Preparation and application of gold nanoparticles and liposomes thereof

Abstract

The present invention belongs to the field of biology and medicine and the field of pharmaceutical preparation technology, and specifically relates to the preparation and application of a gold nanoparticle and its liposome. The present invention prepares an asymmetrically modified gold nanoparticle (HAM) of arginine and maleimide groups, and an active targeted liposome HAM‑αLip encapsulating gold nanoparticles. On the one hand, the large-diameter targeted modified liposomes ensure that they will not be cleared by the reticuloendothelial system in the blood circulation system, ensuring the accumulation of HAM‑αLip in the tumor site; on the other hand, the modified gold nanoparticles are released when they reach the tumor site, achieving deep penetration of the tumor and improving the anti-tumor treatment effect.

Description

Technical Field

[0001] The invention belongs to the fields of biology and medicine and pharmaceutical preparation technology, and specifically relates to the preparation and application of gold nanoparticles and liposomes thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Tumor immunotherapy has opened a new era of anti-tumor treatment. The premise of activating tumor immunity is to induce tumor antigen production in situ or to deliver tumor antigens exogenously to initiate an immune response. Compared with exogenous delivery of antigens, the production of in situ induced antigens has the advantages of a wide variety of antigens, no need to determine the type of antigens, and simple technology. Radiotherapy, certain chemotherapy drugs, photothermal therapy, photodynamic therapy, etc. can all promote the production of tumor antigens in situ after treatment to initiate an immune response and exert a remote effect. The efficacy of the immune response depends on the degree to which antigens activate T cells. Ensuring the production of sufficient amounts of antigens is the key to ensuring T cell activation.

[0004] Nano drug delivery systems have certain targeted delivery capabilities and can be used to achieve precise intratumoral delivery. However, recent studies have found that only 0.7% (median) of nanoparticles are successfully delivered to solid tumors, which greatly limits the efficacy of clinical tumor treatment. Specifically, the abnormal growth and infiltration of tumor cells and the imperfect development of tumor neovascularization cause tumor tissue to exhibit high interstitial pressure and osmotic pressure, which seriously hinders the delivery of drugs. Nanoparticles can only gather around the tumor and kill cells around the tumor, while very few drugs reach the inside of the solid tumor, and it is impossible to produce sufficient antigens inside the tumor tissue. Therefore, effectively improving the ability of nano drugs to penetrate deep into the tumor is the key to improving the immune response.

[0005] At present, a variety of strategies have been developed to improve the penetration of nanomedicines into tumor tissues. By regulating the particle size, the infiltration of nanoparticles into the tumor is improved. Large-sized nanoparticles are more likely to accumulate at the tumor site but difficult to penetrate into the tumor. Small-sized nanoparticles can more easily penetrate into solid tumors but difficult to accumulate in tumor tissues. Based on this, studies have developed nanoparticles with convertible particle sizes in order to effectively accumulate and penetrate at the tumor site. However, after regulating the particle size, nanoparticles still need to enter the tumor tissue through passive diffusion, which is not enough to completely counteract the high interstitial pressure and high permeability of tumor tissue. In recent years, the autonomous movement of emerging nanomotors has provided technical support for achieving deep penetration of tumor tissues. Deep penetration can be achieved by providing a certain driving force with the help of endogenous factors or exogenous factors. Since the physiological characteristics of tumor tissue itself are different from those of normal tissue, the development of nanomotors using endogenous factors is critical to ensure deep tumor penetration and sufficient antigen production.

[0006] Arginine can be decomposed into NO under the action of ROS. NO can not only serve as the driving force of nanomotors, but also improve the abnormality of tumor neovascularization, alleviate the high interstitial pressure and high osmotic pressure inside tumor tissue to a certain extent, and provide double protection for deep tumor penetration. To ensure that the nanomotor has sufficient driving force, asymmetrically modifying arginine on the surface of the nanomotor is an effective strategy. Gold nanoparticles have rich surface active groups and are easy to modify. They can produce ROS and photothermal reactions under laser irradiation, which can provide conditions for the decomposition of arginine into NO and induce tumor cell apoptosis to release tumor antigens. In addition, gold nanoparticles have high biocompatibility and low biotoxicity, and are a suitable nanomotor carrier. In order to achieve effective capture of tumor antigens, maleimide groups are modified on the surface of gold nanoparticles, which can simultaneously capture tumor antigens released by tumor tissue after photothermal reaction under laser irradiation. To ensure that the maleimide groups on gold nanoparticles can avoid binding to proteins in the blood and that small-sized gold nanoparticles are cleared by the reticuloendothelial system in the blood, we have developed a large-particle thermosensitive liposome modified for targeted tumor tissue. The gold nanoparticles can be encapsulated in the liposomes to prevent the maleimide groups from binding to proteins in the blood, while avoiding uptake by the reticuloendothelial system, thereby enhancing accumulation at the tumor site. Under laser irradiation, the thermosensitive liposomes rupture and release small-sized gold nanoparticles, thereby achieving deep penetration into tumor tissues, effectively activating the immune response, and exerting anti-tumor therapeutic effects. Summary of the invention

