CN116196437A - Preparation of erythrocyte-loaded oncolytic virus intravenous delivery preparation and anti-tumor application thereof - Google Patents
Preparation of erythrocyte-loaded oncolytic virus intravenous delivery preparation and anti-tumor application thereof Download PDFInfo
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- CN116196437A CN116196437A CN202310156570.9A CN202310156570A CN116196437A CN 116196437 A CN116196437 A CN 116196437A CN 202310156570 A CN202310156570 A CN 202310156570A CN 116196437 A CN116196437 A CN 116196437A
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Abstract
The preparation method comprises the steps of wrapping the oncolytic virus by using polyethyleneimine, and then adsorbing the oncolytic virus wrapped by the polyethyleneimine on the surface of the erythrocytes through electrostatic effect, so that the erythrocytes are loaded with the oncolytic virus, and the oncolytic virus is used for intravenous injection delivery to achieve the effect of resisting tumor. The oncolytic virus which is wrapped by the polyethylenimine and loaded by the red blood cells can effectively help the oncolytic virus to evade phagocytosis by a mononuclear macrophage system in the internal circulation process, prolong the circulation time of the oncolytic virus in peripheral blood, improve the targeted enrichment of the oncolytic virus on lung metastasis tumor, and further improve the curative effect of the oncolytic virus on lung metastasis; the erythrocyte load can also obviously reduce the blood circulation and the organ toxicity caused by the oncolytic virus, thereby improving the biosafety of intravenous injection of the oncolytic virus as an antitumor drug.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a preparation method of an intravenous delivery preparation of an erythrocyte-loaded oncolytic virus capable of being subjected to intravenous injection, and an anti-tumor application of the preparation.
Background
In recent decades, cancer has become a disease with high morbidity and mortality worldwide, and although various therapeutic strategies such as radical surgery, chemotherapy, radiotherapy, targeting and immunotherapy are applied to cancer, prognosis of cancer is improved to some extent, but there are many malignant tumors with poor therapeutic effects. With rapid developments in modern molecular biology, molecular immunology, etc., biological treatment of tumors has become the fourth largest therapeutic modality following surgery, radiation therapy and chemotherapy. As an important biotherapeutic strategy, oncolytic Viruses (OV) are of great interest due to good tumor killing effect and high selectivity. Oncolytic viruses are a brand new therapeutic drug aiming at malignant tumors clinically at present, and are mainly genetically modified by viruses with weaker pathogenicity in nature, including herpesviruses, poxviruses, adenoviruses and the like. Under the condition of ensuring safety, the oncolytic virus can specifically infect and kill tumor cells so as to achieve the anti-tumor effect. In addition to direct lysis of cells, oncolytic viruses can also stimulate anti-tumor immune responses. In one aspect, oncolytic viruses are capable of directly affecting tumor immunity, and infected tumor cells elicit an Interferon or Toll-like receptor response, or after immune recognition, initiate a cascade of responses through tumor necrosis factor (tumor necrosis factor, TNF) and Interferon IFN (IFN) -related factors, as well as retinoic acid inducing gene 1, thereby activating the JAK/STAT pathway and thus activating protein kinase R. The latter is able to sense intracellular viral components and prevent protein transcription, ultimately promoting apoptosis. Oncolytic viruses, on the other hand, are capable of modulating the tumor microenvironment and enhancing the enrichment of immune cells in tumor tissue. Oncolytic viruses can target capillary endothelial cells, tumor-related fibroblasts and the like which are related to the development of the oncolytic viruses in the tumor microenvironment and cause the oncolytic viruses to lyse, so that the tumor immune microenvironment is directly regulated. Oncolytic viruses are also capable of inducing tumor cell lysis and release specific tumor-associated antigens, promoting T cell recognition of tumor antigens and thus establishing tumor-specific T cell immunity. Meanwhile, oncolytic viruses can inhibit enrichment of regulatory T cells and myeloid-derived suppressor cells in an immune microenvironment, thereby overcoming tumor immunosuppression in a tumor microenvironment. Oncolytic viruses are also capable of recruiting and activating immune cells such as dendritic cells and natural killer cells by inducing cytokines such as IFN-alpha, IL-12, TNF-alpha and IL-6, and factors such as lesion-related molecular patterns (damage associated molecular patterns, DAMPs), pathogen-related molecular patterns (PAMP), and the like, thereby improving infiltration of immune cells in tumor tissues. In addition, the oncolytic virus has the functions of inhibiting tumor angiogenesis, improving the curative effect of cytotoxic T lymphocyte-associated protein 4 (cytotoxin T-lymphocyte-associated protein, CTLA-4) or programmed death receptor 1 (programmed cell death protein, PD-1) inhibitors, and the like. Currently, a variety of oncolytic viruses have been approved for clinical use worldwide, two of which are the more prominent adenovirus type 5 (Ad 5) H101 approved in China in 2005 and the U.S. and European approved T-VEC viruses available for intratumoral and pleuroperitoneal injection.
