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What can we do? 01 02 03 Analyze Seek For Study Literature Drug Reports Professional Advice Opioids opium morphine heroin POPPY Opioids top the list of illicit drugs and cause the most burden of disease and drug-related deaths

  3. What can we do? 01 02 03 Analyze Seek For Study Literature Drug Reports Professional Advice

  4. Opioids opium morphine heroin POPPY ‘ Opioids top the list of illicit drugs and cause the most burden of disease and drug-related deaths worldwide. ’ —— The United Nations World Drug Report (2014)

  5. Harm of opioids D rug hazard index A buse of Drug 60 Heroin Millions of users Cocaine 40 Alcohol ATS 20 Ecstasy 0 Opioids Opiates Cocaine ATS “ Ecstasy ” Mean Physical dependence Global estimate of users of different drugs ， 2012 Psychological dependence Pleasure According to World drug report 2014

  6. Current treatments Naltrexone opioid receptor antagonist Methadone Opioid receptor agonist

  7. Target — Mu-opioid receptor Opioids act primarily as agonists of Opioid Mu-opioid receptor (MOR) Altered gene expression receptors Gi/c CREB CREB MOR is an ideal therapeutic μ Opioid Morphine target for opioid addiction Recetor PKA CREB Adenylate Cyclase PKA

  8. Action of heroin on MOR

  9. Medicine — siRNA A tiny but powerful gene silencing tool

  10. Limitations Technical barriers ： 1. tissue non-specificity 2. immune activation and toxicity 3. brain impermeability

  11. Exosome Exosome Borrowing the body’s own RNA transport service Natural, endogenous nano-sized vesicles

  12. Advantages 01 Specificity 02 Safety 03 Brain accessibility

  13. E xperimental design

  14. E xperimental design Target —— MOR Medicine —— siRNA opioid Agent —— exosome siRNA Silencing device exosome Targeting device Pumping device μ r eceptor Rewarding

  15. Silencing device 5’ 3 ’ Plasmid skeleton

  16. Silencing device exosomes siRNA siRNA plasmid siRNA HEK293 cell

  17. Targeting device Neuron targeting RVG Lamp2b RVG ： Rabies Virus Glycoprotein Lamp2b ： lysosomal-associated membrane protein 2b

  18. Pumping device nsMase2 HEK293 cell HEK293 cell Molecular pump

  19. Assembly of RNAi-Targeting-Pumping devices nsMase2 Molecular pump exosomes Lamp2b-RVG degradation siRNA Acetyl-choline receptor targeting plasmid targeting plasmid HEK293 cell mRNA siRNA plasmid siRNA plasmid neuron

  20. E xperimental results

  21. E xperimental results Exosome modification Targeting and characterization capabil ility vali lidation Silencing Safety val alidation capability validation

  22. Interference efficiency of MOR siRNA Relative levels of MOR mRNA in Neuro2A cells 1.5 1.0 1.0 0.47 0.45 0.42 0.22 0.5 0

  23. siRNA concentration in exosomes siRNA is successfully packaged into exosomes 100 in exosome (pmol/ μg ) siRNA concentration 80 60 40 UD UD 20 0 TEM image of modified exosomes RVG siRNA siRNA-RVG control exosome exosome exosome

  24. nSMase2 as a molecular pump Quantitative RT-PCR analysis control nSMase2 of siRNA loaded into exosomes 4 Relative siRNA level 3 control nSMase2 2 1 0 Nanoparticle tracking analysis of exosome production

  25. E xperimental results Exosome modification Targeting and characterization capabil ility vali lidation Silencing Safety val alidation capability validation

  26. Targeting capability validation （ in vitro ） Ne Neuro ro2A A549 A5 (non-neuro (n ronal) (neuronal) (n C2C1 C2 C12 MCF-7 (non-neuro (n ronal) (n (non-neuro ronal) siRNA siRNA-RVG siRNA siRNA-RVG contr trol contr trol exosome exosome exosome exosome

  27. Targeting capability validation （ in vitro ） siRNA concentration (siRNA/U6) 0.4 0.3 0.2 UD UD UD 0.1 0.0 Neuro2A cell C2C12 cell

