BACKGROUND
botulinum neurotoxins  are produced by the anaerobic clostridium botulinum species of bacteria and are the cause of botulism, a life-threatening neuroparalytic disease.
they are extremely potent food poisons, with a mouse ld <dig> of  <dig>  ng/kg for type a  <cit> .
aerosol exposure of bonts does not occur naturally, but could be attempted by bioterrorists to achieve a widespread effect.
it has been estimated that a single gram of crystalline toxin, evenly dispersed and inhaled, could kill more than one million people  <cit> .
bonts are large proteins with a molecular weight of  <dig> kda.
they are produced as a complex containing the neurotoxins and associated proteins  <cit> .
they are synthesized as inactive single chain protoxins and are activated by protease nicking to form a dichain molecule  and a  <dig> kda heavy chain ) linked through a disulfide bond  <cit> .
the hc is responsible for binding to the target nerve cells  and translocating the lc into the cell cytoplasm   <cit> .
inside the neuronal cytosol, the lc acts as a zn2+-endopeptidase against specific intracellular protein targets present either on the plasma membrane or on the synaptic vesicle, and inhibits neurotransmitter release by disabling the exocytotic docking/fusion machinery  <cit> .
bonts catalyze proteolysis of specific proteins of the soluble nsf attachment protein receptor  complex that have been implicated in the exocytotic machinery  <cit> .
bont/a,/c, and /e cleave a  <dig> kda synaptosomal associated protein .
current therapy for botulism involves respiratory supportive care and the administration of antitoxin.
the antitoxin could be the currently available equine bont antibodies or potentially more effective recombinant multivalent antibodies.
however, only a few antitoxins, which must be administered before toxins reach the nerve cells, are available.
thus, the therapeutic window for using an antitoxin is short.
once the syndrome is developed, the antitoxin is less effective since it cannot penetrate the nerve cell to neutralize the toxin.
the flaccid muscle paralysis caused by bont/a lasts for several months  <cit> .
therefore, patients who have already developed the syndrome must be put under respiratory intensive care during paralysis  <cit> .
should a bioterrorist attack occur, public health crisis could arise due to the lack of effective antidotes against botulism, especially in the absence of reliable presymptomatic diagnostics.
for relief from bont-mediated paralysis, it is important to rescue the poisoned nerve cells through restoration of the neurotransmitter release process.
while drugs have been designed to block the bont endopeptidase activity, which is believed to be responsible for the inhibition of neurotransmitter release, delivery of the drugs specifically to the poisoned nerve terminals remains a major hurdle.
therapeutic targeting is important for two main reasons:  delivering an effective high concentration of the therapeutic compound to the site of toxicity, i.e., nerve terminals for botulism, and  minimizing systemic toxicity, if any, due to treatment compounds.
at present, some examples of the proposed pharmacological antidotes for bont poisoning are a protease inhibitor, a phospholipase a <dig> activator or a modulator of intracellular free ca2+ concentration.
since all of these parameters are involved in normal body functions, a systemic therapeutic approach is inadvisable due to potential toxicity concerns.
therefore, we developed a drug delivery vehicle  comprising the non-toxic recombinant heavy chain of bont-a coupled to a 10-kda amino dextran via the heterobifunctional linker 3--propionyl hydrazide.
the heavy chain served to target botulinum neurotoxin-sensitive cells and promote internalization of the complex, while the dextran served as a platform to deliver model therapeutic molecules to the targeted cells.
RESULTS
structure of ddv
initially we designed a ddv utilizing the recombinant bont/a heavy chain , which is known to specifically bind to the presynaptic nerve terminals and be internalized via endocytosis.
the ddv construct was a modification of that developed by goodnough et al.