With the recent intensive research into oncolytic virus anti-tumor, the development of biological treatment of tumors using oncolytic viruses has been further promoted. Oncolytic viruses need to be delivered to and replicated in tumor tissue by appropriate administration to produce an anti-tumor effect. At present, the main modes of oncolytic virus delivery are percutaneous intratumoral injection and intravenous injection, but the main modes have larger problems; although the oncolytic virus can increase the virus concentration at the tumor part by percutaneous intratumoral injection, the virus is severely limited in penetration capacity under the combined action of the highly compact interstitial tissue of the tumor tissue and the high interstitial pressure of the tumor microenvironment during intratumoral injection, and the treatment effect is difficult to ensure. In addition, percutaneous intratumoral injection administration often requires the use of ultrasound, computed tomography, nuclear magnetic resonance imaging and other techniques to assist in guiding the administration, which is cumbersome and complex to operate, has poor patient compliance, and is costly and risky to operate with bleeding, perforation and the like.
Intravenous injection delivery of oncolytic viruses is the most convenient delivery mode at present, overcomes the practical operation difficulty and poor patient compliance caused by intratumoral injection, and can effectively deliver oncolytic viruses to all parts of the whole body, but has obvious defects; first, as with the intravenous delivery of other drugs, oncolytic virus is easily associated with other systemic organs during transport from the injection site to the tumor site, causing serious side effects such as severe organ toxicity and Cytokine Release Syndrome (CRS). In addition, since oncolytic viruses often need multiple administrations to achieve the purpose of treatment, multiple administrations can easily cause a large amount of virus neutralizing antibodies and complements to be generated in serum of patients, and the neutralization effect of the antibody complements and the phagocytosis and clearance effect of immune cells such as macrophages in the peripheral circulatory system of human bodies on the viruses can greatly influence the long circulation time and efficiency of intravenous delivery of the viruses, thereby influencing the curative effect. In addition, for primary lung cancer and lung metastases from most tumors, oncolytic virus therapy by intratumoral injection is particularly difficult due to the presence of multiple and deep microscopic tumor foci.
Disclosure of Invention
Aiming at the existing delivery mode of injecting oncolytic virus in percutaneous tumor, the existing delivery mode has severely limited penetration capacity, more auxiliary equipment, complicated and complex operation, bleeding and perforation risks and high cost; and the intravenous oncolytic virus delivery mode has the problems that oncolytic viruses are easy to combine with other organs of tissues of the whole body to cause serious side effects, and multiple administration can easily cause a large number of virus neutralizing antibodies and complements in serum of a patient, and the phagocytosis and clearance of immune cells on viruses influence the long circulation time and efficiency of intravenous delivery of the viruses, thereby influencing the curative effect. The invention provides a preparation method and an anti-tumor application of an erythrocyte-loaded oncolytic virus intravenous delivery preparation, which is characterized in that an oncolytic virus intravenous delivery preparation system is designed to comprise oncolytic virus, polyethylenimine (PEI) and erythrocytes, the surface of the oncolytic virus is coated by the polyethylenimine, and then the oncolytic virus coated by the PEI is adsorbed on the surface of the erythrocytes through electrostatic action, so that the oncolytic virus coated by the erythrocytes is prevented from being phagocytized by a mononuclear macrophage system in an in-vivo circulation process, the circulation time of the oncolytic virus in peripheral blood is prolonged, the delivery efficiency of the oncolytic virus to a lung metastatic tumor part is improved, the visceral toxicity and systemic cytokine release syndrome caused by the off-target effect of the oncolytic virus are obviously reduced, and the anti-tumor curative effect of the oncolytic virus is improved. The specific technical scheme is as follows:
a method for preparing an erythrocyte-loaded oncolytic virus intravenous delivery formulation, comprising the steps of:
s1: dissolving polyethyleneimine in PBS solution to prepare polyethyleneimine solution;
s2: adding oncolytic viruses into the polyethyleneimine solution, uniformly mixing, and adsorbing the polyethyleneimine on the surface of the oncolytic viruses through electrostatic action to prepare a polyethyleneimine-coated oncolytic virus suspension;
s3: uniformly mixing the polyethyleneimine-coated oncolytic virus suspension with the erythrocyte suspension, standing, and adsorbing the polyethyleneimine-coated oncolytic virus on the surface of erythrocytes through electrostatic action to prepare the erythrocyte-loaded oncolytic virus suspension, namely the erythrocyte-loaded oncolytic virus intravenous delivery preparation.
In the above method S1, the concentration of the polyethyleneimine contained in the polyethyleneimine solution is 100 to 300ug/ml.
In S1 of the above method, the PBS solution is a phosphate buffer solution.
In the method S1, the molecular weight of the polyethyleneimine is M.W.800-10000.
In S2 of the above method, the oncolytic virus is adenovirus subtype 11 (AD 11).
In the above method S2, the above-mentioned polyethyleneimine solution is mixed with an oncolytic virus, wherein the number of polyethyleneimine molecules is the number of oncolytic virus particles= (5000 to 50000): 1, proportioning.
