CN111150857A - Lipid nanoscale ultrasonic contrast agent targeting tumor-associated macrophages and preparation method and application thereof - Google Patents
Lipid nanoscale ultrasonic contrast agent targeting tumor-associated macrophages and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a lipid nanoscale ultrasonic contrast agent targeting tumor-associated macrophages, and a preparation method and application thereof. The nanoscale ultrasonic contrast agent takes lipid as a shell membrane material, and low-molecular-weight hyaluronic acid and gaseous fluorocarbon are wrapped inside the shell membrane; the lipid comprises dipalmitoylphosphatidylcholine, distearoylphosphatidylethanolamine, and distearoylphosphatidylethanolamine-polyethylene glycol-folic acid; the particle size of the nano-scale ultrasonic contrast agent is 255-497 nm, the nano-scale ultrasonic contrast agent can penetrate through gaps of vascular walls of tumor tissues, and the nano-scale ultrasonic contrast agent has passive targeting and high efficiency for tumor treatment; coupled folic acid is arranged outside a lipid shell membrane through a PEG chain, and low-molecular-weight hyaluronic acid is wrapped inside the contrast agent, so that the nanoscale ultrasonic contrast agent can be ensured to actively target tumor-related macrophages in a folic acid-folic acid receptor mode, and the low-molecular-weight hyaluronic acid acts on the tumor-related macrophages to convert the tumor-related macrophages from M2 type macrophages to M1 type macrophages.
Description
Technical Field
The invention relates to a lipid nanoscale ultrasonic contrast agent targeting tumor-associated macrophages, and a preparation method and application thereof, and belongs to the technical field of ultrasonic molecular imaging.
Background
Macrophages exhibit a continuous state of functional activation under pathological conditions, with types M1 and M2 being used to represent the two extremes of macrophage activation, with the two types of activation playing opposite roles in immune and inflammatory responses. Tumor Associated Macrophages (TAMs) are a special type of M2 type macrophages, and can promote tumor neovascularization and extracellular matrix remodeling, thereby promoting tumor generation and metastasis, being a main immune component in the tumor microenvironment and playing a vital role in the generation of primary tumors, the diffusion, seeding and proliferation of metastatic organs. Furthermore, the density of TAMs is closely related to the progression and prognosis of tumors. Given the plasticity of macrophage function, promoting its conversion from M2-type (pro-tumor) macrophages to M1-type (anti-tumor) macrophages is a better choice than directly killing TAMs. In addition, TAMs are genetically stable compared to tumor cells and therefore less resistant to chemotherapy and radiotherapy. Furthermore, TAMs are located primarily in the limbic region of the tumor stroma and are prone to interact with therapeutic drugs that are reached through the blood circulation. In summary, TAMs are important therapeutic targets in tumor treatment protocols.
Several commonly used molecules or receptors targeting TAMs include IL-4R, mannose receptor and folate receptor β (folate receptor β β), but IL-4R is also present on the surface of other immune cells than TAMs, such as T or B cells, while mannose receptor is also highly expressed in endothelial cell subsets FR β is highly expressed on the surface of human TAMs, but not or under expressed in normal tissues, thus FR β can be selected as a target for targeting TAMs.
As a Drug Delivery System (DDS), nanoscale ultrasound contrast agents have great advantages in tumor targeting therapy, can be monitored in real time in combination with medical diagnostic ultrasound, release drugs at specific times and locations, and enhance the absorption capacity of cells for drugs through ultrasound-targeted nanobubble destruction (UTND). Related researches on TAM targeted nanoscale ultrasound contrast agents are not seen at present.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a lipid nanoscale ultrasonic contrast agent for targeting tumor-associated macrophages (TAM) and a preparation method thereof.
The invention also provides application of the lipid nanoscale ultrasound contrast agent for targeting tumor-associated macrophages (TAM) in promoting conversion of the tumor-associated macrophages (TAM) from M2 type macrophages to M1 type macrophages.
Description of terms:
tumor-associated macrophages: (tumor associated macrophage, TAM), an activated type of macrophage, is the major immune component in the tumor microenvironment. TAM is a specific type of macrophage M2, and promotes tumor neovascularization, extracellular matrix remodeling, and tumor development and metastasis.
Low molecular weight hyaluronic acid: (low molecular weight hyaluronic acid, LMW-HA), hyaluronic acid with molecular weight of 3-10 kDa.
DSPE-PEG-FOL: distearoylphosphatidylethanolamine-polyethylene glycol-folic acid.
