Discussion
Primary cultures of spinal cord represent a convenient and sensitive system to study mechanisms of neurotransmitters release.[11,12] 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.[13–17] 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.[18] 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 4 clearly demonstrated the following facts. The red (Cy3-rHC) (A1-A3) and the blue (Alexa 633-endosome) (C1-C3) 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 (E1-E3) 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 (D1-D3) and those of green and blue fluorescence (F1-F3). In D1-D3, 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-D3, 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% (total in images) minus percentage of co-localization rate (Table 1). In F1-F3, 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-D3, E1-E3 and F1-F3 micrographs, it is interesting to note that intact DDV (D1-D3), rHC (E1-E3) 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.[20]
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.
BMC Pharmacol. 2009;9(12):1-9. © 2009
Cite this: An Efficient Drug Delivery Vehicle for Botulism Countermeasure - Medscape - Oct 27, 2009.
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