  28. Targeting capability validation （ in vivo ） Olfactorius Olfactory brainstem cortex liver lung spleen bulbus medulla epencephalon contr trol siRNA exosome siRNA-RVG exosome Neuronal tissues Non-neuronal tissues

  29. E xperimental results Exosome modification Targeting and characterization capabil ility vali lidation Silencing Safety val alidation capability validation

  30. Silencing capability （ in vitro ） 1.5 Relative level of MOR mRNA lower MOR mRNA and protein levels become 1.0 Neuro2A cell 0.5 MOR GAPDH 0.0 siRNA siRNA-RVG siRNA siRNA-RVG control control exosome exosome exosomes exosome

  31. Silencing capability （ in vivo ） Conditional Place Preference Test Natural preference Morphine-paired preference Morphine Injection

  32. Flow chart Morphine injection Exosome injection Day 1 1 2 3 4 5 6 7 8 9 10 11 12 26 26 28 30 32 33 34 32 34 12 CPP SCORE (sec ) 0 -500 -1000 Day 1 Pr Day 12 12 te Day 26 26 te Day 34 34 te Pre te test test-1 test-2 test-3 control siRNA-RVG exosome siRNA exosome Mobile heatmap control siRNA exosome siRNA-RVG exosome

  33. MOR expression after CPP test 1.5 Relative level of MOR mRNA RVG exosome-delivered siRNA pass through 1.0 BBB to down-regulate MOR expression 0.5 MOR GAPDH 0.0 siRNA siRNA-RVG siRNA-RVG siRNA control control exosome exosome exosome exosome

  34. E xperimental results Exosome modification Targeting and characterization capabil ility vali lidation Silencing Safety val alidation capability validation

  35. Safety validation Forced Swimming Test Mobile time （ s ） 400 Inject exosomes 300 200 100 Record the video and analyze the activation of the mice

  36. Model Model

  37. Modeling Overview Delivery Module Silencing Module Signaling Module

  38. Delivery Module Methods Pharmarcokinetics of exosome 𝑒 𝐶𝑑𝑔 = 𝐿 𝑐𝑚𝑝𝑝𝑒𝑒𝑗𝑡 ∙ 𝐶 𝑑𝑐 − 𝐿 𝑐𝑚𝑝𝑝𝑒𝑐𝑗𝑜𝑒 ∙ 𝐶 𝑑𝑔 − 𝐹𝑢 𝑒𝑢 𝑢𝑗𝑡𝑡𝑣𝑓 𝑒 𝐶𝑑𝑐 = −𝐿 𝑐𝑚𝑝𝑝𝑒𝑒𝑗𝑡 ∙ 𝐶 𝑑𝑐 + 𝐿 𝑐𝑚𝑝𝑝𝑒𝑐𝑗𝑜𝑒 ∙ 𝐶 𝑑𝑔 𝑒𝑢 𝑅 𝑢𝑗𝑡𝑡𝑣𝑓 ∙𝐶 𝑑𝑔 𝑒 𝐹𝑢 𝑒𝑢 = 𝐿 𝑢𝑠𝑏𝑜𝑡𝑐𝑚𝑝𝑝𝑒 ∙ 𝑞𝑏𝑠𝑗𝑢𝑗𝑝𝑜𝑢𝑗𝑡𝑡𝑣𝑓 ∙ − 𝐿 𝑐𝑗𝑜𝑒𝑢𝑗𝑡𝑡𝑣𝑓 ∙ 𝐹𝑢 − 𝐿 𝑛 ∙ 𝐵𝑆 ∙ 𝐹𝑢 𝑅𝑑 exosome Cellular Trafficking of exosome binding internalization d Tb Lysosome dt = 𝐿 𝑐𝑗𝑜𝑒𝑢𝑗𝑡𝑡𝑣𝑓 ∙ E𝑢 + 𝐿 𝑛 ∙ AR ∙ Et − 𝐿 𝑗𝑜𝑢𝑢𝑗𝑡𝑡𝑣𝑓 ∙ Tb degradation d Tissue = 𝐿 𝑗𝑜𝑢𝑢𝑗𝑡𝑡𝑣𝑓 ∙ Tb − 𝐿 𝑓𝑚𝑗𝑛𝑢 ∙ Tissue endosome dt d Enc = 𝐿 𝑑 ∙ 𝐿 𝑗𝑜𝑢 ∙ Tbbrain − 𝐿 𝑓𝑡𝑑𝑓𝑜𝑒𝑤𝑓𝑑 ∙ Enc Endosome RISC complex dt escape