DSPE-PEG-FITC: distearoylphosphatidylethanolamine-polyethylene glycol-fluorescein isothiocyanate.
DPPC: dipalmitoylphosphatidylcholine.
HA-FOL-NB: a folic acid modified nano-scale ultrasound contrast agent carrying low molecular weight hyaluronic acid.
FOL-NB: a folic acid modified nano-scale ultrasound contrast agent.
NB: the nano-scale ultrasonic contrast agent has no targeted modification and is not carried with low molecular weight hyaluronic acid.
Room temperature: having a meaning well known in the art, typically 25. + -. 2 ℃.
The technical scheme of the invention is as follows:
a lipid nanoscale ultrasound contrast agent targeting tumor-associated macrophages takes lipid as a shell membrane material, and low-molecular-weight hyaluronic acid and gaseous fluorocarbon are wrapped inside the shell membrane; the lipid comprises Dipalmitoylphosphatidylcholine (DPPC), Distearoylphosphatidylethanolamine (DSPE), and distearoylphosphatidylethanolamine-polyethylene glycol-folic acid (DSPE-PEG-FOL); the particle size of the nanoscale ultrasonic contrast agent is 255-497 nm.
According to the invention, the mass ratio of the lipid to the low molecular weight hyaluronic acid is (5-10): 1; a further preferred mass ratio is 7.5: 1.
According to the invention, the mass ratio of the Dipalmitoylphosphatidylcholine (DPPC), the Distearoylphosphatidylethanolamine (DSPE) and the distearoylphosphatidylethanolamine-polyethylene glycol-folic acid (DSPE-PEG-FOL) is (1-7): 1-4; a further preferred mass ratio is 7:2: 4.
Preferably according to the invention, the gaseous fluorocarbon is perfluoropropane.
Preferably, according to the present invention, the nanoscale ultrasound contrast agent has an average particle size of 342 nm.
The lipid nanoscale ultrasound contrast agent targeting the tumor-related macrophages is used for targeting combination of the tumor-related macrophages by taking folic acid as a targeting ligand and folic acid receptor β on the surfaces of the tumor-related macrophages as a receptor.
In the invention, the low molecular weight hyaluronic acid in the lipid nanoscale ultrasound contrast agent for targeting tumor-associated macrophages can promote the transformation of the tumor-associated macrophages from M2 type macrophages to M1 type macrophages, and the transformation effect is not influenced by the initial activation state of the macrophages. Hyaluronic acid has excellent properties in terms of water solubility, biocompatibility, and non-immunogenicity.
The preparation method of the lipid nanoscale ultrasound contrast agent for targeting tumor-associated macrophages comprises the following steps:
(1) mixing glycerol and phosphate buffer solution to obtain a hydrated solution, mixing propylene glycol and the hydrated solution, and adding Dipalmitoylphosphatidylcholine (DPPC), Distearoylphosphatidylethanolamine (DSPE) and distearoylphosphatidylethanolamine-polyethylene glycol-folic acid (DSPE-PEG-FOL) according to a certain proportion to obtain a suspension;
(2) introducing gaseous fluorocarbon into the suspension obtained in the step (1) to displace air, and heating in a water bath until the lipid is completely dissolved to obtain emulsion;
(3) and (3) adding low-molecular-weight hyaluronic acid into the emulsion in which the lipid in the step (2) is completely dissolved in proportion, mechanically oscillating for 80-100 s, standing, taking the lower layer solution, diluting the lower layer solution with a phosphate buffer solution, centrifuging at a low speed, and taking the upper layer suspension to obtain the lipid nanoscale ultrasonic contrast agent targeting tumor-related macrophages.
According to the present invention, the volume ratio of glycerol to phosphate buffer in the hydration solution in step (1) is preferably 1: 9.
According to the invention, the volume ratio of the propylene glycol and the hydration liquid in the step (1) is 1: 1.
Preferably according to the invention, the phosphate buffer in steps (1) and (3) has the following composition: na (Na)2HPO48mM，KH2PO42mM，pH 7.2。
According to the invention, the temperature of the water bath heating in the step (2) is 50-70 ℃; further preferably 65 ℃.
According to the invention, the low-speed centrifugation in the step (3) is preferably performed at 200-500 rpm for 3-7 min; more preferably, the centrifugation is carried out at 300rpm for 5 min.
According to the present invention, the volume ratio of the subnatant to the phosphate buffer for dilution in step (3) is preferably 1: 3.
The application of the nanoscale ultrasonic contrast agent in combination with ultrasonic irradiation in promoting the conversion of tumor-associated macrophages from M2 type macrophages to M1 type macrophages is provided.