<cit>  consisting of a targeting molecule, cy <dig> labeled purified  hc linked by a disulfide bond to a drug simulant, which was oregon green  <dig>  labeled  <dig> kda dextran .
the ddv structure was used for further experiments.
to our knowledge, this is the first experimental demonstration of a prospective therapeutic approach to treat botulism in a relevant peripheral neuronal model combined with a feasible targeted drug delivery technology.
rhc was a safe ddv component for delivery of bont antidotes
to exclude any possible toxicity of the rhc component in our ddv construct, we compared the inhibition of  <dig> mm k+ stimulated glycine release due to increasing concentrations of rhc or native bont/a holotoxin by the assay described in methods.
in these experiments, the results obtained using the particular batch of toxin showed that bont/a was quite toxic, as expected, with an ic <dig>  of approximately < <dig> pm and a total inhibition at ~ <dig>  nm.
however, a much higher concentration of rhc, up to  <dig> nm, did not show any inhibition of glycine release under the same assay conditions .
ddv entry into neurons via bont/a receptor mediated endocytosis
the experimental design was to mimic a therapeutic application of the ddv strategy to treat individuals poisoned with bont/a and exhibiting clinical symptoms of botulism.
since the targeted ddv approach is based on the premise of a selective entry of ddv into presynaptic nerve terminals via bont/a receptor mediated endocytosis, we demonstrated by competition experiments that the uptake of the ddv-mas- <dig> was via bont/a receptors.
in these experiments, 3-week old spinal cord cultures were exposed for  <dig> hours to ddv  alone or to ddv plus a 1-, 3-, or 10-fold excess of unlabeled rhc  or bont/a holotoxin  added to cultures simultaneously.
as seen in fig.
<dig>  in the absence of rhc or bont/a, ddv uptake and dextran separation were as expected; however, a 10-fold excess of rhc or bont/a holotoxin completely blocked the uptake of ddv.
these results suggested that ddv entry into neurons occurred by the same route as used by bont/a.
drug carrier could be separated from ddv in a fashion bont/a lc dissociates from the hc in the holotoxin
to determine the efficacy of delivering the therapeutic compound, we studied the separation of the drug carrier from ddv.
confocal microscopy was used to detect the separation of the ddv components.
spinal cord neurons were treated for  <dig> hours with  <dig> nm labeled ddv at 37°c.
the staining pattern of unseparated ddv was orange  and punctate .
the punctate nature of the staining suggested clustering of ddv in vesicles.
the images shown in fig.
<dig> highlight the presence of released drug carrier  in the particles present in the nerve terminal cytosol.
inclusion of endosome staining  indicated that the ddv was intra-endosomal  as expected for material transported by bont hc.
it indicated that the separation of the drug carrier from ddv was in a fashion similar to the dissociation of bont/a lc from the hc in the holotoxin.
separation of drug carrier from ddv was neuronal maturation-dependent
different stages of spinal cell culture growth were used to evaluate the efficacy of separation of drug carrier from ddv.
confocal image analysis revealed that about  <dig>   <dig> and 40% of the drug carrier component separated from ddv and diffused into the cytosol from endosomes in  <dig>   <dig> and  <dig> weeks culture, respectively .
these results indicated that the separation of the drug carrier from ddv is neuronal maturation-dependent.
furthermore, the separations of ddv components occur in a time-dependent manner .
the processes of exocytosis and endocytosis are not tightly coupled
it is possible that in neurons, the processes of exocytosis and endocytosis are tightly coupled, i.e., interruption of exocytosis, as in bont/a poisoning, might halt endocytosis as well.
if true, the ddv approach as presented here would not be a feasible drug delivery system in bont poisoned neurons because the uptake of ddv, via endocytosis, could be blocked as a sequel of exocytosis blockade by bont.
to discount this possibility, we demonstrated uptake of labeled ddv , of which oregon green  <dig> was omitted and and only cy <dig> was used, in spinal cord neurons previously exposed to a high concentration  of alexa 488-labeled bont-a ;  <dig> nm bont-a had completely blocked k+-stimulated glycine release.
to examine ddv-mas- <dig> uptake in these cells, the cells were washed once using warm culture medium and reincubated at 37°c with  <dig> nm ddv for  <dig> hours.
confocal microscopy results indicated that both bont/a and ddv were taken up in the same cell pool, but localized in separate population of endosomes , demonstrating internalization of ddv via endocytosis into bont/a poisoned neurons.
this suggested that the exocytosis and endocytosis are not tightly coupled in bont/a poisoned neurons.
discussion
primary cultures of spinal cord represent a convenient and sensitive system to study mechanisms of neurotransmitters release  <cit> .
