Tissue Transportation,Pharmacokinetic And Immune-Stimulatory Effects Of Different Materials Via Acupoints Injection

Acupuncture and moxibustion have been widely used in clinical practice of traditional Chinese medicine（TCM）for multiple diseases’treatment and prevention by stimulating acupoints with acupuncture needle or moxibustion stick.Acupoint injection,as an innovation and heritage based on acupuncture in TCM,is a successful example of the clinical application of combination of TCM and Western medicine.Guided by the meridian theory,acupoint injection therapy combines the mechanical stimulation of meridians and acupoints with the chemical stimulation of drugs for disease treatment.It has attracted much attention in TCM clinical practice exactly because of its greater overall efficacy than that of conventional acupuncture therapy,intravenous or intramuscular injection.However,it is still unclear how small-molecule drugs or nanomedicines are transported and distributed in the body after injection and how these materials exert in vivo.In this paper,we used materials of different particle sizes for acupoint injection,a small molecule contrast agent ioversol to simulate small molecule drugs,a near infrared II silver sulfite quantum dots（Ag₂S QD）to simulate inorganic nanomaterials and a tetrahedral DNA nanomaterial（tetrahedral DNA-cy5,TDN-cy5）to simulate organic nanomaterials.In order to explore the transmission and pharmacokinetic of different substances after acupoint injection,the tissue transport was observed by X-ray and fluorescence imaging and pharmacokinetic analysis of the materials was analyzed.Furthermore,a nano-vaccine of mimic Novel Corona Virus was designed to study tissue transport and immune-stimulatory effects in vivo after acupoint injection.Details of the research are as follows:（1）In vivo pharmacokinetic and tissue distribution of small molecule ioversol by acupoint injection.First,the small molecule ioversol was injected into the body to simulate small molecule drugs,and the traditional injection method and acupoint injection are used to visualize the transmission process with synchrotron radiation X-ray imaging technology.It was found that ioversol traveled along the local tissue around the injection site after acupoint injection.Among the acupoint injection modalities,the ioversol respectively traveled along the muscles or tendons after injection at Zusanli and Taixi acupoint,while it entered lymphoid tissue in a short time after injection at Yongquan acupoint.When the ioversol was injected intravenous injection,it quickly entered the blood along the blood vessels after,and traveled along the skin after subcutaneous injection.We found that pharmacokinetic characteristics of acupoint injection is similar to subcutaneous and intramuscular injection.There is an absorption phase first,and then the elimination phase occurs.The peak time of ioversol by acupoint injection in the blood is similar to subcutaneous and intramuscular injection,is about 5 min,with distinct the highest blood concentrations.It was found that acupoint injection has higher medicine concentration and bioavailability in serum.In addition,we synthesized an iodine-synchrotron radiation imaging probe that can specifically distinguish neurons.Through the probe synthesis method,the immunoglobulin G primary antibody specifically bind to the antigen of the acupoint can be replaced by the immunoglobulin G secondary antibody,thereby specifically visualizing the acupoint microenvironment at the cellular level.These results of this study will provide a pharmacokinetic basis for the research and clinical application of acupoint injection of small molecule drugs.（2）In vivo metabolism and tissue distribution of nanoparticles by acupoint injection.Next,we used Ag₂S QD with nano-scale and second near-infrared fluorescence characteristics to simulate nano-drug,and explored its distribution in local and various tissues and organs after acupoint injection.The silver distribution in tissue is unique in tissue after different acupoint injection modalities.We found that the fluorescence signal of Ag₂S QD could still be detected in acupoint in situ for 28 days after acupoints injection,causing persistent mechanical stimulation to acupoints.We observed the pharmacokinetic characteristics through the quantitative analysis of the silver content by ICP/MS after injection.From the pharmacokinetic curve of Ag₂S QD after injection by different methods,we found that the pharmacokinetic of acupoint injection was similar to subcutaneous and intramuscular injection,the absorption phase appeared first,and then the elimination phase appeared gradually.The peak time and peak concentration of different modalities of acupoint injection were different,and the shorter time of peak concentration and lower bioavailability of acupoint injection were distinct with other injection modalities.These results indicate that Ag₂S QD with the nanosize effect can stay in acupoints and interstitial space to cause sustained mechanical stimulation for a long time instead of entering the blood in large quantities after acupoint injection.Different from Ag₂S QD,degradable DNA nanomaterials TDN-cy5 could exist at the tissues around injection acupoint for about7 h,which is significantly shorter than the residence time of Ag₂S QD（28 day）.It similarly indicates that the degradability of the nanosize effect of the TDN-cy5material can affect its local transmission.However,compared with small molecule fluorescent dyes cy5,the persistence time of TDN-cy5 nanoparticles by acupoint injection is significantly increased,confirming that nanomaterials have the advantage of longer time to exert drug effect than small molecules.This study provides a scientific basis for the transmission process and disease treatment in vivo and clinical application of nanomedicine by acupoint injection.（3）Acupoint injection of nano-vaccine and its immune amplification effect.Based on the characteristics of persistent and slow transmission,and long retention time in local tissue around the injection site of nanoparticle,we designed a nano-vaccine of mimic Novel Corona Virus to investigate the transmission process,humoral immunity,and cellular immune responses,compared with conventional injection methods.Firstly,imaging was performed by the nano-vaccine PDA NP-S1modified with rhodamine B.It was found that the nano-vaccine was transmitted in a specific direction,and a large quantity of vaccines remained at the injection site after acupoint injection,the fluorescence intensity of local tissue and lymph nodes was significantly increased compared with intramuscular injection.Secondly,the antibody level of nano-vaccine PDA NP-S1 by ST36 acupoint injection was three times to traditional intramuscular injection,similarly in PDA NP-OVA.Finally,the co-stimulating molecules of antigen presentation in the surface of dendritic cells were significantly increased after injection at ST36 acupoint compared with that after intramuscular injection,which effectively promoted the antigen presentation of dendritic cells.The proportion of memory T cells in lymph nodes were also effectively activated and increased by acupoint injection,which is conducive to the rapid response of the body confronting viral invasion and activate the differentiation of B cells into plasma cells.The combination of double acupuncture points could further enhance the immune effect and activate the drainage lymph node response of local acupoints.Acupoint injection provides a universal and attractive means of vaccination for viral infectious diseases,such as COVID-19.In conclusion,it was found that the substances injected at acupoints have unique distribution and metabolic characteristics,which are affected by the size and properties of the injected substances.Acupoint injection of nano-vaccine has a more efficient immune stimulation effect,and provides a more efficient way of immunization for the prevention of viral infectious diseases,such as COVID-19.