From: Tri-partite complex for axonal transport drug delivery achieves pharmacological effect
 | Study | Agent used | Model | Evaluation method | Result | Figure | Impact |
---|---|---|---|---|---|---|---|
A Synthesis, function and stability | |||||||
   I Chemical synthesis | |||||||
 | 1 | Dextran tripartites | Chemical | NMR, PLC, Mass Spec | 30% loading | 2 |  |
 |  | a. CM dex standard |  |  |  |  |  |
 |  | b. CM dex-amino upgraded |  |  |  |  |  |
 |  | c. CM dex-lysine extended |  |  |  |  |  |
 |  | d. WGA-CM dex |  |  |  |  |  |
 |  | e. WGA-CM dex-valacyclovir |  |  |  |  |  |
 |  | f. CM dex-gabapentin |  |  |  |  |  |
 |  | g. WGA-CM dex-gabapentin |  |  |  | 1 | Validate chemical basis of uptake amplification |
 | 2 | Ferrite particle tripartites | chemical | MRI | useful effect on T2 at obtainable conc. |  |  |
 |  | a. dex coated particles precip |  |  |  |  |  |
 |  | b. periodate conjugation |  |  |  |  |  |
 |  | c. affinity purification |  |  |  |  | Validate assembly of nanoparticulate carrier |
 |  | d. DNA adhesion |  |  |  |  |  |
 |  | e. relaxivity assessment |  | test tube cast gels | solenoid coil | 7 | Validate imageability in MRI |
   II Intracellular release and drug activation | |||||||
 | 3 | valacyclovir | BHK-Herpes | plaque count | control |  | Establish cellular capability of uptake and release |
 |  | dex-val | BHK-Herpes | plaque count | steric decrease in uptake |  |  |
 |  | WGA-dex-val | BHK-Herpes | plaque count, assay | improved delivery |  |  |
B Interaction with Axon Terminals and Intra-axonal environment | |||||||
   III Effects of polymer, linker and drug | |||||||
 | 4 | Dextran 10K | symp gang | fluor mic | rapid uptake |  | Evaluate effects of tripartite size on uptake |
 |  | Dextran 70K | symp gang | fluor mic | slower uptake |  |  |
 | 5 | Dextran-FITC neut | symp gang | fluor mic | extensive uptake | 3 | Evaluate effects of tripartite charge on uptake |
 |  | Dextran-FITC pos | symp gang | fluor mic | extensive uptake |  |  |
 |  | Dextran-FITC neg | symp gang | fluor mic | limited uptake |  |  |
 | 6 | Acylation | symp gang | fluor mic | no uptake |  | Evaluate effects of hydrophobicity on uptake |
   IV Effects of Axonal Transport Facilitator | |||||||
 | 7 | WGA-FITC | symp gang, Campenot | fluor mic | WGA and NGF well transported |  | Comparability of physiologic and non-phys ATF in vitro |
 |  | NGF-TR | symp gang, Campenot | fluor mic | WGA and NGF well transported | 4,17,18 |  |
 | 8 | Phage display | Campenot | pfu's | identification of synthetic ATF |  | Phage display for generation of synthetic ATFs |
 | 9 | Transport inhibitor | symp gang, Campenot | fluor mic | blocked by inhibitors of transport |  | Confirmation of role of microtubule based transport |
   V Effects of Intra-axonal Processing | |||||||
 | 10 | Anti-gabapentin Ab | gastroc/ant tib - rat | excise, Ab stain | delivery of gabapentin antigenicity to DRG |  | Confirm delivery of gabapentin antigenicity |
 | 11 | WGA-HRP | axial - M. fascicularis | HRP-TMB backlit | small IM inj to enzyme in cells in primates | 5 | Confirm that enzymatic function survives transport |
 | 12 | WGA-dex-mag | gastroc - rabbit | EM | transport in axoplasm | 6 | Verify axonal transport compartment |
 | 13 | 59-Fe WGA-dex-mag | gastroc/ant tib-rabbit | autoradiography | gross sections |  | Confirm intraneural transport of particle tripartite |
 | 14 | WGA-dex-mag | gastroc/ant tib-rabbit | MRI vs gel chamber | non-lysis of particle | 8,19 | Confirmation of transport of intact magnetite |
 | 15 | WGA-dex-mag | forearm-rabbit | 4.7T high res MRI | demonstration of nerve image | 9 | Confirmation of fast transport of intact magnetite |
C Targeting and pharmacological efficacy | |||||||
   VI Clinical target access | |||||||
 | 16 | WGA-FITC | hindlimb - rat | excise, section, fl mic | ventral horn & intermediolateral | 10 | Assess access to relevant motor & autonomic targets |
 |  | WGA-dex-FITC | hindlimb - rat | excise, section, fl mic | ventral horn & intermediolateral |  |  |
 |  | Dex-FITC | hindlimb - rat | excise, section, fl mic | limited transport |  |  |
 | 17 | WGA-FITC | hindlimb - rat | excise, section, fl mic | DREZ lamina I and II | 11 | Confirm access to dorsal root entry zone |
 |  | WGA-FITC | hindlimb - rat | excise, section, fl mic | DRG axon dendrite |  |  |
 | 18 | WGA-FITC | hindlimb - rat | fluor mic, cell count | good filling in DRG | 12,13 | Assess properties of access to dorsal root ganglia |
 |  | WGA-TRITC | foot pad-rat | fluor mic, cell count | good filling in DRG |  |  |
 | 19 | WGA-FITC | hindlimb - rat | fluor mic, peripherin | nociceptors reached | 14 | Verify access to nociceptors |
 |  | dextran-FITC | hindlimb - rat | fluor mic, peripherin | no transport |  |  |
   VII Distinctive pattern of distribution relative to trans-vascular | |||||||
 | 20 | 125-I WGA | forelimb/hindlimb - rat | dissect, count | saturable, time consistent, selective | 15 | Characterize expected whole body distrib |
 |  | 125-I NGF | hindlimb - rat | dissect, count | differences in distribution due to ATF |  | Evaluate effects of ATF on distribution |
 |  | 59-Fe WGA-dex-mag | forelimb - rat | dissect, count | large particles comparable |  | Evaluate effects of large agent size on distribution |
 | 21 | 131-I WGA | gastroc/ant. tib-rabbit | gamma camera image | voxel size causes averaging and inj site bright |  | Examine local injection area effects |
 | 22 | 14C-gbp-dex | hindlimb - rat | dissect, count | minimal |  | Validate delivery of therapeutic dose |
 |  | 14C-gbp-dex-WGA | hindlimb - rat | dissect, count | transported in high conc. |  |  |
   VIII Unique pharmacologic effects not obtainable by trans-vascular | |||||||
 | 23 | gbp-dex-WGA | foot pad/hindlimb - rat | withdrawal latency | prolonged decrease in hyperalgesia | 16 | Validate pharmacological efficacy |
 |  | gbp-dex | foot pad/hindlimb - rat | withdrawal latency | minimal | 16 |  |
 |  | gbp | foot pad/hindlimb - rat | withdrawal latency | minimal | 16 |  |
 |  | gbp-dex-WGA | cervical fat pad - rat | withdrawal latency | minimal | 16 |  |