The above-mentioned processes for the preparation of the invention, which are not specifically described, are in accordance with the prior art.
The invention has the technical characteristics that:
the nano-scale ultrasonic contrast agent prepared by the invention takes folic acid as a targeting ligand and low molecular weight hyaluronic acid as a medicinal preparation, when the nano-scale ultrasonic contrast agent is prepared, three lipids, namely Dipalmitoylphosphatidylcholine (DPPC), Distearoylphosphatidylethanolamine (DSPE) and distearoylphosphatidylethanolamine-polyethylene glycol-folic acid (DSPE-PEG-FOL), are dissolved in a mixed solution of glycerol, propylene glycol and phosphate buffer solution, because the glycerol is viscous, the glycerol, the propylene glycol and the phosphate buffer solution are directly mixed to hardly obtain a uniform solution, therefore, the invention firstly prepares the hydration liquid of glycerol and phosphate buffer solution, and then mixes the hydration liquid with propylene glycol to obtain the stable and uniform mixed solution, thereby achieving the best lipid dissolution effect; in addition, in order to take account of the nano-range particle size and the ultrasound enhanced imaging function of the prepared ultrasound contrast agent, the lower layer liquid is taken out after mechanical oscillation, the nanoscale ultrasound contrast agent with the overlarge particle size is discarded, the upper layer liquid is taken out through low-speed centrifugation, the nanoscale ultrasound contrast agent with the overlarge particle size is discarded, and the particle size of the obtained nanoscale ultrasound contrast agent is 255-497 nm.
Has the advantages that:
1. the lipid nanoscale ultrasound contrast agent targeting tumor-associated macrophages (TAM) prepared by the invention is prepared by taking lipid as a shell membrane material, wrapping low molecular weight hyaluronic acid (LMW-HA) and perfluoropropane gas in the shell membrane, and HAs the particle size of 255-497 nm and the average particle size of 342nm (PDI:0.155), can penetrate through the gaps of the vascular walls of tumor tissues in a nanoscale range, HAs an enhanced permeation and retention effect, and HAs passive targeting and high efficiency for tumor treatment.
2. The lipid nanoscale ultrasonic contrast agent for targeting tumor-associated macrophages (TAM) prepared by the invention has the advantages of lasting enhanced imaging capability which can reach 20min, high biological safety, and capability of being exploded by a medical ultrasonic diagnostic apparatus to achieve the purposes of releasing the loaded drug at a designated part and promoting the absorption of the drug through UTND.
3. The lipid nanoscale ultrasound contrast agent targeting tumor-associated macrophages (TAM) prepared by the invention HAs the advantages that coupled folic acid is arranged outside a lipid shell membrane through a PEG chain, and low molecular weight hyaluronic acid (LMW-HA) is wrapped inside the lipid contrast agent, so that the nanoscale ultrasound contrast agent can be ensured to actively target the tumor-associated macrophages (TAM) in a folate-folate receptor mode, and the low molecular weight hyaluronic acid (LMW-HA) acts on the tumor-associated macrophages (TAM) to convert the tumor-associated macrophages (TAM) from M2 type macrophages to M1 type macrophages.
4. The invention can realize the target development of the nano-scale ultrasonic contrast agent by using an ultrasonic irradiation mode, thereby monitoring the tumor range in real time.
5. The lipid nanoscale ultrasound contrast agent targeting tumor-associated macrophages (TAM) prepared by the invention is combined with Ultrasound Targeted Nanobubble Destruction (UTND), so that the nanoscale ultrasound contrast agent can be selectively destroyed, and low molecular weight hyaluronic acid in the nanoscale ultrasound contrast agent is released, thereby promoting the tumor-associated macrophages (TAM) to be transformed from tumor-promoting M2 type macrophages to anti-tumor M1 type macrophages, and providing a new strategy for tumor treatment.
