| Background and PurposeNeuroblastoma(NB)is not only a non-benign solid tumor commonly found in young children,but also the most common extracranial tumor in children.It is mainly derived from the adrenal medulla or the immature embryonic cells of the paraspinal sympathetic ganglia.Many studies show that NB tends to occur in children under 5 years old.The morbidity rate of NB which accounts for 8%to 10%of non-benign tumors in all children under 15 years old is the highest except central nervous system related tumors and leukemiaThe most common site of primary NB is the adrenal medulla,followed by retroperitoneal,mediastinal,pelvic and neck.It is often occult,and the symptoms are not specific.Clinical manifestations are often closely related to the primary site,metastasis and local invasion of tumors.Some children may develop neuroendocrine system symptoms due to the release of some active substances such as vasoactive intestinal peptides.Some children with no symptomswere discovered by imaging examination.Due to the high degree of malignancy,distant metastasis may occur at an early stage,and the locations that are easily metastasized include bone marrow,lymph nodes,kidneys,skin,liver,and bones.NB is treated by multidisciplinary joint diagnosis and treatment program Multidisciplinary participation is the main treatment for NB so for,such as combination chemotherapy,radiotherapy,surgical resection and autologous hematopoietic stem cell transplantation.The survival rate of children with NB in the low-risk group was significantly improvedBut for the high-risk group,it’s difficult to treat,and the prognosis of children was still poor,.Therefore,exploring the new mechanism of occurrence and development of NB,and finding new treatment for NB has important clinical and social significance for improving the survival rate of children and reducing mortality.mTOR(Mammalian target of rapamycin)is a mammalian target of rapamycin,and a kind of serine and threonine protein kinase.The mTOR signaling pathway has a variety of biological functions.mTOR participates in the key transcription of a variety of related genes,the efficient translation of different functional proteins,the regulation of the number of ribosomes,and other important molecular processes,while it also regulates the growth of some cell cytoplasm and induces orderly apoptosis of individual cells.More and more clinical and experimental evidence now indicates that the mTOR signaling pathway plays a crucial role in the tumorigenesis and development of NB.Rapamycin is an mTOR-specific inhibitor,an antibiotic obtained from bacteria by scientists in the 1970s,and a type of macrolide.Until now,the antibiotic has been widely used in clinical treatments including kidney transplantation and various drug-related eluting stents,and has been approved by the US Food and Drug Administration(FDA).Its function is to bind to the interaction of tacrolimus binding protein(FKBP)and further to inhibit the action of mTOR,so that the phosphorylation of S6K1 and eIF-4E and the transcription and translation of proteins were inhibited.At last the effects of antibacterial,antitumor and immunosuppressive were achieved.Rapamycin and its analogues can specifically inhibit mTOR to achieve anti-tumor growth.Rapamycin has been used as a novel therapy in the treatment of a variety of malignant tumors.It has a certain inhibitory effect on a variety of malignant tumors such as gastrointestinal neuroendocrine tumors,gastric cancer,lung cancer,head and neck squamous cell carcinoma.At present,some studies have shown that rapamycin also has a certain inhibitory effect on NB.However,the known rapamycin dosage form is relatively simple,only simple injection of powderwhich has some unavoidable disadvantages,including relatively poorer targeting,relatively larger toxic and side effects,and relatively poorer drug compliance,etc.,So it cannot be fully utilized in clinical treatment.The rational use of rapamycin for the treatment of malignant tumors and the reduction of their associated side effects have become the top priority of future research.As a relatively ideal carrier,the hydrogel system has good water absorption capacity and can maintain the original shape.Similarly,when it exists in human tissues,a large amount of water is absorbed by it,which will not cause irritation or related immunity reaction.Meanwhile,because of the many good properties of hydrogels,such as the permeability of water molecules and bio-tissue compatibility,it has good research value in many fields related to biology and medicine.Therefore,it is often used as a release carrier for biologically active materials such as various polypeptides and proteins.The injectable hydrogel system is particularly special which can be easily implanted into the body without surgical intervention.The main process is to use a hydrogel encapsulating the active material to form a gel at the target site,to slow down the speed of releasing drug,to increase the time of drug metabolism.The entire course of treatment is simple,and can alleviate the damage of patient and reduce the cost of treatment.Similarly,while