**9. Efficiency of organ specific delivery of PABA nanomaterials by side chain variation in Drosophila**

To categorize the intensity of fluorescent molecules as an absolute reflection on efficacy of nanoparticle delivery, different internal body parts of the larvae were dissected and visualized 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 nanoparticles based delivery in imaginal discs.

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

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

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.

**9. Efficiency of organ specific delivery of PABA nanomaterials by side**

To categorize the intensity of fluorescent molecules as an absolute reflection on efficacy of nanoparticle delivery, different internal body parts of the larvae were dissected and visualized

of entry in all the stages of development.

462 Application of Nanotechnology in Drug Delivery

life cycle might influence nanoparticles uptake.

**chain variation in Drosophila**

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

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 parts including brains suggests that physio-chemical properties including shapes, surface texture of the C-11 and C-16 particles are the best-fitted materials (Figure 4).

Such accumulation might give rise to artefact in cellular uptake of micro-sized aggregates as found in artifactual intake of HIV TAT peptide at 4°C [23]. Therefore, cellular entry of PABA might depend on the size of the nanoparticles which is mainly guided by the acid side chain.

Organic Nanotubes: Promising Vehicles for Drug Delivery

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

465

Finally, a systematic screening of PABA conjugated library provides sufficient evidences to support the following statements: 1) Two nanomaterials carrying C-11 and C16 acid side chains are best suited for optimal entry in cells and multiple organs. 2) In live tissues, an internal environment might be a useful barrier for improving nanoparticles delivery in multiple organs. 3) Cellular internalization or uptake mechanism of nanomaterials might unravel the clues for smooth entry in human cells and efficient delivery and 4) finally screening of PABA conjugates determine a functional relationship between energy dependent endocytosis and nanomaterial

We have shown that two carbon linker group C-11 and C-16 forms tubular nanomaterials that are best fitted for mass oral delivery in complex multiorgans. The cellular uptake mechanism is energy dependent endocytosis. The detailed endocytosis pathways for nano PABA structure is operated thorough clathrin-coated pits rather than caveolae or lipid rafts. In vivo screening of PABA nanomaterials produced by different acid side chain select the compatible nano structure ideal for oral delivery and establishes energy dependent entry mechanism is of fundamental importance that will facilitate future developments of PABA nanoparticle

, Jagannadh Bulusu3

2 Centre for Chemical Biology, CSIR-Indian Institute of Chemical Technology, Hyderabad,

3 Division of Organic Chemistry-I, Indian Institute of Chemical Technology, Hyderabad, India

4 Ex-Director & CSIR Bhatnagar Fellow, Indian Institute of Chemical Technology, Hyderabad,

We are highly sympathetic due to sudden demise of Dr Jagannath Bulusu and we hope his

1 Functional Genomics & Gene Silencing Group, CSIR-Centre for Cellular & Molecular

and J.S. Yadav4

structure for each organ specific targeting.

transporters for biological delivery application

, Manika Pal Bhadra2

**11. Conclusions**

**Author details**

Biology, Hyderabad, India

Utpal Bhadra1

India

India

soul lies in peace

#### **10. Perspective**

The key parameters of nanomaterials for easy and efficient delivery are shape, size and flexibility to enter and exit cell barrier. Our results clearly demonstrate that the properties of each acid side chain together with common PABA moiety influences size, shape and surface texture of nanomaterials that lead to differential uptakeand specificity in live cell delivery. The physio-chemical modifications of organic nano carriers also affect cell internalization mecha‐ nisms in sub-cellular organelles as found by distinct accumulation pattern of each nanomate‐ rials following same energy dependent endocytosis. In vivo screening also showed that only C 11 and C-16 produce compatible shape and size of nanomaterials that are best fitted for easy delivery of PABA nanomterials. These results suggest that physical structure of nanomaterials and chemical properties of acid side chain required for self assemble procedure and size variation could be the initial step for cellular uptake.

In addition to cultured cells, tissue specific distribution specifically in adult eyes, imaginal discs, alimentary tracks and neuronal tissues was complex and needs more parameter to consider. Our data revealed that a complex interrelationship of PABA conjugates and cell physiological environment is important in live materials delivery. The internal tissue envi‐ ronment might provide additional barriers for nanomaterial entry as depicted by comparing variable accumulation of same nanomaterials in cross species; Drosophila and human cell lines. A similar difference was also noticed when C-11 or C16 accumulation was compared in multiple complex organ of Drosophila. However, nanomaterials compatible for oral delivery do not show any short-term toxicity, impaired growth of Drosophila larvae and adults. We hypothesize that two distinct parameter nano-skeleton frame with conjugated acid chains and live cell physiology are best suited for cell uptake and delivery to internal organs after oral consumption. Our results also differ from the hypothesis that nano-particle uptake in live cells occurred through energy independent non-endocytotic pathway involving insertions and diffusion across the cell membrane. Sub cellular internalization of PABA nanomaterials predominantly takes place by energy dependent endocytosis. Earlier we have found that PABA nanomaterials can penetrate plasma membrane in the human cells and enter into cytoplasm. The variable amount of different nanomaterial accumulation by energy dependent endo-cytosis in same cell type ruled out the possibility that a single internalization mechanism, endocytosis is exclusively required for uptake. However, a marked reduction of different nanomaterials under endocytosis inhibitory conditions believed that such discrepancies are due to sharp differences in size and shape of the self assembled structures. In addition as organic nanomaterials suffer from uncontrolled aggregation to form micron sized particles after prolong storage; thereby ruling out the possibility of insertion, diffusion and penetration mechanisms [22]. PABA nanoparticles have a high tendency to associate with cell membrane (Figure 2, 3).

Such accumulation might give rise to artefact in cellular uptake of micro-sized aggregates as found in artifactual intake of HIV TAT peptide at 4°C [23]. Therefore, cellular entry of PABA might depend on the size of the nanoparticles which is mainly guided by the acid side chain.

Finally, a systematic screening of PABA conjugated library provides sufficient evidences to support the following statements: 1) Two nanomaterials carrying C-11 and C16 acid side chains are best suited for optimal entry in cells and multiple organs. 2) In live tissues, an internal environment might be a useful barrier for improving nanoparticles delivery in multiple organs. 3) Cellular internalization or uptake mechanism of nanomaterials might unravel the clues for smooth entry in human cells and efficient delivery and 4) finally screening of PABA conjugates determine a functional relationship between energy dependent endocytosis and nanomaterial structure for each organ specific targeting.