Drawings
FIG. 1 is a photomicrograph of a nanoscale ultrasound contrast agent; in the figure, A is an optical micrograph, and B is a fluorescence micrograph;
FIG. 2 is a particle size distribution diagram and a potential distribution diagram of a nanoscale ultrasound contrast agent; in the figure, A is a particle size distribution diagram, and B is a potential distribution diagram;
FIG. 3 is a line graph showing the variation of particle size and Polymer Dispersibility Index (PDI) of a nanoscale ultrasound contrast agent;
FIG. 4 shows the expression of mrc1, il10, nos2 and tnf genes in different concentrations of conditioned medium and at different culture times; in the figure, A is the gene expression of the culture medium with the concentration of 100% at different culture times, and B is the gene expression of the culture medium with different concentrations for 24 h;
FIG. 5 is an optical microscope photograph before and after the establishment of a tumor-associated macrophage model; in the figure, A is an optical microscope photograph before the establishment of a tumor-related macrophage model, and B is an optical microscope photograph after the establishment of the tumor-related macrophage model;
FIG. 6 is a graph illustrating the effect of different nanoscale ultrasound contrast agent materials on cell viability; in the figure, a is the effect of lipid on cell viability, with lipid concentration on the abscissa and viability on the ordinate; b is the influence of the low molecular weight hyaluronic acid on the survival rate of the cells, the abscissa is the concentration of the low molecular weight hyaluronic acid, and the ordinate is the survival rate;
FIG. 7 is an in vitro developed image of a nanoscale ultrasound contrast agent at various time points;
FIG. 8 is a graph of relative intensity changes for in vitro enhanced imaging of nanoscale ultrasound contrast agents; in the figure, A is an imaging relative intensity change curve; b is a histogram of relative intensity changes of the image;
FIG. 9 shows the targeting binding of contrast agents to tumor-associated macrophages; in the figure, A and E are fluorescence photographs of targeted combination of folic acid modified nano-scale ultrasonic contrast agent (FOL-NB) and tumor-related macrophages and flow cytometry results, B and F are fluorescence photographs of targeted combination of folic acid modified nano-scale ultrasonic contrast agent (FOL-NB) and tumor-related macrophages and flow cytometry results under the condition of competitive inhibition of free folic acid, C and G are fluorescence photographs of targeted combination of non-targeted nano-scale ultrasonic contrast agent (NB) and tumor-related macrophages and flow cytometry results, D and H are fluorescence photographs of targeted combination of non-targeted nano-scale ultrasonic contrast agent (NB) and tumor-related macrophages and flow cytometry results under the condition of competitive inhibition of free folic acid;
FIG. 10 shows the expression of mrc1, il10, nos2 and tnf genes under different treatment conditions; in the figure, the abscissa indicates the processing condition, "+" indicates the presence of the processing condition, and "-" indicates the absence of the processing condition;
FIG. 11 is a bar graph of the expression of interleukin 10(IL-10) and tumor necrosis factor (TNF- α) under different treatment conditions, wherein A is the bar graph of the expression of interleukin 10(IL-10) on the ordinate of the concentration of IL-10, and B is the bar graph of the expression of tumor necrosis factor (TNF- α) on the ordinate of the concentration of TNF- α;
FIG. 12 shows the results of flow cytometry on the expression level of the M2-type macrophage surface specific receptor CD206 under different treatment conditions.
Detailed Description
The present invention is further illustrated by the following examples, but the scope of the invention is not limited thereto.
Distearoylphosphatidylethanolamine-polyethylene glycol-folic acid (DSPE-PEG-FOL) was purchased from Avanti corporation under product number 880124P; dipalmitoylphosphatidylcholine (DPPC) was purchased from Sigma-Aldrich, product No. P0763; distearoylphosphatidylethanolamine (DSPE) was purchased from Sigma-Aldrich, product number P3531; distearoylphosphatidylethanolamine-polyethylene glycol-fluorescein isothiocyanate (DSPE-PEG-FITC) was purchased from sienna ruixi, product No. R0055; low molecular weight hyaluronic acid (LMW-HA) was purchased from Huaxi Furida Bio Inc., product number 9004-61-9.
Example 1: preparation of lipid nano-scale ultrasonic contrast agent
A preparation method of a lipid nanoscale ultrasound contrast agent targeting tumor-associated macrophages comprises the following steps:
(1) uniformly mixing glycerol and phosphate buffer solution in a volume ratio of 1:9 to prepare a hydrated solution, putting 0.4mL of propylene glycol and 0.4mL of the hydrated solution into a 1.5mL of EP tube, adding lipid, wherein the lipid is 0.7mg of Dipalmitoylphosphatidylcholine (DPPC), 0.2mg of Distearoylphosphatidylethanolamine (DSPE), 0.4mg of distearoylphosphatidylethanolamine-polyethylene glycol-folic acid (DSPE-PEG-FOL), and in addition, in order to observe the structure of the nano-scale ultrasonic contrast agent under a fluorescence microscope, adding 0.1mg of distearoylphosphatidylethanolamine-polyethylene glycol-fluorescein isothiocyanate (DSPE-PEG-FITC) to obtain a suspension;
(2) introducing perfluoropropane into the suspension obtained in the step (1) to displace air, placing an EP tube in a constant-temperature water bath kettle at 65 ℃ and heating until the lipid is completely dissolved to obtain emulsion;
(3) and (3) adding 0.2mL of low molecular weight hyaluronic acid (LMW-HA) solution with the concentration of 1mg/mL into the emulsion after the lipid is completely dissolved in the step (2), replacing air with perfluoropropane again, mechanically oscillating for 90s, standing, taking 0.5mL of lower-layer liquid, transferring into a 15mL centrifuge tube, adding phosphate buffer solution to 2mL, centrifuging at 300rpm for 5min, and taking the upper-layer suspension to obtain the lipid nanoscale ultrasonic contrast agent for targeting tumor-related macrophages.