internalized neurotransmitters by spinal cord neurons in culture were released quantitatively in response to depolarization and ca2+.
this release is inhibited by tetanus toxin and botulinum neurotoxins in a concentration- and time-dependent manner  <cit> .
therefore, this system serves as a suitable model to examine the efficacy of prospective bont countermeasures.
sheridan and adler indicated that the evoked release of neurotransmitters, notably glycine, in this system was time-dependently increased  <cit> .
in our studies, there was a pronounced time-dependent increase of the drug carrier separation from ddv, which paralleled an enhancement of transmitter release.
we postulate that the ability of drug carrier to separate from ddv in our spinal cord cell culture model is dependent upon neuronal maturation at increasing age of the cultures.
regarding ddv uptake and processing in neurons, the results in fig  <dig> clearly demonstrated the following facts.
the red   and the blue   fluorescence signals were strongly present in neurons at any stage of development, however, were always in a distinct punctate localization.
moreover, the red and the blue signals were always co-localized  and the red signals never diffused into the cytosol.
these observations indicated that the ddv was readily internalized into neurons apparently via endocytosis and the rhc component remained in the endosomes as generally believed to be the case in bont endocytosis and trafficking phenomena.
the hc remains localized in the endosome and not released into the cytosol.
these micrographs exhibit both punctuate and diffused distribution of signals with the level of diffusion apparently increasing with increased age of cultures.
this observation may be explained as follows: in mature neurons, the ddv is taken up into endosomes, the dextran component separates from the rhc and then gets released into the cytosol.
this explanation is supported by our results presented in the micrographs showing the overlays of red and green fluorescence  and those of green and blue fluorescence .
in d1-d <dig>  overlap of red and green generated orange indicating intact ddv; the released og488-dextran was green.
it should be noted that the orange was punctate suggesting endosomal localization, whereas the green was diffuse suggesting the release of og488-dextran component of the ddv into the cytosol, which was enhanced with increasing age of cultures.
using the images in d1-d <dig>  we calculated the separation and release rates of og488-dextran from the ddv by utilizing the bio-rad autodeblur and autovisualize software to quantitate fluorescence intensity.
the separation/release rates were expressed as 100%  minus percentage of co-localization rate .
in f1-f <dig>  overlap of bright blue and green generated light or greenish blue indicating og488-dextran remaining in the endosomes; the released og-dextran was green.
on examining the d1-d <dig>  e1-e <dig> and f1-f <dig> micrographs, it is interesting to note that intact ddv , rhc  and unreleased dextran all seem to be localized in the same endosomal pool; this provides additional support to our proposed mechanism of ddv uptake and processing.
relevant to this proposition, most significant was our demonstration that in neurons, the ddv function that required an efficient endocytotic mechanism was developmentally regulated, i.e., neuronal maturation-dependent.
moreover, our results showed that the efficiency in neuroexocytosis was also a function of mature neurons.
to demonstrate the feasibility of delivering a therapeutic compound via the ddv, we examined the separation of the prototype drug carrier dextran from ddv.
the results indicated that the drug carrier components were satisfactorily separated from ddv and diffused into cytosol.
as described in the results section, the targeting component of the ddv, i.e., rhc was nontoxic.
therefore, the ddv approach presented here may be a physiologically compatible and a feasible targeted drug delivery method to counteract botulism.
although it was believed to be the case, our results provided the first experimental evidence that the hc of bont/a upon internalization into neurons remains localized in the endosomes and thus, does not participate in the cytosolic mechanism of bont/a toxicity.
very meaningful was the fact that bont/a poisoned neurons, which were totally incapable of stimulated exocytosis, could still incorporate the hc via endocytosis.
this was apparently due to new or spare receptors available for hc molecule binding on the plasma membrane.
furthermore, the exocytosis and endocytosis phenomena in neurons may not necessarily be coupled tightly.
neale et al.
also reported that bont/a blocked synaptic vesicle exocytosis but not endocytosis at nerve terminal  <cit> .
in conclusion, this report provides new knowledge of endocytosis and exocytosis, as well as of bont trafficking and action.
notably, application of this ddv approach to antagonize botulism is not necessarily limited to the neuronal targeting of bont as shown here, but also should be useful for delivery of other prospective antidotes, such as protease inhibitors to protect the vesicle fusion proteins as applicable.
