Artificial Red Blood Cells Constructed By Replacement Method For Reversing Radioresistance

Radioresistance,caused by tumor hypoxia,is a key factor limiting the efficiency of radiotherapy(RT)and ultimately leads to tumor recurrence and metastasis.In order to alleviate the hypoxic microenvironment of tumors,many studies based on hemoglobin-like oxygen carriers(HBOC)have been carried out,providing new options for cancer patients with radiotherapy resistance.However,the potential auto-oxidative cytotoxicity and nephrotoxicity in hemoglobin(Hb)impede their clinical application,with most studies stalled in clinical phase ?.Therefore,it is highly desirable to find a safe and reliable novel hemoglobin oxygen carrier.In this paper,we constructed a nano-red blood cell(nnRBC)with multiple excellent properties through the " replacement method".To be specific,heme in hemoglobin was replaced with perfluorodecalin(FDC)which is a highly hydrophobic and oxygen-carrying perfluoro compound to obtain the FDC-globin nanocomposite(NPs),then being coated with a layer of red blood cell membrane(RBCm)on the outside to prepare the nnRBCs.The research content of this paper is mainly divided into four parts:The first part.The preparation process and characterization of nnRBCs in vitro.The hemoglobin is used as the raw material,and globin is collected by a classical acidic acetone method.Comparing the size of the particle size after emulsification of FDC with four different emulsifiers of poloxamer,albumin,liposome and globin,it was determined that globin is the most suitable emulsifier.Next,the volume ratio of RBCm and NPs was screened,and it was confirmed that the nnRBCs prepared by co-extruding the membrane at a ratio of 4:1 had the smallest particle size and the best stability.The successful encapsulation of RBCm on NPs was then demonstrated by particle size determination,potentiometric measurement,transmission electron microscopy images and gel electrophoresis images.UV qualitative and quantitative methods were used to determine the complete removal of heme in the system.The loading content of FDC in the system was determined by gas chromatography.Finally,the particle stability of nnRBCs in different media and temperatures was investigated.Our results reflected the advantages of nnRBCs with high drug loading,long-term stability and reliable safety.The second part.Oxygen release behavior and cytotoxicity of nnRBCs in vitro.The oxygen carrying capacity of nnRBCs was measured by an oxygen electrode,and then the hypoxic and normoxic microenvironment in vivo were simulated in vitro.The oxygen electrode was used to monitor the oxygen release process of nnRBCs and several other preparations.Visually,changes in blood oxygen saturation were identified by changes in blood color.The effects of nnRBCs on cell hypoxia,damage to DNA molecules,and radiotherapeutic toxicity to cell were then tested on colon cancer cell models.The results suggested that nnRBCs could effectively improve hypoxia and assist in enhancing cell destruction and damage caused by radiotherapy in vitro.The third part.Study on the alleviation of hypoxia in tumors by nnRBCs.In the CT26 colon cancer mouse model,the changes of hypoxia intensity and HIF-1?protein expression in the tumor after intravenous injection of nnRBCs were studied by immunofluorescence staining and western blot.Then,downstream gene of HIF-la related to tumor development was quantified.Together,these results indicated that nnRBCs could alleviate hypoxia in tumors,then down-regulated the expression of the genes that promote tumor progression caused by hypoxia.The fourth part.The enhancement of radiotherapy and the safety of nnRBCs in vivo.In this part,the CT26 colon cancer subcutaneous tumor model was carried out,nnRBCs combined with RT played a "1+1>2" effect,which significantly restricted tumor growth.The effects of nnRBCs on renal function were appraised by serum biochemical analysis and section staining.The results showed that nnRBCs were highly safe.In summary,nnRBCs provides a new approach to FDC delivery since it has not ever been emulsified by any FDA approved emulsifier.After intravenous delivery,nnRBCs boost the efficiency of RT by effectively reversing tumor hypoxia.It is worth noting that the ingredients of nnRBCs are all derived from organisms or safe substances verified by human experiments,which endows nnRBCs have great potential for rapid entering into clinical trials.