Wherein, the phosphate buffer solution comprises the following components: na (Na)2HPO48mM，KH2PO42mM，pH 7.2。
The prepared nanoscale ultrasonic contrast agent is diluted and then is dripped on a glass slide, the apparent morphology of the contrast agent is observed through an optical microscope and a fluorescence microscope, and the result is shown in figure 1, the nanoscale ultrasonic contrast agent is circular under a 1000X optical microscope (figure 1A), is uniformly dispersed without aggregation, and has a definite core-shell structure; under a fluorescence microscope (fig. 1B), the shell of the nanoscale ultrasound contrast agent appears green, confirming that lipids (including DPPC, DSPE, and DSPE-PEG2000-FOL) constitute the shell of the nanoscale ultrasound contrast agent.
The prepared nanoscale ultrasonic contrast agent is diluted and then the particle size and the potential of the nanoscale ultrasonic contrast agent are detected by using a nanometer laser particle size analyzer, and the result is shown in figure 2, wherein the particle size of the nanoscale ultrasonic contrast agent is 255-497 nm, the average particle size is 342nm (polymer dispersibility index PDI:0.155), and the zeta potential is-10.05 mV.
The prepared nano-scale ultrasonic contrast agent is mixed with an RPMI 1640 culture medium containing 10% fetal calf serum, the mixture is placed at 4 ℃ for standing, the nano-scale ultrasonic contrast agent is taken out at different time points (1, 2, 4, 6, 8 and 10 hours), the particle size and PDI of the nano-scale ultrasonic contrast agent are detected by using a nano laser particle size analyzer after dilution, and the result is shown in figure 3, the particle size of the nano-scale ultrasonic contrast agent is not obviously changed within 4 hours, the nano-scale ultrasonic contrast agent has good stability, and the PDI of the nano-scale ultrasonic contrast agent is within 0.4, which indicates that the particle size of the prepared nano-scale ultrasonic contrast agent is uniform.
Example 2: establishing tumor-related macrophage model
Establishing a tumor-associated macrophage model by culturing RAW264.7 macrophages in a conditioned medium, and in order to verify that RAW264.7 macrophages are successfully activated into tumor-associated macrophages, observing morphological changes of the tumor-associated macrophages by using an optical microscope, and determining whether the expression of tumor-associated macrophage-associated genes mrc1, il10, nos2 and tnf is up-regulated or not by using real-time fluorescence quantitative polymerase chain reaction. The culture supernatant of mouse Lewis lung cancer cells was collected and filtered through a 0.22 μm membrane filter to obtain a conditioned medium. To optimize the conditions for RAW264.7 macrophage activation as tumor-associated macrophages, RAW264.7 macrophages were treated and genetically tested at different time points (0, 24, 48, 72h) using different concentrations (0%, 50%, 100%) of conditioned media.
The expression of related genes of RAW264.7 macrophages treated by the condition culture medium with the concentration of 100% at different time points is shown in figure 4A, the expression of related genes of RAW264.7 macrophages treated by the condition culture medium with different concentrations for 24h is shown in figure 4B, and as can be seen from figure 4, the expression of related genes of tumor-related macrophages is obviously increased along with the increase of the concentration of the condition culture medium and the prolonging of the culture time, which proves that a tumor-related macrophage model is successfully established.
Finally, RAW264.7 macrophage is cultured for 48h by using a condition culture medium with the concentration of 100%, and RAW264.7 macrophage is activated into tumor-related macrophage.
FIG. 5 is an optical micrograph of tumor-associated macrophages before and after modeling, showing that the tumor-associated macrophages are more voluminous and appear more "tentacles" than unactivated macrophages.