[0007] Considering that the barrier effects of high osmotic pressure and interstitial pressure of tumor tissues hinder nanoparticles from penetrating into the tumor and inducing the production of tumor antigens, we designed and prepared a novel nanodrug delivery system for overcoming the high osmotic pressure of tumor tissues and capable of inducing antigen capture. Based on this, we prepared an active targeted liposome HAM-αLip containing hollow gold nanospheres coated with Argine and Maleimide (HAM). The liposome is a thermosensitive liposome composed of dipalmitoylphosphatidylcholine (1,2-DIHEXADECANOYL-RAC-GLYCERO-3-PHOSP, DPPC) and cholesterol. The interior of the liposome is gold nanoparticles modified with asymmetric arginine and maleimide groups. On the one hand, the large-particle targeted modified thermosensitive liposomes ensure that they will not be cleared by the reticuloendothelial system in the blood circulation system, and protect the maleimide groups on the gold nanoparticles from being bound by proteins in the blood, ensuring the accumulation of HAM-αLip in the tumor site; on the other hand, when reaching the tumor site, the thermosensitive liposomes rupture under laser irradiation, releasing the modified gold nanoparticles, and the small-particle size and nitric oxide (Nitric Oxide, NO) can ensure that gold nanoparticles penetrate deep into tumor tissues. At the same time, NO improves the abnormality of tumor neovascularization, relieves the high permeability and high interstitial pressure of tumor tissues, and further promotes the deep penetration of gold nanoparticles into tumors. After achieving deep tumor penetration, gold nanoparticles induce photothermal reaction under laser irradiation, promote the production of antigens inside tumor tissues, and can capture antigens, migrate to lymph nodes to initiate immune response, and improve the anti-tumor treatment effect.

[0008] The invention provides a thermosensitive liposome encapsulating gold nanoparticles, wherein the gold nanoparticles are asymmetrically modified with arginine and maleimide groups, and the thermosensitive liposomes include DPPC, cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol 2000-RGD (DSPE-PEG 2000 -RGD).

[0009] Furthermore, the modification ratio of arginine and maleimide groups of the gold nanoparticles is that the mass ratio of hollow gold nanospheres (HGN), S-acetylthioglycolic acid N-succinimidyl ester-polyethylene glycol 2000-arginine (SATA-PEG2000-Arg) and S-acetylthioglycolic acid N-succinimidyl ester-polyethylene glycol 2000-maleimide (SATA-PEG2000-Mal) is 1:0.5~1.5:0.5~1.5

[0010] Further, the preparation of the HAM is as follows:

[0011] (1) Sodium citrate, cobalt chloride and polyvinyl pyrrolidone are added to pre-deoxygenated water under nitrogen protection, and sodium borohydride is added under continuous stirring to form uniform cobalt nanospheres.

[0012] (2) When no bubbles are generated in the solution, chloroauric acid is added to form a gold shell on the surface of the cobalt nanospheres. The reaction solution is then exposed to air to oxidize the internal cobalt nanospheres, thereby obtaining the desired hollow gold nano-HGNs.

[0013] (3) The glass slide with positive charge on the silanized surface was immersed in the hollow gold nano-HGN solution with negative charge on the surface, so that HGN self-assembled on the glass slide surface through electrostatic adsorption, and then the glass slide adsorbed with HGN was immersed in the activated S-acetylthioglycolic acid N-succinimidyl ester-polyethylene glycol 2000-arginine solution and left to stand overnight. The activated S-acetylthioglycolic acid N-succinimidyl ester-polyethylene glycol 2000-arginine was combined with the hollow gold nano-HGN through the gold-sulfur bond, so that arginine was modified on the surface of the gold nanoparticles.

[0014] (4) The arginine-modified gold nanoparticles were separated from the glass slide by ultrasound, and activated S-acetylthioglycolic acid N-succinimidyl ester-polyethylene glycol 2000-maleimide was added for secondary modification to obtain asymmetrically modified gold nanoparticles.

