**7. Uptake of PABA nanomaterials by clathrin dependent endocytosis**

To rule out the possibility of cellular uptake of PABA conjugates via caveolae or lipid rafts pathway, we pre-treated the cells with drug filipin and nystatin, which disrupt cholesterol distribution within the cell membrane. In contrast to clathrin blocking experiments, pretreatment of the drugs had a negligible blockage on the cellular uptake, which suggests a little or no involvement of the caveolae dependent cell entry. In a similar control experiment we studied the uptake of fluroscent labelled cholera toxin B (CTX-B) which is a multivalent ligand protein known to be internalised by caveolae depenent endocytic pathway (Figure 3E). The CTX-B showed a significant inhibition in cell entry in the presence of filipin and nystatin. Taken together, the results verify that cellular internalization of PABA conjugates is mediated through the clathrin-dependent endocytosis pathway.

### **8. Oral uptake of variable PABA nanomaterials in Drosophila**

Organic nano-assemblies have negligible adverse effect on cellular physiology, behaviour, sensitivity to adult sex and other pharmacokinetics parameters of Drosophila. We have screened nanoparticles conjugated with variable side chains for organ specific targeting in Drosophila. Different sets of larvae, pupae and adult flies were grown with sole feeding of nanoparticle containing media. The accumulation to various tissues, selective organ uptake and their clearance was also monitored by imaging the fluorescence signals during the stages of development in Drosophila. In live insects, oral feeding of nanomaterials causes systemic spreading of signals through the gut by peristaltic movement tocross the cell membrane barrier. In general, majority of the nanoparticles carrying unsaturated side chains (C-11U, C-18U) showed a low level of incorporation in all stages of Drosophila life cycle although C-18U showed a comparatively high level of incorporation in two different life stages, larvae and pupae (Additional File 2 Figure S16A-B). We further investigated the efficacy of in vivo targeted delivery among nanoparticles that emits intrinsic green and nanoparticles with intercalated rhodamine B in the wall. Intrinsic green nanostructures carrying C-16 side chain showed a maximum amount of incorporation through cell membranes, compared to C-18, and C-11 that showed a variable amount of incorporation in different developmental stages. Animals fed with C-18 selfassembled particles exhibit a maximum incorporation during larval stage as compared to other tested stages. Animals fed with C11 showed an overall low level of entry in all the stages of development.

under fluorescence microscope. A wide range of variation in fluorescence intensity was observed in different larval body parts, for example mouth, brain, larval neural ganglia, salivary glands, alimentary canal and malpighian tubules etc (Figure 4; Additional File 2 Figure S16). A clear contrast was observed in the delivery of nanomaterials in the salivary glands. C-14 and C-18 containing nanostructures incorporated at a massive level in the glands but shows an intermediate level of incorporation in both neural tissues and organism itself (Figure 4). Surprisingly, we observed that malphigian tubules absorbed more nano-particles that emit intrinsic fluorescence. Therefore nanoparticles with different side chains showed a distinct distribution in various internal tissues in the larvae. Nanoparticle entry showed a clear variation in rapidly dividing cells of mature larval imaginal discs (the precursor organs of adult wings, eyes and legs). PABA conjugated with C-16 side chain showed a higher intensity of uptake in all three discs tested. However the intensity of fluorescence is moderate in C-11U, C-12 and C-18 particles (Figure 4). It suggests that the structure and surface texture of C-16 side chain is the most effective cargo for delivery in precursor and rapid dividing cells, though we cannot rule out other unmet criteria in the tracking process (Figure 4). As described above, the delivery of C-12 structure in all the stages of development is ideal compared to C-14 and nanomaterials with unsaturated side chains in C-11U and C-18U. Surprisingly a differential uptake of nanomaterials produced by C18 and C18U specially in leg discs that possess same number of carbon bonds interprets that length of the side chain is not an only criteria for nano-

Organic Nanotubes: Promising Vehicles for Drug Delivery

http://dx.doi.org/10.5772/58412

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The conjugated side chains of PABA nanostructures were also screened for delivery to complex adult body parts derived from same sets of larval imaginal discs. Entry of nanomaterials was analysed in adult eyes, halters and legs. Incorporation in adult eyes is complicated and novel from other body parts. Two different fluorescent tags showed distinct uptake through eye ommatidia (Figure 4) raising the possibility that difference in fluorophore emission and structure make their entry visible and distinct in adult eyes. The intrinsic green showed a poor emission through ommatidia. Only a trace amount of green colour was visualized whereas rhodamine B showed a greater intensity with a maximum incorporation of C-16 in the eyes. However, the incorporation pattern of nanomaterials conjugated with variable side chains in halters and legs is distinct from their distribution in eyes. Among all possible nanostructure, C-11U and C-18U were targeted orally at a maximal level to the legs while C-11, C-12 and C-16 in the halters showed an equal but greater amount of incorporation, which suggests that the unsaturated carbon chains have advantage for selective entry in the accessory organs of Drosophila. Taken together, the delivery of nanoparticles associated with variable side chains in the culture cells and in

Furthermore distribution of numerous neurons and other cells make brain more compact and the delivery of therapeutic agents in the neuronal tissues is the most challenging task. In spite of complicated entry in brain, two nanoparticles, C-11 and C-16 containing particles show a considerable amount of entry when incorporation of other particles is nominal in the brain (Figure 4). Truly, greater dissemination of nanostructures in adults, larvae and different body

particles based delivery in imaginal discs.

vivo uptake by oral delivery in different body parts is different.

Delivery of rhodamine B embedded nanoparticlesC-12 showed an equal and maximal level of incorporation in all stages of development. The intensity was conspicuously greater than the nano-structure carrying C-14 chain. Taken together, specific carbon chains and associated morphologies of nanostructures brought a potential difference in entry through gut cell walls. These results suggest the possibility that the physiology of gut cells in different stages of the life cycle might influence nanoparticles uptake.

For in vivo tracking, fluorescence dyes attached to nanoparticles suffer with multiple problems including photo-bleaching and ability to interrogate multiple targets etc. The aftermath effect of such limitations of fluorescence imaging in live objects was described earlier. In all cases, during in vivo delivery there was no photobleaching of the nanomaterials through all stages of development providing a better advantage in tracking in live systems. But the fluorescence intensity was reduced conspicuously after extending the culture on an average of 18-25 days and nearly eliminated within 40-45 days allowing a total clearance of fluores-cence from the live tissues. We further screened the efficacy of nano-particles inheritance through germ cells. The adult flies emerging from sole feeding of nanoparticle containing media were cultured in normal food media for another 7 days. The fertilized eggs from different batches of flies after nanoparticle feeding emits only trace amount of fluorescent as a background effect. Therefore, this ineffective route of gem cell based heritable transmission prevents nanomaterials con‐ tamination in the environment and their natural entry into the food chain via eco-consumers.