Example 3: evaluation of biological safety of nano-scale ultrasonic contrast agent
The tumor-associated macrophages obtained in example 2 were measured at 1X 104Inoculating into 96-well plate, adding culture medium, and culturing at 37 deg.C with 5% CO2After 24h incubation, two experiments were performed, the first, in which the medium for tumor-associated macrophages was replaced with fresh medium containing lipidsThe concentrations of lipids in fresh medium were 25, 50, 100, 200, 300 μ g/mL, respectively, and the lipids contained were divided into two cases, one was Dipalmitoylphosphatidylcholine (DPPC), Distearoylphosphatidylethanolamine (DSPE) and distearoylphosphatidylethanolamine-polyethylene glycol-folic acid (DSPE-PEG-FOL) in a mass ratio of 7:2:4, and one was Dipalmitoylphosphatidylcholine (DPPC) and Distearoylphosphatidylethanolamine (DSPE) in a mass ratio of 7: 6; in the second group of experiments, the culture medium of the tumor-associated macrophages is replaced by a fresh culture medium containing low molecular weight hyaluronic acid (LMW-HA), and the concentrations of the low molecular weight hyaluronic acid (LMW-HA) in the fresh culture medium are respectively 10, 50, 100, 500, 1000 and 2000 mug/mL; after medium exchange, 5% CO at 37 ℃2And (3) incubating for 48h, replacing the culture medium with a fresh culture medium containing 10 mu L of CCK-8, continuing incubating for 1.5h, detecting the light absorption value of the culture medium at the wavelength of 450nm by using an enzyme-labeling instrument, and calculating the cell survival rate.
The calculation result is shown in fig. 6, and the result shows that when the lipid concentration is more than or equal to 200 mug/mL, the cell activity is obviously inhibited, and when the common lipid concentration is 50 mug/mL, no obvious cytotoxicity (the cell survival rate is more than 90%) appears, which indicates that the biological safety of the lipid material nano-scale ultrasonic contrast agent is high; in addition, the low molecular weight hyaluronic acid (LMW-HA) HAs no obvious toxicity to tumor-related macrophages, and the survival rate of the low molecular weight hyaluronic acid exceeds 80 percent.
Example 4: in vitro imaging capability determination of nano-scale ultrasonic contrast agent
The nano-scale ultrasound contrast agent prepared in example 1 was placed in a finger cut from a plastic glove and clamped with a dialysis clamp to maintain tension, and then the plastic finger was fixed in a container containing degassed water at 37 ℃ and observed in a contrast mode of a gelogqe 9 ultrasound diagnostic apparatus, frequency 9.0MHz, Mechanical Index (MI)0.16, dynamic range 60dB, two-dimensional and ultrasound contrast modes in synchronization with degassed water as a control group.
Ultrasound contrast enhanced imaging was performed for 30 minutes using the nanoscale ultrasound contrast agent prepared in example 1, and video of the contrast enhanced imaging was recorded at different time points (0, 1, 2, 5, 10, 20, 30 minutes). At 30 minutes, the nanoscale ultrasound contrast agent was blasted by ultrasound irradiation using a "blast" button (flash) on the ultrasound scanner.
10 seconds of video were recorded at each time point and analyzed by a "TIC analysis" function in the ultrasound scanner. The in vitro enhanced imaging ability of the nanoscale ultrasound contrast agent prepared in example 1 is expressed by relative intensity, i.e., the dB value of the region of interest in the same frame image minus the dB value of the background.
The in vitro development results of the nanoscale ultrasound contrast agent at different time points are shown in fig. 7, the relative intensity change of in vitro enhanced imaging of the nanoscale ultrasound contrast agent is shown in fig. 8, the nanoscale ultrasound contrast agent has higher contrast intensity within 10 minutes of continuous ultrasound irradiation, and the contrast intensity is slightly reduced compared with the initial intensity after 10 minutes, which indicates that the prepared nanoscale ultrasound contrast agent has excellent in vitro contrast imaging capability. At the 30 th minute, after pressing a 'burst' (flash) button on the ultrasonic scanner, the contrast intensity of the image of the region of interest is obviously enhanced and lasts for several seconds, and then the image is rapidly changed into a state with almost no enhancement, which shows that after the 'burst', the contrast intensity of the image is obviously lower than that of the 'burst' and before the 'burst', and most of the nano-scale ultrasonic contrast agents are proved to be damaged by ultrasonic.