  39. Delivery Module Results RVG modification helps exosomes pass through the BBB and target into brain Control siRNA-RVG exosome Control siRNA-RVG exosome

  40. Silencing Module Methods d R dt = 𝐿 𝑓𝑡𝑓𝑜𝑒𝑤𝑓𝑑 ∙ 𝐹 𝑜𝑑 − 𝐿 𝑒𝑗𝑡 RISC ∙ R + 𝐿 𝑔𝑝𝑠𝑛 𝑆𝐽𝑇𝐷 · (𝑠𝑢𝑝𝑢 + 𝐿 𝑒𝑗𝑡 RISC · 𝑆 − C)·Can+ 𝐿 𝑑𝑚𝑓𝑏𝑤𝑏𝑕𝑓 · C −𝐿 𝑒𝑓𝑕 RISC· (R + C) − 𝐿 𝑔𝑝𝑠𝑛 RISC m ·R·M d 𝐷na = −𝐿 𝑒𝑗𝑡 RISC ∙ R + 𝐿 form RISC ∙ ( rtot + 𝐿 𝑒𝑗𝑡 RISC·R − R − C)·Cna dt −𝐿 𝑒𝑓𝑕inna ·Cna d 𝐷 dt = 𝐿 form RISC ∙ R ∙ M − 𝐿 dis 𝑆𝐽𝑇𝐷m ∙ C − 𝐿 𝑒𝑓𝑕 RISC· (R + C) − 𝐿 𝑑𝑚𝑓𝑏𝑤𝑏𝑕𝑓 · C d M dt = 𝐿 𝑔𝑝𝑠𝑛m𝑆𝑂𝐵 + 𝐿 dis 𝑆𝐽𝑇𝐷m ∙ C − 𝐿 𝑒𝑓𝑕m𝑆𝑂𝐵 ·M −𝐿 𝑔𝑝𝑠𝑛 RISC m ·R·M d P dt = 𝐿 𝑔𝑝𝑠𝑛prot ·M − 𝐿 degprot ∙ P Deterministic Model of Silencing Process

  41. Silencing Module Results MOR mRNA MOR protein Injection of exosomes significantly reduce the level of MOR mRNA and protein

  42. Signaling Module Methods — Activation of MOR Diagrams and ODEs are created using Matlab Simbiology d Cabg_R = − ReactionFlux1 + ReactionFlux5 dt G αβγ -R Opiates d Cabg_R_𝑛𝑝𝑠𝑞ℎ𝑗𝑜𝑓 = ReactionFlux1 − ReactionFlux2 + ReactionFlux8 dt d 𝑛𝑝𝑠𝑞ℎ𝑗𝑜𝑓_𝑆 = ReactionFlux2 + ReactionFlux6 − ReactionFlux8 dt G αβγ d 𝐻𝑏_𝐻𝑈𝑄 G αβγ -Opiates-R = ReactionFlux2 − ReactionFlux3 + ReactionFlux7 G αβγ + R dt d 𝐻𝑏_𝐻𝐸𝑄 = ReactionFlux3 − ReactionFlux4 GTP Opiates dt Opiates- d 𝐻𝑏𝑐𝑕 = ReactionFlux4 − ReactionFlux5 + ReactionFlux7 − ReactionFlux dt R G αβγ GDP d 𝑆 dt = − ReactionFlux5 − ReactionFlux6 G αβγ G α -GTP G α -GDP d 𝐻𝑐𝑕 GTP G βγ = ReactionFlux2 − ReactionFlux4 + ReactionFlux7 dt

  43. Signaling Module Stochastic methods using Gillespie's Algorithm GABA GABA GABA synthesized docked release GABA inhibited

  44. Signaling module results Counteractive impacts On inhibit of GABA release On decrease of cAMP level On activation of MOR

  45. Human Human practice practice

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