In the above method S3, the red blood cells are human or mouse red blood cells.
In the S3 of the method, the oncolytic virus suspension coated by polyethyleneimine is mixed with the erythrocyte suspension according to the ratio of the number of the erythrocytes to the number of oncolytic virus particles=1 (10-20).
In the above method S3, the standing time is 40 to 60 minutes, and the standing temperature is room temperature.
The erythrocyte-loaded oncolytic virus intravenous delivery preparation prepared by the method is used for intravenous injection delivery of oncolytic viruses to achieve the anti-tumor effect.
The prepared erythrocyte-loaded oncolytic virus intravenous delivery formulation was tested using the following method:
(1) Erythrocyte-loaded oncolytic viruses were characterized using a Zeta/laser particle sizer, scanning electron microscope, and laser confocal microscope.
(2) In vitro level the effect of erythrocyte-loaded oncolytic viruses against cellular uptake of the virus was verified by cell uptake experiments.
(3) And (3) researching the circulation condition of the erythrocyte-loaded oncolytic virus in vivo and the biological distribution condition in vivo by adopting a drug metabolism detection experiment, a qPCR experiment and a model mouse living body imaging experiment.
(4) The biochemical detection of blood and the in-vivo safety detection experiment are adopted to research the strength of toxic reaction caused by the preparation in vivo, and the improvement effect of erythrocyte load on the biological safety of oncolytic viruses in the in-vivo circulation process is verified.
(5) The antitumor effect of the preparation was evaluated by examining the fluorescence intensity of the metastasis.
Compared with the prior art, the preparation method and the antitumor application of the erythrocyte-loaded oncolytic virus intravenous delivery preparation have the beneficial effects that:
1. according to the invention, erythrocytes are used as vectors of oncolytic viruses, and firstly, compared with the few DC, NK or T cells, the erythrocytes are easy to obtain and have sufficient quantity for transformation. Second, in case of allogeneic cell therapy, the immunogenicity of erythrocytes is determined only by blood type. The Rh-O type red blood cells with rare blood types can be infused into other various blood types, the application range reaches more than 95%, and rejection reaction is basically not needed to be considered. Third, the distribution of erythrocytes is concentrated and essentially limited to the vascular system and reticuloendothelial system such as the liver and spleen. Therefore, it is an excellent targeting vector for drugs that need to act in blood or liver and spleen. Fourth, erythrocytes have a defined life span (120 days), with a long duration of action. Fifth, the mature red blood cells do not have cell nuclei and do not have the capability of cell proliferation, so that the risk of canceration is avoided; the deletion of organelles and cytoskeleton simplifies the internal structure of the red blood cells, changes more capacity and space, and can carry more oncolytic viruses, thereby improving the transportation capacity of the red blood cells. When erythrocytes are used as oncolytic virus vectors, when oncolytic viruses are attached to the surfaces of erythrocytes in a non-firm combination manner to participate in blood circulation, the enrichment degree of oncolytic viruses in the lung can be remarkably increased. In addition, erythrocytes are used as a body component and have complete biocompatibility and complete biodegradability. Compared with the nanotechnology with the general safety problem, the technology has incomparable safety advantages naturally.
2. According to the method, the surface of the oncolytic virus is coated by the polyethyleneimine, and erythrocytes are used for loading, and the experiment of in-vitro anti-macrophage uptake and the experiment of drug metabolism detection of the preparation in plasma after in-vivo administration prove that the oncolytic virus loaded by the erythrocytes after the polyethyleneimine coating has higher capacity of escaping from phagocytosis of macrophages, and the polyethyleneimine coating and the erythrocyte loading have good shielding effect on the virus, so that the drug quantity reaching a focus and the drug effect are better.
3. The invention proves that the toxicity of the polyethyleneimine coated and erythrocyte loaded oncolytic virus delivered by vein is obviously reduced and the biological safety is higher through the blood biochemical detection and the safety detection experiments after the internal injection of preparations such as visceral organ safety pathological section and the like.
4. According to the invention, through the distribution detection of the erythrocyte-loaded oncolytic virus intravenous delivery preparation and the curative effect detection of the preparation aiming at a metastatic tumor model, the anti-tumor capability of the erythrocyte-loaded oncolytic virus in vivo level is proved to be very good. Compared with common oncolytic viruses, the erythrocyte-loaded oncolytic virus intravenous delivery preparation has longer circulation time in peripheral blood, stronger enrichment capacity on lung metastasis sites and better anti-metastasis curative effect after intravenous injection.
In conclusion, the polyethyleneimine is adopted for wrapping, and erythrocytes serving as oncolytic virus carriers can effectively help oncolytic viruses to escape from phagocytosis by a mononuclear macrophage system in an in vivo circulation process, so that the circulation time of the oncolytic viruses in peripheral blood is prolonged, the targeted enrichment of the oncolytic viruses on lung metastasis tumors is improved, and the anti-lung metastasis curative effect of the oncolytic viruses is further improved; meanwhile, as the red blood cells are used as a part of the self-constituent sources, the blood circulation and organ toxicity caused by the oncolytic virus can be obviously reduced, so that the biosafety of intravenous injection of the oncolytic virus as an antitumor drug is improved. Therefore, the new strategy of intravenous administration of the oncolytic virus, which is simple and easy to implement and can simultaneously improve the biocompatibility and the treatment effect of the oncolytic virus, has great significance in specifically treating the primary and metastatic lung cancer.