Example 5: targeted ability analysis of nanoscale ultrasound contrast agents
The tumor-associated macrophages obtained from the culture of example 2 were increased by 1X 106Inoculating each hole in a 6-hole plate, adding a folic acid modified nano-scale ultrasonic contrast agent (FOL-NB) when the cells grow to 50-70% of the density, incubating and culturing for 30min, and washing away the unbound nano-scale ultrasonic contrast agent by using a phosphate buffer solution; meanwhile, free folic acid is used as a competitive inhibitor, and a non-targeted nano-scale ultrasound contrast agent (NB) is used as a control; the targeting ability of the nano-scale ultrasonic contrast agent is analyzed by a fluorescence microscope and a flow cytometer respectively.
Wherein, the folic acid modified nano-scale ultrasonic contrast agent (FOL-NB) is prepared according to the preparation method of the nano-scale ultrasonic contrast agent of example 1, except that low molecular weight hyaluronic acid (LMW-HA) is not added; a targetless nanoscale ultrasound contrast agent (NB) was prepared according to the method of example 1, except that distearoylphosphatidylethanolamine-polyethylene glycol-folic acid (DSPE-PEG-FOL) and low molecular weight hyaluronic acid (LMW-HA) were not added.
Fluorescence microscope and flow cytometer results show: targeted binding of folate-modified nanoscale ultrasound contrast agent (FOL-NB) to tumor-associated macrophages was stronger compared to targetless nanoscale ultrasound contrast agent (NB) (fig. 9A, 9E, 9C, 9G, 10.6% v.s.4.31%); targeted binding of folate-modified nanoscale ultrasound contrast agent (FOL-NB) to tumor-associated macrophages was reduced after competitive inhibition by free folate (fig. 9A, 9E, 9B, 9F, 10.6% v.s.6.45%), whereas binding of untargeted nanoscale ultrasound contrast agent (NB) to tumor-associated macrophages was not affected by free folate (fig. 9C, 9G, 9D, 9H, 4.31% v.s.4.19%). The folic acid modified nano-scale ultrasonic contrast agent (FOL-NB) can actively target tumor-associated macrophages through the combination of folic acid on the shell of the nano-scale ultrasonic contrast agent and a folic acid receptor.
Example 6: transformation of tumor-associated macrophages from M2-type macrophages to M1-type macrophages is promoted by combining nanoscale ultrasonic contrast agent with ultrasonic irradiation
The tumor-associated macrophages obtained from the culture of example 2 were increased by 1X 106Inoculating each cell/well in a 6-well plate, and performing different treatments when the cells grow to 50% -70% of the density, wherein the treatment comprises the following steps: the medium was changed to fresh medium containing low molecular weight hyaluronic acid (LMW-HA group); the medium was replaced with fresh medium containing the nanoscale ultrasound contrast agent prepared in example 1 (HA-FOL-NB group); the medium was replaced with fresh medium containing low molecular weight hyaluronic acid and subjected to ultrasonic irradiation (US + LMW-HA group); the medium was replaced with fresh medium containing the nanoscale ultrasound contrast agent prepared in example 1 and subjected to ultrasound irradiation (US + HA-FOL-NB group); fresh medium was used as CONTROL (CONTROL group), and the ultrasonic irradiation parameters were: sound intensity 1.0W/cm2And time 30 s. After medium replacement, 5% CO at 37 ℃2Continuously culturing for 48h, and detecting M1 type and M2 type macrophage related by real-time fluorescence quantitative polymerase chain reactionAnd (3) detecting the change of the expression level of the related protein by using a flow cytometry and enzyme-linked immunosorbent assay kit.
The expression of macrophage-related genes of M1 type and M2 type is shown in FIG. 10, wherein nos2 and tuf are macrophage-related genes of M1 type, and mrc1 and il10 are macrophage-related genes of M2 type, and the results show that the gene expression of nos2 and tnf in the US + HA-FOL-NB group is obviously increased, and the gene expression of mrc1 and il10 is obviously reduced.
The results of enzyme-linked immunosorbent assay of M2 type macrophage-associated cytokine interleukin 10(IL-10) are shown in FIG. 11A, and the results of enzyme-linked immunosorbent assay of M1 type macrophage-associated cytokine tumor necrosis factor (TNF- α) are shown in FIG. 11B, and the results show that US + HA-FOL-NB can significantly reduce the content of M2 type macrophage-associated cytokine interleukin 10 and increase the content of M1 type macrophage-associated cytokine tumor necrosis factor when treating tumor-associated macrophages.
The expression level of the M2 type macrophage surface specific receptor CD260 is detected by adopting flow cytometry, the detection result is shown in figure 12, and the expression of the specific receptor CD206 of the US + HA-FOL-NB group is obviously reduced.