[0015] Furthermore, the liposome can release gold nanoparticles under near-infrared light.

[0016] Furthermore, a method for preparing liposomes is provided, comprising the following steps:

[0017] The lipid part is prepared using DPPC, cholesterol and DSPE-PEG2000-RGD (5% molar ratio) as materials, which are weighed and dissolved in methanol, and then rotary evaporated to remove the organic solvent to form a lipid film. Then, HAM solution is added and ultrasonication is performed to obtain the final liposome HAM-αLip.

[0018] Another aspect of the present invention provides an application of gold nanoparticles in tumor treatment drugs.

[0019] Another aspect of the present invention provides a use of liposomes in promoting deep penetration of drugs into tumors.

[0020] Another aspect of the present invention provides a use of liposomes in anti-tumor immune drugs.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) In the present invention, arginine and maleimide groups are modified on the surface of gold nanoparticles through SATA-PEG, and asymmetric modification is achieved. Arginine can be decomposed into NO under the action of ROS. NO can not only generate driving force, but also improve the abnormality of tumor neovascularization, alleviate the high interstitial pressure and high osmotic pressure inside the tumor tissue to a certain extent, and provide double protection for ensuring deep tumor penetration.

[0023] (2) The present invention loads HAM into liposomes and modifies tumor tissue targeting factors, which can increase the accumulation of HAM in the tumor site, avoid being cleared by the reticuloendothelial system, prevent the maleimide groups modified on the Au nanoparticles from capturing proteins in the blood, ensure their capture of tumor antigens, and improve the anti-tumor effect.

[0024] (3) The present invention achieves deep penetration of tumor tissue. In a melanoma model, tumor sections showed that HAM-αLip can achieve deep penetration of the tumor site and vascular remodeling, which can enhance the anti-tumor effect and improve the immune response.

[0025] (4) The concentrations of phospholipids and cholesterol, and the HAM loading method in the present invention are all optimized data, and the therapeutic effect is good. The raw materials used in the present invention are simple, safe and reliable. The carrier materials phospholipids and cholesterol are both endogenous substances in the human body, safe, non-toxic and biodegradable, and have good biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0027] Figure 1 : Particle size and electron microscope image of HAM;

[0028] Figure 2 : Particle size and electron microscopy image of HAM-αLip;

[0029] Figure 3 :Cell uptake photography and flow cytometry results;

[0030] Figure 4 : Motion trajectory and MSD fitting results;

[0031] Figure 5 : Results of deep penetration slices of HAM;

[0032] Figure 6 : Section results of tumor vascular remodeling. DETAILED DESCRIPTION

[0033] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0034] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0035] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in combination with specific embodiments and comparative examples.

[0036] Example 1. Preparation and characterization of HAM

[0037] First, hollow gold nanospheres (HGN) were prepared. Sodium citrate (0.1 mM), cobalt chloride (0.1 mM) and polyvinyl pyrrolidone (PVP) (0.1 μM) were added to pre-deoxygenated water (100 mL) under nitrogen protection, and sodium borohydride (0.8 mM) was added under continuous stirring to form uniform cobalt nanospheres. When no bubbles were generated in the solution, 1% chloroauric acid (430 μL) was added to form a gold shell on the surface of the cobalt nanospheres. The reaction solution was then exposed to air to oxidize the internal cobalt nanospheres to obtain the desired HGN. The silanized glass slide was immersed in the HGN solution and allowed to stand overnight, and then the glass slide adsorbed with HGN was immersed in the activated SATA-PEG2000-Arg solution and allowed to stand overnight. Then, the arginine-modified gold nanoparticles were separated from the glass slide by ultrasound, and activated SATA-PEG2000-Mal (the mass ratio of HGN, SATA-PEG2000-Arg and SATA-PEG2000-Mal was 1:0.6:0.6) was added and stirred overnight at room temperature to obtain HAM. The particle size of HAM was measured by DLS, and the morphology of HAM was characterized by transmission electron microscopy ( Figure 1 The particle size of HAM is 50.49 ± 3.45 nm, the particle size is uniform, and the hollow structure can be clearly observed ( Figure 1 ).