finally, the success in the ddv strategy against botulism shown here may open new avenues in developing technologies to treat other neurological disorders that require a targeted delivery of therapeutics to affected neurons or tissues.
before the actual studies are conducted, we can only speculate on a possible route of administration of the ddv as a therapeutic in a patient.
oral or inhalation routes are inadvisable.
the oral administration may result in ddv degradation in the gastrointestinal system.
the inhalation administration may result in a slower ddv absorption and may also require a high ddv concentration, which could possibly trigger a cell-mediated immune response.
based on these considerations, we propose the intravenous route which should rapidly achieve a high ddv level in the circulation for an effective drug delivery into bont poisoned nerve terminals.
CONCLUSIONS
an effective botulinum neurotoxin-based drug delivery vehicle can be used to directly deliver toxin inhibitors into the intoxicated nerve terminal cytosol.
the concept may possibly be utilized for drug delivery for other neuronal and neuromuscular disorders.
besides a bont therapeutic approach, this report also provides new fundamental knowledge of endocytosis and exocytosis as well as of bont trafficking in neurons.

 botulinum neurotoxins are produced by the anaerobic clostridium botulinum species of bacteria and are the cause of botulism, a life-threatening neuroparalytic disease. they are extremely potent food poisons, with a mouse ld of ng/kg for type a. aerosol exposure of bonts does not occur naturally, but could be attempted by bioterrorists to achieve a widespread effect. it has been estimated that a single gram of crystalline toxin, evenly dispersed and inhaled, could kill more than one million people. bonts are large proteins with a molecular weight of kda. they are produced as a complex containing the neurotoxins and associated proteins. they are synthesized as inactive single chain protoxins and are activated by protease nicking to form a dichain molecule and a kda heavy chain ) linked through a disulfide bond. the hc is responsible for binding to the target nerve cells and translocating the lc into the cell cytoplasm. inside the neuronal cytosol, the lc acts as a zn2+-endopeptidase against specific intracellular protein targets present either on the plasma membrane or on the synaptic vesicle, and inhibits neurotransmitter release by disabling the exocytotic docking/fusion machinery. bonts catalyze proteolysis of specific proteins of the soluble nsf attachment protein receptor complex that have been implicated in the exocytotic machinery. bont/a,/c, and /e cleave a kda synaptosomal associated protein. current therapy for botulism involves respiratory supportive care and the administration of antitoxin. the antitoxin could be the currently available equine bont antibodies or potentially more effective recombinant multivalent antibodies. however, only a few antitoxins, which must be administered before toxins reach the nerve cells, are available. thus, the therapeutic window for using an antitoxin is short. once the syndrome is developed, the antitoxin is less effective since it cannot penetrate the nerve cell to neutralize the toxin. the flaccid muscle paralysis caused by bont/a lasts for several months. therefore, patients who have already developed the syndrome must be put under respiratory intensive care during paralysis. should a bioterrorist attack occur, public health crisis could arise due to the lack of effective antidotes against botulism, especially in the absence of reliable presymptomatic diagnostics. for relief from bont-mediated paralysis, it is important to rescue the poisoned nerve cells through restoration of the neurotransmitter release process. while drugs have been designed to block the bont endopeptidase activity, which is believed to be responsible for the inhibition of neurotransmitter release, delivery of the drugs specifically to the poisoned nerve terminals remains a major hurdle. therapeutic targeting is important for two main reasons: delivering an effective high concentration of the therapeutic compound to the site of toxicity, i.e., nerve terminals for botulism, and minimizing systemic toxicity, if any, due to treatment compounds. at present, some examples of the proposed pharmacological antidotes for bont poisoning are a protease inhibitor, a phospholipase a activator or a modulator of intracellular free ca2+ concentration. since all of these parameters are involved in normal body functions, a systemic therapeutic approach is inadvisable due to potential toxicity concerns. therefore, we developed a drug delivery vehicle comprising the non-toxic recombinant heavy chain of bont-a coupled to a 10-kda amino dextran via the heterobifunctional linker 3--propionyl hydrazide. the heavy chain served to target botulinum neurotoxin-sensitive cells and promote internalization of the complex, while the dextran served as a platform to deliver model therapeutic molecules to the targeted cells.  