the efficacy is significantly enhanced,the adverse effects are correspondingly reduced.Therefore,as a new drug-related delivery system,injectable hydrogel systems are receiving more and more attention.PLGA-PEG-PLGA is a natural high molecular polymer which has many properties including degradability,biocompatibility,non-toxicity,etc.,and as an active carrier,it can increase the stability of components,change the way of administration,accelerating the absorption of drugs and improving bioavailability.Then we will achieve targeted therapy and controlled release.The PLGA-PEG-PLGA polymer designed in this paper can fully encapsulate the rapamycin system,and the speed of release of rapamycin can be gradually regulated through continuous clinical application.The controlled concentration is maintained between the minimum and the limit,and the metabolic duration is increased,and toxic side effects is reduced.It can be applied to tissue injections around the tumor after surgery,to inhibit regeneration of cancer cells and reduce the probability of tumor recurrence.In addition,it can be applied to intratumoral injection to reduce adverse reactions by targeted medication injection,so that the purpose of cancer treatment is achieved.This study was to investigate the role of rapamycin gel injection in inhibiting NB cells and animal xenograft models.The study was divided into three parts:firstly,it was confirmed that rapamycin significant inhibited the growth and proliferation of NB cell Secondly,a rapamycin gel injection was prepared based on the temperature sensitive material of PLGA-PEG-PLGA polymer and the model drug of rapamycin.The physicochemical properties and the corresponding conditions of releasing in vitro were carefully studied.Finally,it was confirmed by in vivo experiments that the dosage form has less toxic and side effects,has excellent tumor suppressing activity,and is a relatively effective and safe anti-tumor drug.And this study can also be used as a method model,not only in the chemotherapy and biotherapeutics of other related tumors,but also in the design and theoretical data of controlled release systems for targeted drugs in the future.The main content and structure of this study are as follows:MethodsPart Ⅰ:Biological effects of rapamycin on NB cellsSK-N-SH and SH-SY5Y cells of NB were treated with different concentrations of rapamycin.The effect of rapamycin on the proliferation of SK-N-SH and SH-SY5Y cells of NB was examined by CCK-8 assay.The effect of rapamycin on the cell cycle of SK-N-SH and SH-SY5Y of NB was detected by flow cytometry.The morphological changes of SK-N-SH and SH-SY5Y cells of NB were observed by electron microscope.Western Blot was used to detect the expression of autophagy-related proteins in SK-N-SH and SH-SY5Y cells after rapamycin treatment.The main purpose of this experiment is to determine the effects of rapamycin on biological behaviors such as proliferation and autophagy of NB cell.Part Ⅱ:Construction and characterization of rapamycin gel injectionThe PLGA-PEG-PLGA rapamycin gel was prepared.And the characterization,viscosity properties and phase transition temperature of the preparation were fully investigated and recorded.At the same time,the encapsulation efficiency and drug loading rate of the gel for rapamycin were determined by high-performance liquid chromatography.Part Ⅲ:Effects of rapamycin gel injection on NBNB SH-SY5Y cells were cultured,NB nude mice xenograft model was established,and rapamycin gel injection was injected into the tumors.Then,in order to explore the anti-tumor activity of the preparation in the animal,and gradually explore the drug-related side effects and in vivo safety,the effect of rapamycin gel in vivo and the non-specific toxicity of rapamycin gel injection in nude mice were observed.Results1.Rapamycin can significantly inhibit the proliferation of SK-N-SH and SH-SY5Y in NB cell line.The effects depend on time and concentration and can block cell cycle at G0/G1.Electron microscopy showed that autophagic vesicles appeared in the cytoplasm of the cells.Meanwhile,rapamycin can induce autophagy in SK-N-SH and SH-SY5Y cells by mediating the mTOR signaling pathway which mainly regulates the process of autophagy through mTOR,Beclin-1,p62,and LC3Ⅰ/Ⅱpolyproteins.2.The successfully constructed rapamycin gel injection has good drug-loading and drug-releasing properties,and the preparation can slowly release.3.Rapamycin gel injection can effectively increase the metabolic duration of rapamycin in vivo and maintain long acting function of anti-tumor.What’s more,it can enhance the effect of inhibiting tumor activity.Meanwhile,there is no obvious organ toxic reaction,showing its good safety.Conclusion1.Rapamycin can inhibit the growth and proliferation of NB and induce autophagy,and the mechanism may be related to the mTOR signaling pathway.2.Rapamycin gel injection can inhibit the growth of NB.The preparation has good performance in anti-tumor,relatively small toxic and side effects,and can be used as an effective and safe anti-tumor preparation. |
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