Drawings
FIG. 1 is a schematic diagram of a process for preparing an erythrocyte-loaded oncolytic virus intravenous delivery formulation: the Polyethylenimine (PEI) is dissolved in Phosphate Buffer Solution (PBS) to form PEI solution, the PEI solution is incubated with adenovirus 11 subtype (AD 11) to enable electropositive PEI to be adsorbed on the surface of the negatively charged AD11 to obtain PEI-coated AD11 (hereinafter referred to as PEI-AD 11), and then PEI-AD11 is incubated with red blood cell suspension (RBC) to enable electropositive PEI-AD11 to be adsorbed on the surface of electronegative red blood cells to obtain red blood cell-loaded PEI-AD11 (hereinafter referred to as RBC-PEI-AD 11).
FIG. 2 is a schematic representation of the construction and characterization detection of erythrocyte-loaded AD11; wherein, diagram a: detecting particle size diagrams of ordinary AD11 (NakedAD 11) and PEI-AD11 by using a Zeta/laser particle sizer; graph B: the laser confocal microscope confirms a successful electron microscope image of the load of red blood cells to AD11, wherein AD11 is marked by red fluorescent Cy5.5 dye, and the red blood cells are cell groups in a biconcave disc shape under a bright field; graph C: detecting a successful load pattern of red blood cells on AD11 in a flow mode; graph D: scanning Electron Microscopy (SEM) images of the erythrocyte-loaded oncolytic virus intravenous delivery formulation were taken, confirming successful formulation preparation; * P <0.001.
FIG. 3 is a bar graph of the in vitro functional assay of erythrocyte-loaded AD11, taken up by macrophages; after incubating 3 groups of formulations (NakedAD 11, PEI-AD11 and RBC-PEI-AD 11) with RAW264.7 cells (murine macrophage cell line), respectively, the uptake of each group of formulations by macrophages was examined, and the results showed minimal uptake of RBC-PEI-AD11 by macrophages; * P <0.05, P <0.01.
FIG. 4 is an in vivo functional assay and in vivo profile of an erythrocyte-loaded oncolytic virus intravenous delivery formulation; wherein, diagram a: after intravenous injection of the 3 groups of preparations (NakedAD 11, PEI-AD11 and RBC-PEI-AD 11), the virus content in peripheral blood at each moment, and the ID% represents "initial%; graph B: quantitative detection of distribution patterns of each group of preparations in TC-1 lung metastasis mice by qPCR experiments; graph C: quantitative detection of distribution patterns of each group of preparations in the lungs of TC-1 lung metastasis mice is carried out by qPCR experiments; graph D: AD11 is marked by using Cy5.5-NHS, AD11-Cy5.5 is constructed, distribution patterns of each group of preparations in the lungs of mice with TC-1 lung metastasis are detected, and G1: RBC-PEI-AD11; and G2: PEI-AD11; and G3: nakedAD11; * P <0.01, P <0.001, "ns" represents no statistical difference.
Figure 5 is an in vivo safety level assay for erythrocyte-loaded oncolytic virus intravenous delivery formulations. Graphs a-C: group 3 formulations (nakedAD 11, PEI-AD11 and RBC-PEI-AD 11) showed no statistical difference in blood levels of various inflammatory factors in mice after 72h of intravenous injection with P <0.05, P <0.01, ns.
FIG. 6 is a graph showing the detection of the anti-pulmonary metastasis efficacy of each group of preparations; wherein, diagram a: the pharmacodynamic experimental flow chart of the anti-lung metastasis of each group of preparations. Graph B: the in vivo imaging system of small animals detects the growth of lung metastasis. Graph C: end point lung metastasis solid pictures of each group of pharmacodynamic experiments. Graph D: quantification of pulmonary metastatic nodules in model mice of each group; g1: RBC-PEI-AD11; and G2: PEI-AD11; and G3: nakedAD11; and G4: PBS.
Detailed Description
The invention will be further described with reference to specific embodiments and figures 1-6, but the invention is not limited to these embodiments.
Nouns and methods of:
ordinary AD11, nakedAD11 for short;
red blood cell-loaded AD11 (RBC-PEI-AD 11) suspension;
the PEI solution and the AD11 are incubated together to enable electropositive PEI to be adsorbed on the surface of the AD11 with negative electricity, so that a PEI-coated AD11 (PEI-AD 11) intravenous delivery preparation, namely a PEI-AD11 preparation for short, is obtained.