structure of ddv initially we designed a ddv utilizing the recombinant bont/a heavy chain, which is known to specifically bind to the presynaptic nerve terminals and be internalized via endocytosis. the ddv construct was a modification of that developed by goodnough et al. consisting of a targeting molecule, cy labeled purified hc linked by a disulfide bond to a drug simulant, which was oregon green labeled kda dextran. the ddv structure was used for further experiments. to our knowledge, this is the first experimental demonstration of a prospective therapeutic approach to treat botulism in a relevant peripheral neuronal model combined with a feasible targeted drug delivery technology. rhc was a safe ddv component for delivery of bont antidotes to exclude any possible toxicity of the rhc component in our ddv construct, we compared the inhibition of mm k+ stimulated glycine release due to increasing concentrations of rhc or native bont/a holotoxin by the assay described in methods. in these experiments, the results obtained using the particular batch of toxin showed that bont/a was quite toxic, as expected, with an ic of approximately < pm and a total inhibition at ~ nm. however, a much higher concentration of rhc, up to nm, did not show any inhibition of glycine release under the same assay conditions. ddv entry into neurons via bont/a receptor mediated endocytosis the experimental design was to mimic a therapeutic application of the ddv strategy to treat individuals poisoned with bont/a and exhibiting clinical symptoms of botulism. since the targeted ddv approach is based on the premise of a selective entry of ddv into presynaptic nerve terminals via bont/a receptor mediated endocytosis, we demonstrated by competition experiments that the uptake of the ddv-mas- was via bont/a receptors. in these experiments, 3-week old spinal cord cultures were exposed for hours to ddv alone or to ddv plus a 1-, 3-, or 10-fold excess of unlabeled rhc or bont/a holotoxin added to cultures simultaneously. as seen in fig. in the absence of rhc or bont/a, ddv uptake and dextran separation were as expected; however, a 10-fold excess of rhc or bont/a holotoxin completely blocked the uptake of ddv. these results suggested that ddv entry into neurons occurred by the same route as used by bont/a. drug carrier could be separated from ddv in a fashion bont/a lc dissociates from the hc in the holotoxin to determine the efficacy of delivering the therapeutic compound, we studied the separation of the drug carrier from ddv. confocal microscopy was used to detect the separation of the ddv components. spinal cord neurons were treated for hours with nm labeled ddv at 37°c. the staining pattern of unseparated ddv was orange and punctate. the punctate nature of the staining suggested clustering of ddv in vesicles. the images shown in fig. highlight the presence of released drug carrier in the particles present in the nerve terminal cytosol. inclusion of endosome staining indicated that the ddv was intra-endosomal as expected for material transported by bont hc. it indicated that the separation of the drug carrier from ddv was in a fashion similar to the dissociation of bont/a lc from the hc in the holotoxin. separation of drug carrier from ddv was neuronal maturation-dependent different stages of spinal cell culture growth were used to evaluate the efficacy of separation of drug carrier from ddv. confocal image analysis revealed that about and 40% of the drug carrier component separated from ddv and diffused into the cytosol from endosomes in and weeks culture, respectively. these results indicated that the separation of the drug carrier from ddv is neuronal maturation-dependent. furthermore, the separations of ddv components occur in a time-dependent manner. the processes of exocytosis and endocytosis are not tightly coupled it is possible that in neurons, the processes of exocytosis and endocytosis are tightly coupled, i.e., interruption of exocytosis, as in bont/a poisoning, might halt endocytosis as well. if true, the ddv approach as presented here would not be a feasible drug delivery system in bont poisoned neurons because the uptake of ddv, via endocytosis, could be blocked as a sequel of exocytosis blockade by bont. to discount this possibility, we demonstrated uptake of labeled ddv, of which oregon green was omitted and and only cy was used, in spinal cord neurons previously exposed to a high concentration of alexa 488-labeled bont-a; nm bont-a had completely blocked k+-stimulated glycine release. to examine ddv-mas- uptake in these cells, the cells were washed once using warm culture medium and reincubated at 37°c with nm ddv for hours. confocal microscopy results indicated that both bont/a and ddv were taken up in the same cell pool, but localized in separate population of endosomes, demonstrating internalization of ddv via endocytosis into bont/a poisoned neurons. this