The above results show that compared with the simple application of low molecular weight hyaluronic acid (LMW-HA), the combination of ultrasonic irradiation and low molecular weight hyaluronic acid (LMW-HA) only promotes the down-regulation of IL0 gene and specific receptor CD206, while HA-FOL-NB only down-regulates the expression of cytokine IL-10. Therefore, ultrasonic irradiation or HA-FOL-NB is not used for obviously promoting the transformation of the tumor-associated macrophage, but the lipid nanoscale ultrasonic contrast agent (HA-FOL-NB) targeting the tumor-associated macrophage prepared by the invention can be combined with the ultrasonic irradiation to obviously promote the transformation of the tumor-associated macrophage from M2 type macrophage to M1 type macrophage.
Claims (10)
1. A lipid nanoscale ultrasound contrast agent targeting tumor-associated macrophages is characterized in that lipid is used as a shell membrane material, and low-molecular-weight hyaluronic acid and gaseous fluorocarbon are wrapped inside the shell membrane; the lipid comprises Dipalmitoylphosphatidylcholine (DPPC), Distearoylphosphatidylethanolamine (DSPE), and distearoylphosphatidylethanolamine-polyethylene glycol-folic acid (DSPE-PEG-FOL); the particle size of the nanoscale ultrasonic contrast agent is 255-497 nm.
2. The lipid nanoscale ultrasound contrast agent for targeting tumor-associated macrophages according to claim 1, wherein the mass ratio of the lipid to the low molecular weight hyaluronic acid is (5-10): 1.
3. The lipid nanoscale ultrasound contrast agent for targeting tumor-associated macrophages as claimed in claim 1, wherein the mass ratio of Dipalmitoylphosphatidylcholine (DPPC), Distearoylphosphatidylethanolamine (DSPE) and distearoylphosphatidylethanolamine-polyethylene glycol-folic acid (DSPE-PEG-FOL) is (1-7): (1-4).
4. The lipid nanoscale ultrasound contrast agent for targeting tumor-associated macrophages according to claim 1, wherein said gaseous fluorocarbon is perfluoropropane.
5. The lipid nanoscale ultrasound contrast agent for targeting tumor-associated macrophages according to claim 1, wherein the nanoscale ultrasound contrast agent has an average particle size of 342 nm.
6. The preparation method of the lipid nanoscale ultrasound contrast agent targeting tumor-associated macrophages, according to any one of claims 1 to 5, comprising the following steps:
(1) mixing glycerol and phosphate buffer solution to obtain a hydrated solution, mixing propylene glycol and the hydrated solution, and adding Dipalmitoylphosphatidylcholine (DPPC), Distearoylphosphatidylethanolamine (DSPE) and distearoylphosphatidylethanolamine-polyethylene glycol-folic acid (DSPE-PEG-FOL) according to a certain proportion to obtain a suspension;
(2) introducing gaseous fluorocarbon into the suspension obtained in the step (1) to displace air, and heating in a water bath until the lipid is completely dissolved to obtain emulsion;
(3) and (3) adding low-molecular-weight hyaluronic acid into the emulsion in which the lipid in the step (2) is completely dissolved in proportion, mechanically oscillating for 80-100 s, standing, taking the lower layer solution, diluting the lower layer solution with a phosphate buffer solution, centrifuging at a low speed, and taking the upper layer suspension to obtain the lipid nanoscale ultrasonic contrast agent targeting tumor-related macrophages.
7. The method according to claim 6, wherein the volume ratio of glycerol to phosphate buffer in the aqueous solution in step (1) is 1: 9;
preferably, the volume ratio of the propylene glycol to the hydration liquid in the step (1) is 1: 1.
8. The method according to claim 6, wherein the phosphate buffer in steps (1) and (3) has a composition of: na (Na)2HPO48mM，KH2PO42mM，pH 7.2；
Preferably, the temperature of the water bath heating in the step (2) is 50-70 ℃.
9. The method according to claim 6, wherein the low-speed centrifugation in the step (3) is performed at 200 to 500rpm for 3 to 7 min;
preferably, the volume ratio of the subnatant to the phosphate buffer for dilution in step (3) is 1: 3.
10. Use of the nanoscale ultrasound contrast agent as claimed in any one of claims 1 to 5 in combination with ultrasound irradiation to promote the conversion of tumor-associated macrophages from M2-type macrophages to M1-type macrophages.
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Application publication date: 20200515 |