Source: IOWA STATE UNIVERSITY submitted to NRP
THE DESIGN AND TESTING OF A DELIVERY VEHICLE FOR SELECTIVE INTRACELLULAR TARGETING OF BONT/A ANTIDOTE
Sponsoring Institution
Cooperating Schools of Veterinary Medicine
Project Status
COMPLETE
Funding Source
STATE
Reporting Frequency
Annual
Accession No.
0194226
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Jul 1, 2002
Project End Date
Jun 30, 2004
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
IOWA STATE UNIVERSITY
S. AND 16TH ELWOOD
AMES,IA 50011
Performing Department
VETERINARY MEDICINE
Non Technical Summary
A post-exposure pharmacological treatment for botulism has been pursued for over a decade but has met with only limited success. The purpose of this research is to describe the design of a delivery vehicle for selective intracellular targeting of BoNT A antidote.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
71240101150100%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1150 - Toxicology;
Goals / Objectives
The clostridial neurotoxins (CNTs) tetanus toxin (TeNT) and the family of botulinum toxins (BoNT) are related toxins responsible for the clinical symptoms of tetanus and botulism respectively. The recent interest in botulinum neurotoxins comes from their potential use as biological weapons. Extremely minute quantities of toxin could be introduced into the food supply and have a tremendous impact on our ability to guarantee safe, wholesome food. The toxins act intracellularly and their effects are extremely difficult to reverse. As a result we do not currently have the ability to easily and effectively treat individuals with botulism. There is a high case fatality rate and those individuals that survive often require months of intensive care with artificial respiration. Willful introduction of botulinum toxin into the food supply could have a devastating effect on all aspects of agriculture and our food supply system. The toxins are synthesized as a 150 kDa polypeptide that is subsequently cleaved into two chains (heavy and light). The heavy chain is known to bind specifically to neuronal cells. This binding facilitates transport of the 50kDa light chain into the neuronal cell. The light chain is the cleaves proteins involved in exocytosis and inhibits neuromuscular transmission. Current treatment of BoNT intoxication is largely symptomatic, directed at maintaining respiratory and cardiovascular function. Since botulism leads to protracted paralysis with long-lasting consequences, it is important to develop effective pharmacological treatments for BoNT toxicity. The objective is to develop a unique method for delivery of specific antidotes for botulinum toxins into the cells targeted by these toxins.
Project Methods
The present proposal represents a rational approach towards the use of the nontoxic heavy chain fragment of tetanus toxin (TeNT Hc) to deliver a well characterized selective metalloprotease inhibitors for BoNT A to neurons both in vitro and in vivo. The following steps are involved in the synthesis of delivery vehicle (DV) FITC TeNT Hc dextran glycoprotein: oxidation of the hydroxyl groups in the dextran to allow attachment of the linker, labeling of the dextran with Oregon green 488, and coupling of the labeled dextran and FITC TeNT Hc. Coupling requires disulfide exchange of one of four sulfhydryls of the FITC TeNT Hc with the functional group of the linker to constitute the complete DV. Cells will be exposed to DV, the FITC TeNT Hc, or Oregon green 488 labeled dextran in growth medium for 24 h. The ability of DV to undergo retrograde transport in the rat nervous system will be investigated. 3 to 4 week old rats will be injected in the tongue with FITC TeNT Hc fragment or FITC TeNT Hc fragment or DV. The ability of the complex to be transported to the hypoglossal nucleus within the brainstem will be visualized after 24 h. The inhibitor ICD 1578, is the most potent inhibitor of BoNT, will be assessed for its ability to inhibit the catalytic activity of BoNT A LC using a sensitive fluorescence assay. Since there will be no chemical modification of the ICD 1578, the inhibitory ability of the drug will be preserved to ensure the efficacy of the proposed neuronal targeting DV system. The pH sensitivity of the cross-linking groups of these will induce swelling of the microstructures upon endocytosis and thereby release the entrapped inhibitory drug molecules to the targeted sites. In vitro evaluation of the effect of DV ICD 1578 on BoNT A induced inhibition of exocytosis will be done on cortical neurons. Neuron cultures will be exposed to BoNT A for 18 to 72 h as appropriate. The effect of BoNT A on the levels of SNAP25 will be confirmed by immunocytochemistry. BoNT A cultures will be stimulated for 2 min with 50mM K in order to determine the degree of glutamate release inhibition evoked by BoNT A pretreatment. Levels of glutamate will be determined in perfusate using HPLC. Additional experiments with BoNT A exposed cultures will be done 24, 48 and 72 h post exposure. These data will be used as control to determine the efficacy of ICD 1578 inhibition of BoNT A. DV ICD 1578 will be added to the cultures 24, 48 and 72 h following BoNT A exposure. In vivo evaluation of the effect of DV ICD 1578 on BoNT A induced paralysis will be tested on long digital extensor muscle. Extensor digitorum longus (EDL) muscles in rats will be injected locally with 5 mouse LD50 units of BoNT A. Muscle tensions will be elicited by supramaximal stimulation of the peroneal nerve to monitor effect of BoNT intoxication. DV ICD 1578 complex will be injected after total paralysis developed. DV ICD 1578 complex will be injected locally into the EDL as well systemically.