suggested that the exocytosis and endocytosis are not tightly coupled in bont/a poisoned neurons. discussion primary cultures of spinal cord represent a convenient and sensitive system to study mechanisms of neurotransmitters release. internalized neurotransmitters by spinal cord neurons in culture were released quantitatively in response to depolarization and ca2+. this release is inhibited by tetanus toxin and botulinum neurotoxins in a concentration- and time-dependent manner. therefore, this system serves as a suitable model to examine the efficacy of prospective bont countermeasures. sheridan and adler indicated that the evoked release of neurotransmitters, notably glycine, in this system was time-dependently increased. in our studies, there was a pronounced time-dependent increase of the drug carrier separation from ddv, which paralleled an enhancement of transmitter release. we postulate that the ability of drug carrier to separate from ddv in our spinal cord cell culture model is dependent upon neuronal maturation at increasing age of the cultures. regarding ddv uptake and processing in neurons, the results in fig clearly demonstrated the following facts. the red and the blue fluorescence signals were strongly present in neurons at any stage of development, however, were always in a distinct punctate localization. moreover, the red and the blue signals were always co-localized and the red signals never diffused into the cytosol. these observations indicated that the ddv was readily internalized into neurons apparently via endocytosis and the rhc component remained in the endosomes as generally believed to be the case in bont endocytosis and trafficking phenomena. the hc remains localized in the endosome and not released into the cytosol. these micrographs exhibit both punctuate and diffused distribution of signals with the level of diffusion apparently increasing with increased age of cultures. this observation may be explained as follows: in mature neurons, the ddv is taken up into endosomes, the dextran component separates from the rhc and then gets released into the cytosol. this explanation is supported by our results presented in the micrographs showing the overlays of red and green fluorescence and those of green and blue fluorescence. in d1-d overlap of red and green generated orange indicating intact ddv; the released og488-dextran was green. it should be noted that the orange was punctate suggesting endosomal localization, whereas the green was diffuse suggesting the release of og488-dextran component of the ddv into the cytosol, which was enhanced with increasing age of cultures. using the images in d1-d we calculated the separation and release rates of og488-dextran from the ddv by utilizing the bio-rad autodeblur and autovisualize software to quantitate fluorescence intensity. the separation/release rates were expressed as 100% minus percentage of co-localization rate. in f1-f overlap of bright blue and green generated light or greenish blue indicating og488-dextran remaining in the endosomes; the released og-dextran was green. on examining the d1-d e1-e and f1-f micrographs, it is interesting to note that intact ddv, rhc and unreleased dextran all seem to be localized in the same endosomal pool; this provides additional support to our proposed mechanism of ddv uptake and processing. relevant to this proposition, most significant was our demonstration that in neurons, the ddv function that required an efficient endocytotic mechanism was developmentally regulated, i.e., neuronal maturation-dependent. moreover, our results showed that the efficiency in neuroexocytosis was also a function of mature neurons. to demonstrate the feasibility of delivering a therapeutic compound via the ddv, we examined the separation of the prototype drug carrier dextran from ddv. the results indicated that the drug carrier components were satisfactorily separated from ddv and diffused into cytosol. as described in the results section, the targeting component of the ddv, i.e., rhc was nontoxic. therefore, the ddv approach presented here may be a physiologically compatible and a feasible targeted drug delivery method to counteract botulism. although it was believed to be the case, our results provided the first experimental evidence that the hc of bont/a upon internalization into neurons remains localized in the endosomes and thus, does not participate in the cytosolic mechanism of bont/a toxicity. very meaningful was the fact that bont/a poisoned neurons, which were totally incapable of stimulated exocytosis, could still incorporate the hc via endocytosis. this was apparently due to new or spare receptors available for hc molecule binding on the plasma membrane. furthermore, the exocytosis and endocytosis phenomena in neurons may not necessarily be coupled tightly. neale et al. also reported that bont/a blocked synaptic vesicle exocytosis but not endocytosis at nerve terminal. in conclusion, this report provides new knowledge