Example 1: selecting oncolytic adenovirus 11 subtype (AD 11) as a representative, and encapsulating the AD11 with PEI and then carrying out erythrocyte loading;
comparative example 1: the difference with example 1 is that AD11 is not carried out with red blood cell load, and is directly mixed with PEI solution to obtain PEI-coated AD11 (PEI-AD 11) suspension;
erythrocyte suspension from mice: mouse-derived Red Blood Cells (RBCs) were isolated from mouse whole blood, and the procedure was as follows: c57BL/6 male mice pick eyeballs to obtain blood, after anticoagulation of whole blood with heparin sodium, centrifuging at 4 ℃ for 5min to collect red blood cells, adding PBS solution to resuspend the red blood cells, repeating the above steps for 3 times, collecting bottom red blood cells, and preparing the red blood cells into red blood cell suspension by PBS.
Human derived erythrocyte suspension: red blood cells (hrbcs) are human red blood cell suspensions directly from clinical trials.
Experimental data statistical methods used in the following examples: multiple sets of comparisons were performed using one-step ANOVA, and two sets of comparisons were performed using a two-sided t-test.
Example 1
A method for preparing an erythrocyte-loaded oncolytic virus intravenous delivery formulation, comprising the steps of:
first, a certain amount of PEI (M.W.10000) was weighed and dissolved in PBS to prepare a PEI solution with a concentration of 254ug/ml. PEI-coated AD11 (PEI-AD 11) was prepared by mixing the PEI solution with the oncolytic virus in a ratio of the number of polyethylenimine molecules to the number of oncolytic virus particles=15000:1. And then uniformly mixing the AD11 coated by PEI with the red blood cell suspension according to the ratio of the number of red blood cells to the number of oncolytic virus particles=1:15, and standing at room temperature for 50min to obtain a red blood cell-loaded AD11 (RBC-PEI-AD 11) preparation, wherein the preparation process is schematically shown in figure 1.
In order to facilitate the next step of in vitro characterization and in vivo pharmacokinetic and pharmacodynamic experiments, RBC-PEI-AD11 (mouse) and hRBC-PEI-AD11 (human) were prepared by the methods described above, without specific designation.
The erythrocyte-loaded oncolytic virus intravenous delivery preparation prepared by the method is used for intravenous injection delivery of oncolytic viruses to achieve the anti-tumor effect.
The prepared erythrocyte-loaded oncolytic virus intravenous delivery formulation was tested in this example using the following method:
(1) Erythrocyte-loaded oncolytic viruses were characterized using a Zeta/laser particle sizer, scanning electron microscope, and laser confocal microscope.
(2) In vitro level the effect of erythrocyte-loaded oncolytic viruses against cellular uptake of the virus was verified by cell uptake experiments.
(3) And (3) researching the circulation condition of the erythrocyte-loaded oncolytic virus in vivo and the biological distribution condition in vivo by adopting a drug metabolism detection experiment, a qPCR experiment and a model mouse living body imaging experiment.
(4) The biochemical detection of blood and the in-vivo safety detection experiment are adopted to research the strength of toxic reaction caused by the preparation in vivo, and the improvement effect of erythrocyte load on the biological safety of oncolytic viruses in the in-vivo circulation process is verified.
(5) The antitumor effect of the preparation was evaluated by examining the fluorescence intensity of the metastasis.
The specific detection and effect are as follows:
1. verification and characterization detection of erythrocyte-loaded oncolytic viruses:
the potential/laser particle size measurement showed that the particle size of the particles increased significantly after coating with PEI, indicating successful PEI-AD11 construction, compared to the NakedAD11 average particle size of 104.9+ -4.7 nm and PEI-AD11 average particle size of 235.4 + -2.3 nm (FIG. 2A).
In order to conveniently detect the loading condition of red blood cells on AD11, the red fluorescent dye Cy5.5-NHS is utilized to mark the AD11 in the preparation process, the AD11-Cy5.5 is constructed, and then the red blood cell loaded oncolytic virus preparation (RBC-PEI-AD 11) with the Cy5.5 fluorescence is successfully prepared according to the preparation method. The results of the photographs with a laser confocal microscope showed that red fluorescence in RBC-PEI-AD11 overlapped 100% of the biconcave disk-shaped erythrocytes, all co-located, indicating successful loading of AD11 by erythrocytes (fig. 2B).
The binding of RBCL to AD11 was detected by flow cytometry, and likewise, red fluorescent dye Cy5.5-NHS was used to label AD11 to prepare RBC-PEI-AD11, which showed 98.9% of the erythrocytes were fluorescently labeled, again indicating successful preparation of the erythrocyte-loaded AD11 preparation (FIG. 2C).
Then, we successfully prepared red cell-loaded AD11 (RBC-PEI-AD 11 and hbc-PEI-AD 11) by the above method, fixed the red cell-loaded AD11 with 2.5% glutaraldehyde solution, washed in an increasing ethanol gradient, and then chemically dried with hexamethyldiazine. Finally, after the sample is subjected to sputter coating before imaging, the combination of AD11 and red blood cells is confirmed by a scanning electron microscope, so that the AD11 particles coated by PEI are adhered to the surface of the red blood cells, and the red blood cells loaded with the AD11 particles still maintain good biconcave discoid cell morphology. Morphological characterization of erythrocyte-loaded oncolytic viruses by scanning electron microscopy again demonstrated the success of erythrocyte-loaded oncolytic virus technology (fig. 2D).
