**5. Relative uptake of nanomaterials in insect and human cell lines**

We have carried out a systematic and comparative study on the relative uptake and estimation of the accumulated nanomaterials of varying composition and size inside the subcellular organelles by fluorescence microscopy, confocal laser scanning microscopy, and fluorometry. Three model cell lines representing different physiological function (insect and human) were chosen for the investigation. Drosophila S2 was chosen as a model as they are phagocytic cells and have a high cotransfection rate and for the mammalian cell lines HeLa cancer cells and the nonneoplastic Human Embryonic Kidney (HEK-293) were chosen for the study. The cell lines were cultured in media containing different concentrations of the nanomaterial; 10 μg/ml, 30 μg/ml and 60 μg/ml in 0.01% DMSO. In all cases, nanomaterial containing media to a final concentration 60 μg/ml in 0.01% DMSO showed no adverse effect on cell physiology.

In general, basic cell physiology and cell surveillance do not allow easy accessibility of foreign particles inside the cells. Exhaustive efforts are being carried out for engineering smooth delivery vehicles, synthesized from biocompatible and biodegradable materials. Though use of nano-materials has been successful in in vitro cultured cells [26], in practice, its adaptability in in vivo organ tracking by repeated injections is more challenging because of its limited selflife, delivery hurdles, and compatibility to fragile cell environment and potent immunogenic‐ ity [19]. Major improvements on chemical modifications of nano-materials play a fundamental role in cell uptake and live tissue distribution [27]. The surface texture by using small mole‐ cules, side chains and other conjugates alter the biological properties of nanoparticles [20]. We therefore hypothesized that such variation could increase smooth transition to shuttle inside live cells. To date, efforts for surface modifications of organic nanostructures have been rare. It is mainly due to lack of self-assembled organic molecules and compatibility of small molecules with nanoskeleton [29. 30].

Accumulation of nanomaterials varied widely based on the side chains of PABA conjugates inside both insect (Drosophila S2) and human (HEK293, HeLa) cells. It was observed that nanoparticles, which emit intrinsic green fluorescence (C-11, C-16 and C-18) accumulate almost equally in all three cell types despite the differences in the length of carbon side chains (Figure 4). These results suggest that the tubular shape of all three nanostructures is more important than the length of the acid chains for cell entry. The accumulation increased proportionately to the concentration of incubated nanoparticles and time. Moreover, uptake of nanotubes C-12 and C-14, are more intense relative to unsaturated acid chains (C-11U and C-18U) in human cells. It is possible that PANA nanomaterials with unsaturated side chain might hinder the cellular entry. In contrast, a distinct internal cell environment of Drosophila S2 cells increases the uptake of unsaturated C-11U particles. These results demonstrated that three major factors; shape, properties associated with unsaturated side chain and cross species cell physiology are involved in the rate of cellular uptake.

**Figure 3.** Physico-chemical properties and microscopic views of seven PABA anomaterials. elative Uptake of several nanomaterials in insect (Drosophila S2) and human tumour cells (HeLa) were shown. The differences in chemical struc‐ ture, shape and surface texture of nanomaterials leads to a variation in cell uptake. Scale-250 nm (SEM), 50 μm (cells)

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Since rhodamine was not covalently bonded with nanostructures C11U, C12, C14 and C18U, we cannot rule out the possibility that they might leach the dye from the nanostructures during cell uptake. Total fluorescence intensity in cells following exposure to the nanoparticle solutions could be due to the presence of nanoparticles through the cell, rather than correctly assigned to either a combination of free-dye and nanoparticle-bound dye, or even entirely to free dye, we incubated both the insect and human cells with rhodamine dye as well as

using blue diode option (maximum excitation 477 nm) was much stronger and found in 510 nm than that of the non-fluorescent monomer (studied in CH2Cl2, where it does not aggregate)

We have carried out a systematic and comparative study on the relative uptake and estimation of the accumulated nanomaterials of varying composition and size inside the subcellular organelles by fluorescence microscopy, confocal laser scanning microscopy, and fluorometry. Three model cell lines representing different physiological function (insect and human) were chosen for the investigation. Drosophila S2 was chosen as a model as they are phagocytic cells and have a high cotransfection rate and for the mammalian cell lines HeLa cancer cells and the nonneoplastic Human Embryonic Kidney (HEK-293) were chosen for the study. The cell lines were cultured in media containing different concentrations of the nanomaterial; 10 μg/ml, 30 μg/ml and 60 μg/ml in 0.01% DMSO. In all cases, nanomaterial containing media to a final

**5. Relative uptake of nanomaterials in insect and human cell lines**

concentration 60 μg/ml in 0.01% DMSO showed no adverse effect on cell physiology.

In general, basic cell physiology and cell surveillance do not allow easy accessibility of foreign particles inside the cells. Exhaustive efforts are being carried out for engineering smooth delivery vehicles, synthesized from biocompatible and biodegradable materials. Though use of nano-materials has been successful in in vitro cultured cells [26], in practice, its adaptability in in vivo organ tracking by repeated injections is more challenging because of its limited selflife, delivery hurdles, and compatibility to fragile cell environment and potent immunogenic‐ ity [19]. Major improvements on chemical modifications of nano-materials play a fundamental role in cell uptake and live tissue distribution [27]. The surface texture by using small mole‐ cules, side chains and other conjugates alter the biological properties of nanoparticles [20]. We therefore hypothesized that such variation could increase smooth transition to shuttle inside live cells. To date, efforts for surface modifications of organic nanostructures have been rare. It is mainly due to lack of self-assembled organic molecules and compatibility of small