of endocytosis and exocytosis, as well as of bont trafficking and action. notably, application of this ddv approach to antagonize botulism is not necessarily limited to the neuronal targeting of bont as shown here, but also should be useful for delivery of other prospective antidotes, such as protease inhibitors to protect the vesicle fusion proteins as applicable. finally, the success in the ddv strategy against botulism shown here may open new avenues in developing technologies to treat other neurological disorders that require a targeted delivery of therapeutics to affected neurons or tissues. before the actual studies are conducted, we can only speculate on a possible route of administration of the ddv as a therapeutic in a patient. oral or inhalation routes are inadvisable. the oral administration may result in ddv degradation in the gastrointestinal system. the inhalation administration may result in a slower ddv absorption and may also require a high ddv concentration, which could possibly trigger a cell-mediated immune response. based on these considerations, we propose the intravenous route which should rapidly achieve a high ddv level in the circulation for an effective drug delivery into bont poisoned nerve terminals.  an effective botulinum neurotoxin-based drug delivery vehicle can be used to directly deliver toxin inhibitors into the intoxicated nerve terminal cytosol. the concept may possibly be utilized for drug delivery for other neuronal and neuromuscular disorders. besides a bont therapeutic approach, this report also provides new fundamental knowledge of endocytosis and exocytosis as well as of bont trafficking in neurons. 
an effective medical countermeasure strategy would require developing a drug that could rescue poisoned neuromuscular synapses and include its efficient delivery specifically to poisoned presynaptic nerve terminals. the drug delivery vehicle consisted of the non-toxic recombinant heavy chain of botulinum neurotoxin-a coupled to a 10-kda amino dextran via the heterobifunctional linker 3--propionyl hydrazide. once internalized into neurons, the drug carrier component separated from the drug delivery vehicle in a fashion similar to the separation of the bont-a light chain from the holotoxin. our results indicated that the drug delivery vehicle entry into neurons was via bont-a receptor mediated endocytosis. this drug delivery vehicle could be used to deliver bont-a antidotes into bont-a intoxicated cultured mouse spinal cord cells. an effective bont-based drug delivery vehicle can be used to directly deliver toxin inhibitors into intoxicated nerve terminal cytosol. here we report a drug delivery strategy that could directly deliver toxin inhibitors into the intoxicated nerve terminal cytosol. this report also provides new knowledge of endocytosis and exocytosis as well as of bont trafficking. the heavy chain served to target botulinum neurotoxin-sensitive cells and promote internalization of the complex, while the dextran served as a platform to deliver model therapeutic molecules to the targeted neurons. this approach can potentially be utilized for targeted drug delivery to treat other neuronal and neuromuscular disorders.
 botulinum neurotoxin is the most potent poison known to mankind. currently no antidote is available to rescue poisoned synapses. an effective medical countermeasure strategy would require developing a drug that could rescue poisoned neuromuscular synapses and include its efficient delivery specifically to poisoned presynaptic nerve terminals. here we report a drug delivery strategy that could directly deliver toxin inhibitors into the intoxicated nerve terminal cytosol.  a targeted delivery vehicle was developed for intracellular transport of emerging botulinum neurotoxin antagonists. the drug delivery vehicle consisted of the non-toxic recombinant heavy chain of botulinum neurotoxin-a coupled to a 10-kda amino dextran via the heterobifunctional linker 3--propionyl hydrazide. the heavy chain served to target botulinum neurotoxin-sensitive cells and promote internalization of the complex, while the dextran served as a platform to deliver model therapeutic molecules to the targeted neurons. our results indicated that the drug delivery vehicle entry into neurons was via bont-a receptor mediated endocytosis. once internalized into neurons, the drug carrier component separated from the drug delivery vehicle in a fashion similar to the separation of the bont-a light chain from the holotoxin. this drug delivery vehicle could be used to deliver bont-a antidotes into bont-a intoxicated cultured mouse spinal cord cells.  an effective bont-based drug delivery vehicle can be used to directly deliver toxin inhibitors into intoxicated nerve terminal cytosol. this approach can potentially be utilized for targeted drug delivery to treat other neuronal and neuromuscular disorders. this report also provides new knowledge of endocytosis and exocytosis as well as of bont trafficking. 