Progress 01/01/03 to 12/31/03

Outputs
The botulinum toxins (BoNTs) are best known for their role in mediating food-borne botulism. Although an effective vaccine is available, the relatively low incidence of botulism makes widespread vaccination impractical. A post-exposure pharmacological treatment has problems of low efficacy. The heavy chain (HC) is known to bind specifically to presynaptic neuronal cells, and the light chain (LC) penetrates the cell to inhibit neurotransmitter release. There are three potential targets for antagonism of BoNT action: 1. the ectoacceptor on the surface of nerve terminals, 2. the exocytotic apparatus that mediates toxin internalization and 3. the active site of the LC. Attempts to inhibit the active site of the BoNT LC have been confined to the use of zinc chelators and the metalloprotease inhibitors. The chelators were found to be only marginally effective with high systemic toxicity. This proposal describes the design of a delivery vehicle for selective intracellular targeting of BoNT/A antidote. The delivery vehicle for selective intraneuronal delivery of BoNTx antidote consist of the TeNT heavy chain (HC) fragment. The specificity of the HC fragment of TeNT for neuronal cells is high. An MCM-41 type mesoporous silica nanosphere-based (MSN) controlled release delivery system has been synthesized and characterized using surface-derivatized cadmium sulfide (CdS) nanocrystals as chemically removable caps. The biocompatibility and delivery efficiency of the MSN system with astrocytes in vitro were demonstrated. In contrast to many current delivery systems, the molecules of interest were encapsulated inside the porous framework of the MSN. We envision that this new MSN system could play a significant role in developing new generations of site-selective, controlled release delivery nanodevices. The delivery vehicle (DV) consists of the isolated heavy chain of TeNT-Hc coupled to a MSN via the heterobifunctional linker. The TeNT-Hc served to target BoNT-sensitive cells and promote internalization of the complex, while the MSN is to serve as a platform to deliver therapeutic molecules to the targeted cells. To demonstrate that cultured neurons internalize TeNT-Hc cortical cells were exposed to FITC- TeNT-Hc. Cortical neurons internalized FITC-TeNT Hc in high percentage. The staining pattern of the dye was punctate and perinuclear. The punctate nature of the staining suggests clustering of FITC-TeNT-Hc in vesicles. Exclusion of nuclear staining indicates that the DV is intracellular, as expected for material transported by BoNT- Hc. Staining of soma and processes was of approximately equal intensity.

Impacts
The problems of low efficacy were solved by new synthetic approaches, the specific neuronal targeting of drugs is needed for selective intracellular delivery of sufficiently high concentrations to inhibit the proteolytic activity of the light chain of botulinum toxin.

Publications

  • Lai C,-Y., D. M. Jeftinija, K. Jeftinija, S. Jeftinija, S. Xu, and V. Lin. 2003 Mesoporous silica nanosphere-based carrier system with chemically removable CdS nanoparticle caps for stimuli responsive controlled release of neurotransmitters and drug molecules. JACS, 125:4451-4459.


Progress 01/01/02 to 12/31/02

Outputs
New project: no progress report at this time.

Impacts
(N/A)

Publications

  • No publications reported this period