[0038] Example 2. Preparation and characterization of HAM

[0039] First, hollow gold nanospheres (HGN) were prepared in the same manner as in Example 1. The silanized glass slide was immersed in the HGN solution and allowed to stand overnight, and then the glass slide adsorbed with HGN was immersed in the activated SATA-PEG2000-Arg solution and allowed to stand overnight. Then, the arginine-modified gold nanoparticles were separated from the glass slide by ultrasound, and activated SATA-PEG2000-Mal (the mass ratio of HGN, SATA-PEG2000-Arg and SATA-PEG2000-Mal was 1:1:1) was added, and stirred overnight at room temperature to obtain HAM. It can be observed with the naked eye that the HAM solution is clear and uniform without coagulation.

[0040] Example 3. Preparation and characterization of HAM

[0041] First, hollow gold nanospheres (HGN) were prepared in the same manner as in Example 1. The silanized glass slide was immersed in the HGN solution and allowed to stand overnight, and then the glass slide adsorbed with HGN was immersed in the activated SATA-PEG2000-Arg solution and allowed to stand overnight. Then, the arginine-modified gold nanoparticles were separated from the glass slide by ultrasound, and activated SATA-PEG2000-Mal (the mass ratio of HGN, SATA-PEG2000-Arg and SATA-PEG2000-Mal was 1:1.5:1.5) was added, and stirred overnight at room temperature to obtain HAM. It can be observed by the naked eye that the HAM solution is clear and uniform without coagulation.

[0042] Example 4. Preparation and characterization of HAM-αLip

[0043] HAM-αLip was prepared by a thin film dispersion method. The lipid part was prepared using DPPC (20 mg), cholesterol (2.5 mg) and DSPE-PEG2000-RGD (5% molar ratio) as materials, which were weighed and dissolved in methanol (2 mL). The organic solvent was removed by rotary evaporation at 40°C for 30 minutes to form a lipid film. Then, HAM solution (2 mL) was added and ultrasonicated for 30 minutes to obtain HAM-αLip. The particle size of HAM-αLip was determined by DLS, and the morphology of HAM-αLip was characterized by transmission electron microscopy ( Figure 2 The particle size of HAM-αLip is 134.80 ± 1.67 nm, and the shape is round. It can be observed that HAM is encapsulated inside the liposome ( Figure 2 ).

[0044] Example 5: Evaluation of cell-targeted uptake ability

[0045] The targeting ability of RGD on human umbilical vein endothelial cells (HUVEC) was verified by cell uptake experiments. 1×106 HUVEC cells were seeded in a 12-well plate and cultured overnight. Then, free coumarin 6 (C6), non-targeted nanoformulation containing C6 (C6-Lip), and targeted nanoformulation containing C6 (C6-αLip) were added to each well, respectively, and the concentration of C6 was 100 ng / mL. After co-incubation for 0.5 h and 2 h, the cells were rinsed with PBS, and 4% tissue cell fixative was added to fix the cells. The fluorescence analysis microplate reader was used to take pictures and flow cytometry was used to evaluate the cell uptake. The results show that at 0.5 h and 2 h, the uptake of C6-αLip by HUVEC cells was significantly higher than that of C6-Lip, proving that RGD has good targeting ability for tumor neovascularization ( Figure 3 ).

[0046] Example 6: Sports performance evaluation

[0047] H2O2 was used to simulate the ROS environment in the tumor site. Freshly prepared H2O2 solution was added to hollow gold nanospheres coated with Maleimide (HM) and HAM solution (the final concentration of H2O2 was 4.9 mM), and the motion trajectories of HM and HAM in H2O2 solution and HAM in aqueous solution were tracked and recorded at room temperature using the nanoparticle tracking instrument Zetaview. The recording time was 9 seconds and 10 frames per second. The speed of the nanomotor was calculated by MSD fitting. The results show that compared with the motion trajectories of the nanoparticles in the HM+H2O2 group and the HAM group, the HAM+H2O2 group showed good mobility, proving that the nanomotor HAM uses the NO gas produced by the reaction of arginine and ROS as a driving force to achieve self-motion. The results of the MSD fitting curve further prove this conclusion ( Figure 4 ).

[0048] Example 7: Evaluation of deep penetration ability

[0049] The nanomotors were modified with Cy5.5. Free HA-Cy5.5, HM-Cy5.5-αLip, and HA-Cy5.5-αLip (Cy5.5 dose of 30 μg / mouse) were injected into mice bearing B16F10 tumors via the tail vein and left overnight. The next day, the mice were irradiated with near-infrared light (808 nm, 1.2 W / cm 2, 5 min). After 4 h of light exposure, the mice were killed, the tumor tissues were removed, fixed with 4% tissue cell fixative, and the tumor tissues were sliced. The cell nuclei were labeled with DAPI, and then the slices were photographed fluorescently to observe the penetration of each nanoparticle in the tumor tissue. The results showed that compared with the free group, the accumulation of gold nanomotors in the tumor site in the liposome group with targeting ability was more significant, and compared with the HM-Cy5.5-αLip group without motility, the HA-Cy5.5-αLip group had motility and was able to reach the inside of the tumor, so the fluorescence signal inside the tumor was stronger, proving that HAM has the ability to penetrate deep into the tumor site ( Figure 5 ).

