Anew study by a team of Indian medical researchers on accessing the peripheral nervous system (PNS) by topically applied nanoparticles clearly suggests that nanoparticles with neurotropic targeting ligands can be utilised for quickly delivering nanoparticles to neuronal cell bodies via axonal transport mechanisms. This novel approach with clinical applications in several PNS disorders, studied by researchers at Center for Nanosciences and Molecular Medicine, Amrita Institute of Medical Sciences and Research Center, Amrita Vishwa Vidyapeetham University, Kochi, was published in the January issue of Scientific Reports.
According to the researchers, this new approach attempted to target dorsal root ganglia (DRG) neurons, via their axons, by topical application of lectin-functionalized gold nanoparticles (IB4-AuNP) since the skin is richly innervated by long peripheral axons that arise from cell bodies located distally within ganglia.
Neurons of the peripheral nervous system are involved in the processing and propagation of electrical signals to and from the central nervous system. Strategies to deliver therapeutic molecules to the peripheral neurons have the potential to be used in several clinical conditions, such as neuropathic pain, neuropathies, nerve injuries and regeneration. The cell bodies of peripheral neurons give out axons that form distinct bundles within nerves and innervate limbs, organs, and other tissues. Although axons are an important therapeutic target for various nerve disorders, the neuronal cell body itself can also be potentially targeted for the alteration of gene and protein expression.
Novel Delivery System
Attempts to deliver drug molecules to the peripheral neurons have been difficult, primarily because of the restrictive neuroanatomical distribution of the peripheral nerves, and also due to the presence of the tight blood-nerve barrier that protects the nerve endoneurial microenvironment. The systemic route of drug delivery is associated with high drug concentration-related off-target effects, while local delivery around a nerve, as used in regional and nerve block anaesthesia, are quite limited in their application.
To circumvent many of these limitations related to nerve-drug delivery, nanoparticles have been utilised to specifically target neural tissue. Attempts to deliver specialized nanoparticles of various sizes and surface decorations to neural tissue have yielded mixed results. Though nanoparticles are small enough to be passively transported through most cell junctions, the blood-nerve barrier still limits nanoparticle entry. Direct administration of nanoparticles locally around a peripheral nerve has been shown to result in partial entry of nanoparticles into the nerve substance, though their distribution specifically within axons and cell body is not known. Additionally, direct application of nanoparticles around the nerve requires expertise in the neuroanatomical location of the nerve and the procedure itself can be technically challenging. In the midst of these limitations, a possible alternative strategy to access peripheral axons and consequently the neuronal cell bodies could be from the skin. The skin is innervated by a vast number of axonal terminals, which are the branched endings of peripheral nerves.
Several neurotropic viruses, such as the rabies virus, are known to utilise peripheral axons to gain access into individual neurons and translocate themselves to neuronal cell bodies,
and even to the brain.
In this study, the researchers took motivation from such neurotropic viruses and attempted to deliver gold nanoparticles via skin nerve endings to neuronal cell bodies. It has been previously reported that topically applied metallic nanoparticles not only penetrate skin layers, but also tend to aggregate in the nerve fibre rich, epidermal-dermal junction. Here the researchers demonstrate that the epidermal-dermal fibres of sensory neurons can be used to deliver nanoparticles to their respective cell bodies, which potentially has wide applications in treating conditions such as neuropathic pain and other neuropathies.
“The use of nanoparticles for delivering therapeutic molecules to the nervous system has immense potential for future clinical strategies. However, once administered, nanoparticles face a daunting task in reaching their target areas in the nervous system, primarily due to barriers that are unique to the brain and nerve. In this study, we set out to explore a new route to deliver nanoparticles to the cell bodies of neurons, that could supplement, if not replace the existing routes of delivery to the nervous system,” says Dr Sahadev A Shankarappa, the lead scientist and author of the paper.
He added that; “For this study we drew motivation from the fascinating rabies virus that is well known to enter cell bodies of neuron via their axons. Axons are long extensions of neuron cell bodies that innervate distant regions of the body. As a proof of concept, we attempted to deliver gold nanoparticles to sensory neurons whose axons are richly innervated in the skin.”
This route of delivering nanoparticles potentially has significant implications in treating disorders such as neuropathic pain, neuritis, and amyotrophic lateral sclerosis, Dr Shankarapa said in an interview with Future Medicine.
Least Explored Target
Peripheral nerve fibres innervating the skin are arguably the least explored targets for delivering therapeutic molecules and their carriers. Barring topical anaesthetics that are used for blocking nociceptive receptor activity on peripheral axons, clinically there are almost no other drug molecules or delivery systems that target peripheral nerve fibres. In this study, 105.3 ± 1.0 nm-sized AuNP were functionalized with IB4 to target axonal terminals and promote binding and uptake. Surface functionalisation of AuNP with IB4 resulted in the reduction of total surface charge by almost − 20 mV, and an SPR peak shift that was most likely due to the positively charged lysine and tryptophan-rich sites on IB4.
