases the drug uptake by cells.

Example 5 tumor xenograft mouse model and determination of mouse biodistribution

All in vivo mouse experiments were approved by the university of Shandong animal protection and use Committee. At 1 × 10⁶The number of the cells was C57BL/6 mice (6-8 weeks old) inoculated subcutaneously on the right flank with B16-F10 cells. When the tumor volume reaches 200mm³In this case, B16-F10 tumor-bearing mice were randomly grouped (n ═ 3), and free DiR, DiR-HEP/DOTAP (DiR-) DOTAP @ heparin/Protamine), DiR-DOTAP and DiR-HA/DOTAP (DiR-DOTAP @ hyaluronic acid/Protamine) were injected intravenously at a dose of 100. mu.g/kg.

Mice were anesthetized at 1 hour, 4 hours, 12 hours, and 24 hours post-dose. Real-time images were obtained by the Xenogen IVIS luminea system (Caliper Life Sciences, Waltham, MA, USA). Mice were sacrificed 24 hours later and heart, lung, liver, spleen, kidney and tumors were harvested for ex vivo imaging. Images were analyzed in Living Image 4.1 software (Caliper Life Sciences, Waltham, MA, USA). The results are shown in FIG. 8, with the DiR-HEP/DOTAP and DiR-HA/DOTAP groups having good long-circulating ability at 24 hours and some accumulation at the tumor site. The results also showed that free DiR, DiR-DOTAP and DiR-HA/DOTAP had more major organ accumulation, while DiR-HEP/DOTAP had less liver accumulation (FIG. 8 b)).

(1) In vivo antitumor study

To evaluate the antitumor efficacy and safety of the nanoparticles, 1 × 10 was used⁶B16-F10 cells were injected subcutaneously into each C57BL/6 mouse. When the tumor volume reaches 100mm³When mice were randomized into 8 groups (n-8), and 100 μ LP was injected intravenouslyBS, blank NP (DOTAP @ heparin/Protamine), free PTX, free alloferon-1, free combination, PTX NP (PTX-DOTAP @ heparin/Protamine), alloferon-1 NP (DOTAP @ alloferon-1-heparin/Protamine), and PTX/alloferon-1 NP (PTX-DOTAP @ alloferon-1-heparin/Protamine) paclitaxel at a dose of 10mg/kg, and alloferon-1 at a dose of 1.25 mg/kg. Tumor volume and weight were measured every 3 days to observe anti-tumor efficacy and toxicity in vivo. After dosing was complete, tumors and organs (heart, liver, spleen, lung and kidney) were collected and fixed for TUNEL, Ki67 and heparanese-1 staining. Mice were analyzed for survival time after Kaplan-Meier survival score using GraphPad Prism 7.0. The research results show that PTX-DOTAP @ Alloferon-1-Heparin/Protamine can enhance tumor inhibition and delay tumor growth.

(2) Immune-related in vivo studies

To explore NK cell and CD8+ T cell activation in vivo, this example evaluated alloferon-1 induced anti-tumor immune responses by granzyme B, NKG2D, CD94, IFN- γ and TNF- α. Mice were sacrificed after intravenous injection of different formulations using C57BL/6 mice carrying B16-F10 as a model, tumor tissue was obtained for immunohistochemical staining, and peripheral serum immune factors were tested using the corresponding ELISA kits. Anti-mouse mabs were used in this study. Anti-granzyme B, anti-CD 8+, anti-NKG 2D, anti-CD 94, anti-IFN- γ, anti-TNF- α, IFN- γ and TNF- α ELISAs were purchased from Abcam (Cambridge, UK) and HuaBio (Hangzhou, China). Images were acquired using a laser scanning confocal microscope (LSM 780, Carl Zeiss, Oberkochen Germany). Among them, the changes in IFN-. gamma.and TNF-. alpha.levels in peripheral blood of mice are shown in FIGS. 9a) and b), and the immunofluorescence results for NKG2D and CD94 in tumor sections are shown in FIGS. 9c) and d).

(3) Toxicity study of PTX-DOTAP @ alloferon-1-heparin/Protamine on major organs

To examine the safety of nanoparticles in vivo, fixed vital organs (heart, liver, spleen, lung and kidney) were embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Stained sections were observed and photographed using a VS120 virtual slide microscope (Olympus corp., Tokyo, Japan). Peripheral serum markers were tested using alanine Aminotransferase (ALT) ELISA kit, aspartate Aminotransferase (AST) ELISA kit, and Blood Urea Nitrogen (BUN) ELISA kit, with the results shown in table 5 and fig. 10.

TABLE 5 serum AST, ALT, and BUN levels (n. 3) for the different drug groups

ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen

(4) Statistical analysis

Data are presented as mean ± standard deviation. One-way and two-way anova were used for multiple comparisons. Bonferroni post-test was performed when all groups were compared, and a two-tailed t-test was used when both groups were compared. In vivo tumor treatment studies were repeated in two independent experiments to ensure adequate sample size and reproducibility. All statistical analyses were performed using GraphPad Prism and SPSS 19.0 software. Statistical significance was as follows: p <0.05, p <0.01, and p < 0.001.

The above description is only a preferred embodiment of the present disclosure and is not intended to limit the present disclosure, and various modifications and changes may be made to the present disclosure by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present disclosure should be included in the protection scope of the present disclosure.

Claims

1. A nanoparticle for co-delivery of paclitaxel with Alloferon-1; in the nanoparticle, the paclitaxel is in a paclitaxel cation micelle form, and the Alloferon-1 is in a polyelectrolyte complex form;

the paclitaxel cationic micelle is prepared from paclitaxel and DOTAP;

the raw materials of the polyelectrolyte compound comprise Alloferon-1, protamine sulfate and heparin sodium;

the nano-particles comprise the following raw materials in percentage by weight: 1.200-1.600% of paclitaxel, 15.000-19.000% of DOTAP, 9.000-13.000% of protamine sulfate, 78-0.450% of Alloferon-10.150 and 60.000-80.000% of heparin sodium.

2. The nanoparticle of claim 1, wherein the nanoparticles are prepared from the following raw materials in the following amounts: 1.300-1.500% of paclitaxel, 16.000-18.000% of DOTAP, 10.000-12.000% of protamine sulfate, 0.400% of alloferon-10.200 and 65.000-75.000% of heparin sodium.

3. The method for preparing nanoparticles according to any one of claims 1-2, wherein the preparation method comprises the preparation of paclitaxel cationic micelles, Alloferon-1 polyelectrolyte complexes and nanoparticles;

the preparation method of the paclitaxel cationic micelle comprises the following steps: dissolving paclitaxel and DOTAP in chloroform, removing solvent by rotary evaporation at room temperature, and ultrasonic hydrating;

the preparation method of the Alloferon-1 polyelectrolyte compound comprises the following steps: mixing protamine sulfate and Alloferon-1, slowly dripping the mixture into a heparin sodium solution, and stirring the mixture to obtain the finished product;

the preparation method of the nanoparticle comprises the step of dropwise adding the prepared paclitaxel cationic micelle into the Alloferon-1 polyelectrolyte compound at room temperature and stirring to obtain the paclitaxel cationic micelle.

4. The preparation method of the nanoparticles according to claim 3, wherein the hydration temperature of the ultrasound is 20-40 ℃ and the ultrasound time is 30-60 s.

5. The method of claim 3, wherein the excess Alloferon-1-Heparin/protamine is removed by dialysis.

6. Use of the nanoparticles according to any of claims 1-2 for the preparation of antitumor drugs.

7. The use of claim 6, wherein the anti-neoplastic drug is an anti-melanoma drug.