[0050] Example 8 Evaluation of Tumor Vascular Remodeling Ability

[0051] The mice bearing subcutaneous tumors were randomly divided into 4 groups (5 mice / group). When the tumors grew to about 80 mm 3 At 14:50 pm, mice were injected with NS, HM-αLip, HAM-Lip, and HAM-αLip by tail vein. On the second day after injection, the HM-αLip, HAM-Lip, and HAM-αLip groups were irradiated with laser (808 nm, 1.2 W / cm 2 , 5 min), and every three days was a treatment cycle. 3 Or 16 days later, the mice were killed, the tumor tissues were removed and paraffin sections were prepared. The tumor sections were stained with CD31 / DAPI, CD31 / NG2 and HIF-α / DAPI to observe angiogenesis, pericyte coverage and tumor tissue hypoxia. The results showed that HM-αLip and NS tumor angiogenesis were similar. Compared with NS, HAM-Lip and HAM-αLip could promote vascular remodeling in the tumor site, and the HAM-αLip group with targeting ability had the best vascular remodeling effect and could effectively alleviate hypoxia ( Figure 6 ).

[0052] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A liposome HAM-αLip, characterized in that: It comprises blank liposomes and gold nanoparticles, wherein the blank liposomes comprise dipalmitoylphosphatidylcholine, cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol 2000-RGD, and the gold nanoparticles are asymmetrically modified with arginine and maleimide groups; The modification ratio of the arginine and maleimide groups of the gold nanoparticles is that the mass ratio of the hollow gold nanoparticles HGN, S-acetylthioglycolic acid N-succinimidyl ester-polyethylene glycol 2000-arginine and S-acetylthioglycolic acid N-succinimidyl ester-polyethylene glycol 2000-maleimide is 1:0.5~1.5:0.5~1.5; The preparation method of the gold nanoparticles is as follows: (1) adding sodium citrate, cobalt chloride and polyvinyl pyrrolidone to pre-deoxygenated water under nitrogen protection, and adding sodium borohydride under continuous stirring to form uniform cobalt nanospheres; (2) When no bubbles are generated in the solution, chloroauric acid is added to form a gold shell on the surface of the cobalt nanospheres; the reaction solution is then exposed to air to oxidize the internal cobalt nanospheres to obtain the desired hollow gold nano-HGNs; (3) Immerse the glass slide with a positively charged surface after silanization treatment in a solution of hollow gold nanoparticles HGN with a negatively charged surface, so that HGN self-assembles on the surface of the glass slide through electrostatic adsorption, and then immerse the glass slide adsorbed with HGN in an activated S-acetylthioglycolic acid N-succinimidyl ester-polyethylene glycol 2000-arginine solution and let it stand overnight; the activated S-acetylthioglycolic acid N-succinimidyl ester-polyethylene glycol 2000-arginine combines with the hollow gold nanoparticles HGN through gold-sulfur bonds, so that arginine is modified on the surface of the gold nanoparticles; (4) separating the arginine-modified gold nanoparticles from the glass slide by ultrasound, and adding activated S-acetylthioglycolic acid N-succinimidyl ester-polyethylene glycol 2000-maleimide for secondary modification to obtain asymmetrically modified gold nanoparticles; The preparation method of the liposome HAM-αLip comprises the following steps: The blank lipid part was prepared using dipalmitoylphosphatidylcholine, cholesterol and 5% molar ratio of distearoylphosphatidylethanolamine-polyethylene glycol 2000-RGD as materials, which were weighed and dissolved in methanol to form a lipid film. Then, the asymmetrically modified gold nanoparticle solution was added and the final liposome HAM-αLip was obtained by ultrasound.

2. The liposome HAM-αLip according to claim 1, characterized in that: The liposome HAM-αLip can release gold nanoparticles under near-infrared light.

3. Use of the gold nanoparticles as claimed in claim 1 in preparing tumor therapeutic drugs.

4. Use of the liposome HAM-αLip as claimed in claim 1 or 2 in the preparation of a drug for promoting deep penetration of tumors.

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