IB4-AuNP were found to bind sensory neurons dissociated from rat DRGs. It is worth noting that IB4 was selected from a list of known neurotropic ligands, including rabies virus glycoprotein (RVG), tetanus toxin C, and wheat germ agglutinin. Though RVG29 has been used to target Neuro 2A cells and mouse brain cells in culture, we were unable to detect RVG29 binding and uptake in DRG-derived sensory neurons. IB4 was selected based on its selectivity in binding sensory neurons, and for possible future application in pain mitigation. In addition, IB4 has been reported to bind almost 70% of small-diameter sensory neurons, which are known to predominately carry nociceptive signals and express pain-associated proteins such as Nav1.8, Nav1.9, and TRPV family of receptors. The study observed IB4-AuNP bound to the cell membrane of sensory neurons, and additionally found AuNP excitation scatter within neurons, suggestive of uptake. Here, it is important to note that the study has utilised the intrinsic property of AuNP to scatter light, for direct visualization under a confocal microscope. It is well-known that at specific wavelengths of the incident light, the surface plasmon resonance phenomena of 100 nm-sized AuNP cause light to be reflected in the 650–700 nm range.
IB4-AuNP were found bound to axonal fibres in the dermal-epidermal regions, and also to a few intra- epidermal nerve fibres in thin skin sections. Individual nerve fibres in the skin measure about 1–4 microns in diameter, which allows for the possibility of nanoparticles to bind and enter terminal axons. Previously, researchers had observed that gold nanoparticles in the size ranges of 20 nm to 180 nm, easily penetrate all layers of thick skin, and appear in the bloodstream within 4 days. In addition, topically applied gold nanoparticles accumulate in the nerve fibre rich, epidermal-dermal regions, thereby significantly increasing the chances of nanoparticle-nerve fibre contact. The in vitro studies very clearly showed that IB4-AuNP bind axonal terminals and undergo retrograde axonal transport to the cell bodies. Previous in vitro studies show that quantum dots and iron oxide nanoparticles undergo endosome-associated transport within axons via the dynein-microtubule system.
“Our results agree quite well with this observation since disruption of microtubule polymerization by colchicine, prevented the axonal transport of AuNP. Interestingly our in vitro data also shows a slight decrease in fluorescence intensity at the 4th-hour time-point and an increase in data variability at subsequent time points,” says the authors.
This is curious, because it has been well reported that metallic nanoparticles undergo cellular exocytosis from various cell-lines, though such phenomenon has not been reported in primary sensory neurons. “It is plausible that the reduction in fluorescence intensity at the soma could be due to differences in the rate of nanoparticle transport and nanoparticle exocytosis. More focused studies are warranted to better explore this phenomenon in differentiated neurons,” they suggested in the paper.
Further, the researchers added in the report that the in vivo experiments clearly showed a larger amount of topically applied IB4-AuNP accumulating in the ipsilateral lumbar DRG as compared to the contralateral control. This observation was quite similar to what was observed in cultured axons, where IB4-AuNP accumulated in the cell soma after application at the axonal end.
The study has also observed that the nanoparticles in sciatic and DRG tissue harvested from rats that were topically exposed to AuNP, albeit in very low amounts. Taken together, this data strongly supports the hypothesis that topically applied IB4-AuNP accumulates in the DRG, in large part, by utilising the axonal innervation from the topical site. Since DRG neurons synapse with second-order neurons in the dorsal horn of the spinal cord, it is plausible that topically applied AuNP may translocate to the spinal cord, but this was not checked in this study.
Considering the study observation that AuNP were detected in contralateral DRG and sciatic nerve, it is possible that part of the total nanoparticles detected in the ipsilateral DRG of rats exposed to IB4-AuNP could also be due to implantation from the systemic circulation. It has been shown that nanoparticles as large as 180 nm can penetrate across intact rat hind-paw skin and appear in the bloodstream 4 days later, the report suggests.
Another alternative possibility for IB4-AuNP accumulating in the ipsilateral DRG, could be due to the localized distribution of nanoparticles in the ipsilateral limb, resulting in axonal entry and nanoparticle translocation via nerve fibres innervating the muscle. However, the predominant nerve fibres innervating the muscle tend to be motor with nerve terminals ending in the spinal cord, thus making this route of AuNP delivery less probable.
In the study, it was also interesting to note that even though rats were exposed to an almost similar amount of topical AuNP in the IB4-functionalized and non-IB4 functionalized groups, the total amount of IB4-AuNP that were absorbed through the skin was much lower compared to non-functionalized AuNP. This is of much relevance because IB4 functionalisation seemed to reduce the number of particles actually crossing the skin, even though the total amount of AuNP accumulating in the ipsilateral DRG was much higher. One probable explanation for the reduced permeability of IB4-AuNP compared to citrate capped AuNP could be the uneven surface distribution of lectin on the IB4-AuNP surface. Simulation studies suggest that nanoparticles with homogeneously distributed binding moieties on their surface, similar to our citrate-capped AuNP, dramatically enhance permeability, compared to nanoparticles with random or heterogeneously distributed ligands. Furthermore, the presence of AuNP within the sciatic nerve in amounts as seen in this study, did not affect nerve function, suggesting the benign nature of this approach.
“Our experiments also showed that the concentration of nanoparticles used in the study did not detrimentally effect the functioning of the nerve, though further studies need to done to confirm complete safety,” says Neeraj Katiyar, the first author of the paper.
“We haven’t also found any aberrant behavioural alterations that could indicate any sensory dysfunction. However, further safety studies need to be performed to determine the functional and cellular effects of nanoparticles within axons. But, we could clearly demonstrate that metallic nanoparticles can be specifically translocated to neuronal cell bodies using axonal terminals located in the skin via simple topical application,” concluded the researchers.