2. Examination of the in vitro anti-macrophage uptake ability of erythrocyte-loaded oncolytic virus intravenous delivery formulations:
RAW264.7 was applied at 10 per well 6 Individual cells were plated in 12-well plates. NakedAD11, PEI-AD11 and RBC-PEI-AD11 were added separately with a starting MOI (multiplicity of infection) of 10 pfu/cell. After 4h incubation, the cell supernatant medium and remaining preparations were discarded, the cells were washed with PBS and the cell DNA was collected with Fast-pureCell/TissueDNAIsolationMiniKit (Vazyme). The number of AD11 copies was determined by qPCR to determine the amount of 3 groups of AD11 preparations taken up by RAW264.7 cells. The results show that the quantity of RBC-PEI-AD11 phagocytosed by macrophages is far lessNakedAD11 group and PEI-AD11 group (FIG. 3).
3. In vivo circulation study of erythrocyte-loaded oncolytic viruses:
each group of prepared AD11 preparations (NakedAD 11, PEI-AD11 and RBC-PEI-AD 11) was intravenously injected into C57 mice, blood was taken from the submaxillary vein at different time points (5, 30min,1,2,4,8 h), genomic DNA in whole blood was extracted using a whole blood DNA extraction kit (AxyGEN, china), and the content of residual AD11 in blood was detected by qPCR method. The results show a significant increase in the cycle time of RBC-PEI-AD11 compared to NakedAD11 and PEI-AD11 (FIG. 4A).
4. Tissue distribution study of erythrocyte-loaded oncolytic viruses:
for tissue distribution studies, a mouse derived lung cancer TC-1 cell was used to construct a lung metastasis model mouse, 10 6 The TC-1 cells were intravenously injected into C57 mice to create a model of lung metastasis. And after the mice model for detecting lung metastasis by the living animal imaging system is successfully constructed, carrying out subsequent experimental operation. Each group of preparations (NakedAD 11, PEI-AD11 and RBC-PEI-AD 11) was intravenously injected into mice, and after 4 hours, the main organs (heart, liver, spleen, lung, kidney) of the mice were taken, weighed and homogenized, and the organs and solid tumor DNA were collected by Fast-pureCell/TissueDNAIsolationMiniKit (Vazyme), with the organs of the blank mice not injected with any AD 11-related preparations as a standard. The results showed that the lung AD11 copy number was significantly higher in the RBC-PEI-AD11 group than in the NakedAD11 and PEI-AD11 groups, approximately 13.16 times the other groups (FIGS. 4B and 4C). This result demonstrates that erythrocyte-loaded oncolytic viruses have better lung metastatic site targeting ability.
Tissue distribution was further studied by using a small animal living body imaging method, and a C57 mouse lung metastasis model was constructed by the same method. AD11 is marked by using Cy5.5-NHS to construct AD11-Cy5.5, and then each group of preparations (NakedAD 11, PEI-AD11 and RBC-PEI-AD 11) with Cy5.5 fluorescence are successfully prepared according to the preparation method to carry out in vivo tissue distribution research of the fluorescence preparations. The preparation of each group is injected into the C57 pulmonary metastasis mice in an intravenous way at equal dose, the lungs of each mouse of different groups are taken out in vitro after 4 hours, a fluorescence image of the lungs of the pulmonary metastasis mice is shot under a small animal living body fluorescence/bioluminescence imaging system (figure 4D), then fluorescence intensity quantification is carried out by using small animal living body fluorescence/bioluminescence imaging analysis software, as shown by a fluorescence quantification result of figure 4D, the fluorescence intensity of the preparation of the lungs of the mice of the RBC-PEI-AD11 group is obviously higher than that of the NakedAD11 group and the PEI-AD11 group, and the erythrocyte-loaded oncolytic virus has the best targeted enrichment effect of the pulmonary metastasis.
5. Research on biosafety improving effect of erythrocyte-loaded oncolytic virus:
for biosafety studies of each group of formulations, C57 mice were given a dose of 10 by intravenous injection of NakedAD11, PEI-AD11 or RBC-PEI-AD11 8 pfuAD11. Each group of formulations was bled from the submaxillary vein of each mouse 72 hours after injection and the index of several inflammatory factors (IFN-. Gamma., IL-6, TNF-. Alpha.) in the blood was determined. As shown in FIG. 5, the level of both IFN-. Gamma.and IL-6 inflammatory factors in blood was significantly lower in the RBC-PEI-AD11 group than in the NakedAD11 and PEI-AD11 groups. These two inflammatory factors are considered to be the primary inflammatory factors of the antiviral response in vivo. While for TNF-. Alpha.there was no significant difference in the three groups of mice. The results indicate that the erythrocyte-loaded oncolytic virus has the lowest degree of inflammatory response compared with the other two groups, and has the best biological safety.
