Living Red Blood Cells-Carried Dexamethasone Sodium Phosphate Long-Circulation Delivery Systems:Preparation And Preliminary Evaluation

Objective:Red blood cells(RBC)are the largest number of blood cells in the human body.They exceed 30 trillion in normal adults,and the life cycle in the blood circulation can be as long as 120 days.Many drugs will interact with red blood cells after entering the human body.Red blood cells play a role as a natural carrier in the transport and metabolism of these drugs.Therefore,the researchers thought of actively using red blood cells as a drug carrier to build a long-circulation drug delivery system(DDS)based on living red blood cells.The erythrocyte membrane acts as a bilayer of phosphoester,which has a certain degree of elasticity under different osmotic pressures.It can reversibly open the pores in the cell membrane under lower osmotic pressure,and can return to normal under plasma osmotic pressure.Using this property of red blood cells,drugs can be easily loaded into red blood cells.The glucocorticoid Dexamethasone(DS)can effectively slow down the development of lung diseases such as cystic fibrosis,but long-term use can cause many serious adverse effects,such as hypertension,growth disorders,glucose intolerance and bone poor quality,etc.Researchers at Ery Del in Italy,relying on their proprietary Red Cell Loader?,encapsulated the non-diffusible prodrug Dexamethasone Sodium Phosphate(DSP)in human living red blood cells-carried,and then returned it to the patients.The inactive DSP encapsulated in the red blood cells slowly dephosphorylates under the action of enzymes in the red blood cells,transforms into diffusible active DS,and is slowly released from the red blood cells.This technology can process 50-70ml of whole blood and return it to the patient at one time,maintaining DS at a low concentration level for a long time,and the drug effect can be maintained for 2-3 weeks,effectively avoiding the side effects caused by long-term use of glucocorticoids.This article relies on the research team’s early red cell loading device specifically modeled on Red Cell Loader?to prepare a DSP-based live red blood cell long-circulation drug delivery system and carry out relevant evaluation research.Method:First,establish the HPLC drug content determination method of the RBC-containing DSP long-circulation drug delivery system,using chromatographic peak retention time,resolution,symmetry factor and theoretical plate number as the inspection indicators.The detection wavelength,chromatographic column,mobile phase,column temperature,flow rate and other chromatographic conditions were screened,and methodological verification was conducted through experiments such as specificity,linearity,precision,stability and recovery;and dexamethasone-21 was prepared based on the red blood cell drug-specific equipment imitated by the research group The sodium-phosphate red blood cell(DSP-RBC)long-circulation drug delivery system optimizes the process in terms of drug loading time,blocking solution composition,blocking time,drug solution osmotic pressure,etc;then changes in hematological parameters,morphology,surface antigen and to characterize and evaluate the DSP-RBC long-circulation drug delivery system in terms of the survival of the drug-loaded RBC;finally,through the preliminary pharmacokinetic study in rats,the DSP-RBC long-circulation drug delivery system was investigated for its sustained release ability.Results:The DSP-RBC long-circulation drug delivery system established in this article was used to determine the content of the HPLC main drug DSP.The DSP showed a good linear relationship with the peak area ratio in the concentration range of 2.54μg/ml～100.04μg/ml,and the recovery rate was>98%,The test solution is stable at 4℃and the room temperature for 24 h,the precision meets the requirements,and can be used for the determination of DSP content in DSP-RBC.The DSP-RBC long-circulation drug delivery system was successfully prepared and the preparation process was optimized by relying on self-made special equipment for loading red blood cells.The DSP-RBC hematological parameters were basically normal by the automatic blood cell analyzer;the DSP-RBC morphology was basically normal by scanning electron microscope observation;the experimental results of osmotic brittleness and turbulence brittleness showed that the drug loading process had little effect on the elasticity of the red blood cell membrane.The flow cytometric analysis of Na~+/K~+-ATPase and Phosphatidyl Serine(PS)on the surface of erythrocyte membrane revealed that the drug loading process decreased the level of Na~+/K~+-ATPase on the surface of erythrocyte membrane and markedly increased the PS rise.The survival rate of DSP-RBC in rats was investigated.Compared with unloaded red blood cells,the survival rate of drug-loaded red blood cells decreased at 24 hours,and the survival rate was equivalent at 11 days.The preliminary pharmacokinetic experiments in rats verified that the DSP-RBC long-circulation drug delivery system prepared in this paper has obvious sustained release effect in vivo.Conclusion:This article is the first domestic research on RBC-encapsulated DSP based on special equipment for red blood cell drug loading,which optimizes the prescription and preparation process,and characterizes the optimized prescription.The pharmacokinetic experiments verify the sustained-release effect.