6. Study of anti-tumor effect of erythrocyte-loaded oncolytic virus on metastasis:
will 10 6 The TC-1-hCD46-luc cells were intravenously injected into C57 mice to create a model of lung metastasis. After 2d, the successfully constructed bioluminescent lung metastatic mice were randomly divided into 4 groups, each 5×10 every other day 7 The NakedAD11, PEI-AD11 or RBC-PEI-AD11 doses of pfuAD11 or equal amounts of PBS were intravenously injected 6 times (3, 5,7,9, 11, 13 days) and the entire metastatic pharmacodynamic flow chart is shown in FIG. 6A. Bioluminescence imaging of mice at intervals (2, 6, 10, 14, 18 days) using a small animal bioluminescence imaging system (PerkinElmer, USA) to monitor growth of lung metastases, and the in vivo tumor metastasis bioluminescence image (FIG. 6B) of the treated mice clearly shows progression of lung metastases in each group of mice, can be usedIn order to see that metastasis of PBS group mice progressed rapidly, the tumor cell bioluminescence area and intensity increased gradually with time, compared to the lung metastasis of nakeda d11 and PEI-AD11 group mice progressed relatively rapidly, although not as fast as PBS group mice. The lung metastasis progress speed of the mice in the RBC-PEI-AD11 group is obviously lower than that of the mice in the NakedAD11 group and the mice in the PEI-AD11 group, the lung metastasis basically does not obviously progress, and even at the time of the pharmacodynamics monitoring end point of the lung metastasis bioluminescence image, the bioluminescence of the lung metastasis part of the mice is basically disappeared. The fluorescence intensity of the bioluminescence images of each group of mice on day 21 was quantitatively analyzed to draw a bar graph, and the results show that compared with the other groups, the fluorescence of the lung metastasis of the mice in the RBC-PEI-AD11 group is disappeared, which indicates that the RBC-PEI-AD11 preparation basically cures the lung metastasis of the mice in the lung metastasis model (figure 6B). Animals were sacrificed after bioluminescence imaging monitoring on day 18, hearts were perfused, lungs of each group of mice were removed, and after dip fixation with Bouin's staining solution, images of lung metastasis entity were photographed for each group of mice (fig. 6C), and it was seen that RBC-PEI-AD11 group of mice had significantly fewer lung surface metastasis nodules than PBS, nadedad 11, and PEI-AD11 group of mice. The results of the counting of pulmonary metastasis nodules in each group of model mice are shown in FIG. 6D, where there is no statistical difference between the number of pulmonary metastasis nodules in the PBS, nakedAD11 and PEI-AD11 groups, while the number of pulmonary metastasis nodules in the RBC-PEI-AD11 group is significantly less than in the first three groups, with minimal pulmonary metastasis nodules. The results show that the RBC-PEI-AD11 group preparation has very remarkable curative effect on metastatic tumors relative to other groups.
Example 2
A method for preparing an erythrocyte-loaded oncolytic virus intravenous delivery formulation, as described in example 1, which differs from example 1 in that: PEI (M.W.8000); the concentration of PEI solution was 282ug/ml; number of polyethylenimine molecules oncolytic virions = 14000:1; number of erythrocytes oncolytic virus particles = 1:10, standing for 40min at room temperature.
Compared with the control example (AD 11 is not subjected to erythrocyte loading) with the same PEI solution concentration, the preparation prepared in the embodiment has longer circulation time in peripheral blood, stronger enrichment capacity on lung metastasis sites and better anti-metastasis curative effect. The detection method is the same as that of embodiment 1, and will not be described in detail.
Example 3
A method for preparing an erythrocyte-loaded oncolytic virus intravenous delivery formulation, as described in example 1, which differs from example 1 in that: PEI (M.W.5000); the concentration of PEI solution is 125ug/ml; number of polyethylenimine molecules oncolytic virions = 12000:1; number of erythrocytes oncolytic virus particles = 1:14, standing for 45min at room temperature.
Compared with the control example (AD 11 is not subjected to erythrocyte loading) with the same PEI solution concentration, the preparation prepared in the embodiment has longer circulation time in peripheral blood, stronger enrichment capacity on lung metastasis sites and better anti-metastasis curative effect. The detection method is the same as that of embodiment 1, and will not be described in detail.
Example 4
A method for preparing an erythrocyte-loaded oncolytic virus intravenous delivery formulation, as described in example 1, which differs from example 1 in that: PEI (M.W.3500); the concentration of PEI solution is 100ug/ml; number of polyethylenimine molecules oncolytic virions = 10000:1; number of erythrocytes oncolytic virus particles = 1:16, standing at room temperature for 50min.
Compared with the control example (AD 11 is not subjected to erythrocyte loading) with the same PEI solution concentration, the preparation prepared in the embodiment has longer circulation time in peripheral blood, stronger enrichment capacity on lung metastasis sites and better anti-metastasis curative effect. The detection method is the same as that of embodiment 1, and will not be described in detail.
Example 5
A method for preparing an erythrocyte-loaded oncolytic virus intravenous delivery formulation, as described in example 1, which differs from example 1 in that: PEI (m.w.800); the concentration of PEI solution is 300ug/ml; number of polyethylenimine molecules oncolytic virions = 16000:1; number of erythrocytes oncolytic virus particles = 1:20, standing at room temperature for 55min.
Compared with the control example (AD 11 is not subjected to erythrocyte loading) with the same PEI solution concentration, the preparation prepared in the embodiment has longer circulation time in peripheral blood, stronger enrichment capacity on lung metastasis sites and better anti-metastasis curative effect. The detection method is the same as that of embodiment 1, and will not be described in detail.
Example 6
A method for preparing an erythrocyte-loaded oncolytic virus intravenous delivery formulation, as described in example 1, which differs from example 1 in that: PEI (M.W.5000); the concentration of PEI solution is 120ug/ml; number of polyethylenimine molecules oncolytic virions = 5000:1; number of erythrocytes oncolytic virus particles = 1:12, standing at room temperature for 50min.
Compared with the control example (AD 11 is not subjected to erythrocyte loading) with the same PEI solution concentration, the preparation prepared in the embodiment has longer circulation time in peripheral blood, stronger enrichment capacity on lung metastasis sites and better anti-metastasis curative effect. The detection method is the same as that of embodiment 1, and will not be described in detail.
Example 7
A method for preparing an erythrocyte-loaded oncolytic virus intravenous delivery formulation, as described in example 1, which differs from example 1 in that: PEI (M.W.8000); the concentration of PEI solution is 260ug/ml; number of polyethylenimine molecules oncolytic virions = 50000:1; number of erythrocytes oncolytic virus particles = 1:18, standing at room temperature for 55min.
Compared with the control example (AD 11 is not subjected to erythrocyte loading) with the same PEI solution concentration, the preparation prepared in the embodiment has longer circulation time in peripheral blood, stronger enrichment capacity on lung metastasis sites and better anti-metastasis curative effect. The detection method is the same as that of embodiment 1, and will not be described in detail.
Claims (10)
1. A method for preparing an erythrocyte-loaded oncolytic virus intravenous delivery formulation, comprising the steps of:
s1: dissolving polyethyleneimine in PBS solution to prepare polyethyleneimine solution;
s2: adding oncolytic viruses into the polyethyleneimine solution, uniformly mixing, and adsorbing the polyethyleneimine on the surface of the oncolytic viruses through electrostatic action to prepare a polyethyleneimine-coated oncolytic virus suspension;
s3: uniformly mixing the polyethyleneimine-coated oncolytic virus suspension with the erythrocyte suspension, standing, and adsorbing the polyethyleneimine-coated oncolytic virus on the surface of erythrocytes through electrostatic action to prepare the erythrocyte-loaded oncolytic virus suspension, namely the erythrocyte-loaded oncolytic virus intravenous delivery preparation.
2. The method for preparing an erythrocyte-loaded oncolytic virus intravenous delivery formulation according to claim 1, wherein in S1, the concentration of polyethylenimine in the polyethylenimine solution is 100-300 ug/ml.
3. The method of claim 1, wherein in S1, the PBS solution is a phosphate buffer solution.
4. The method of claim 1, wherein in S1, the molecular weight of the polyethyleneimine is m.w.800-10000.
5. The method of claim 1, wherein in S2, the oncolytic virus is adenovirus subtype 11 (AD 11).
6. The method for preparing an erythrocyte-loaded oncolytic virus intravenous delivery preparation according to claim 1, wherein in S2, the polyethylenimine solution is mixed with oncolytic virus according to the ratio of polyethylenimine molecular number: oncolytic virus particle number= (5000-50000): 1.
7. The method of claim 1, wherein in S3, the erythrocytes are derived from human or mouse erythrocytes.
8. The method for preparing an oncolytic virus intravenous delivery preparation for erythrocyte loading according to claim 1, wherein in S3, the oncolytic virus suspension coated by polyethylenimine is mixed with the erythrocyte suspension according to the ratio of the number of the erythrocyte to the number of the oncolytic virus particles=1 (10-20).
9. The method for preparing an erythrocyte-loaded oncolytic virus intravenous delivery formulation according to claim 1, wherein in S3, the standing time is 40-60 min, and the standing temperature is room temperature.
10. An erythrocyte-loaded oncolytic virus intravenous delivery preparation, prepared according to the preparation method of the erythrocyte-loaded oncolytic virus intravenous delivery preparation of claim 1, wherein the erythrocyte-loaded oncolytic virus intravenous delivery preparation is used for intravenous injection delivery of oncolytic virus to achieve the anti-tumor effect.
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Application publication date: 20230602 Assignee: Shenyang Beike Chuangwei Technology Transformation Research Institute Assignor: THE FIRST HOSPITAL OF CHINA MEDICIAL University Contract record no.: X2024980013861 Denomination of invention: Preparation and anti-tumor application of red blood cell loaded oncolytic virus intravenous delivery preparation Granted publication date: 20240412 License type: Exclusive License Record date: 20240904 |