Accumulation of nanomaterials varied widely based on the side chains of PABA conjugates inside both insect (Drosophila S2) and human (HEK293, HeLa) cells. It was observed that nanoparticles, which emit intrinsic green fluorescence (C-11, C-16 and C-18) accumulate almost equally in all three cell types despite the differences in the length of carbon side chains (Figure 4). These results suggest that the tubular shape of all three nanostructures is more important than the length of the acid chains for cell entry. The accumulation increased proportionately to the concentration of incubated nanoparticles and time. Moreover, uptake of nanotubes C-12 and C-14, are more intense relative to unsaturated acid chains (C-11U and C-18U) in human cells. It is possible that PANA nanomaterials with unsaturated side chain might hinder the cellular entry. In contrast, a distinct internal cell environment of Drosophila S2 cells increases the uptake of unsaturated C-11U particles. These results demonstrated that three major factors; shape, properties associated with unsaturated side chain and cross species

under the same 0.3 wt % concentration.

452 Application of Nanotechnology in Drug Delivery

molecules with nanoskeleton [29. 30].

cell physiology are involved in the rate of cellular uptake.

**Figure 3.** Physico-chemical properties and microscopic views of seven PABA anomaterials. elative Uptake of several nanomaterials in insect (Drosophila S2) and human tumour cells (HeLa) were shown. The differences in chemical struc‐ ture, shape and surface texture of nanomaterials leads to a variation in cell uptake. Scale-250 nm (SEM), 50 μm (cells)

Since rhodamine was not covalently bonded with nanostructures C11U, C12, C14 and C18U, we cannot rule out the possibility that they might leach the dye from the nanostructures during cell uptake. Total fluorescence intensity in cells following exposure to the nanoparticle solutions could be due to the presence of nanoparticles through the cell, rather than correctly assigned to either a combination of free-dye and nanoparticle-bound dye, or even entirely to free dye, we incubated both the insect and human cells with rhodamine dye as well as

rhodamine bound nanomaterials (C-11U and C-14) separately under same experimental conditions. After equal period of incubation, cells from both conditions were processed and viewed under confocal microscope. Cells cultured with only rhodamine showed accumulation at the outer periphery with negligible amount inside, while an intense fluorescence was seen inside the cells cultured with rhodamine containing nanoparticles indicating that rhodamine dye did not leached and was entrapped or contained in the nanotubes and nanocubes. (can we make this statement If all of the dye is nanoparticle bound then the nanoparticles are localized in sub-cellular oraganelles and where as if these system contains a significant amounts of free dye then fluorescence is distributed throughout the cell across cell barriers

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**Figure 5.** Uptake of raw rhodamine were compared to rhodamine containing nanoparticles uptake in HEK-293 cul‐ tured cell lines. Three separate sets of HEK-293 cells were cultures with raw rhodamine, C-11U and C-14 at 60 μg/ml

The fruit fly, Drosophila melanogaster, is a good model for study cell biology with emphasis on toxicology (Peterson RT, Nass R, Boyd WA, Freedman JH, Dong K, Narahashi T. Use of non-mammalian alternative models for neurotoxicological study. Neurotoxicology. 29,546– 555,2008). Here, we employed the Drosophila model to investigate nanoparticle interactions at different hierarchical scales of organization on Drosophilla at the egg, larval, and adult stages. PABA nanotubes and nanomaterials were mixed with yeast the standard Drosophila food at different concentrations, The food was seeded with 50 eggs of Drosophila melanogast‐

concentration. The cells were fixed, processesd and viewed in a confocal microscope. Scale 50 μm.

and into the cytoplasm.)

**Figure 4.** Biocompatibility of nanoparticles in nonneoplastic (HEK-293) Human Embryonic Kidney cells. Specificity of cellular uptake of different nanoparticles in HEK-293 cells was shown. The cells were incubated in 0.1% DMSO (con‐ trol) and 60mg/ml of each nanoparticle containing culture media separately for 12 hrs prior to process. The cells were counterstained with DAPI. The DIC images and merge figures were shown in the left and right sides of the panel. Scale 40μm

rhodamine bound nanomaterials (C-11U and C-14) separately under same experimental conditions. After equal period of incubation, cells from both conditions were processed and viewed under confocal microscope. Cells cultured with only rhodamine showed accumulation at the outer periphery with negligible amount inside, while an intense fluorescence was seen inside the cells cultured with rhodamine containing nanoparticles indicating that rhodamine dye did not leached and was entrapped or contained in the nanotubes and nanocubes. (can we make this statement If all of the dye is nanoparticle bound then the nanoparticles are localized in sub-cellular oraganelles and where as if these system contains a significant amounts of free dye then fluorescence is distributed throughout the cell across cell barriers and into the cytoplasm.)

**Figure 5.** Uptake of raw rhodamine were compared to rhodamine containing nanoparticles uptake in HEK-293 cul‐ tured cell lines. Three separate sets of HEK-293 cells were cultures with raw rhodamine, C-11U and C-14 at 60 μg/ml concentration. The cells were fixed, processesd and viewed in a confocal microscope. Scale 50 μm.

The fruit fly, Drosophila melanogaster, is a good model for study cell biology with emphasis on toxicology (Peterson RT, Nass R, Boyd WA, Freedman JH, Dong K, Narahashi T. Use of non-mammalian alternative models for neurotoxicological study. Neurotoxicology. 29,546– 555,2008). Here, we employed the Drosophila model to investigate nanoparticle interactions at different hierarchical scales of organization on Drosophilla at the egg, larval, and adult stages. PABA nanotubes and nanomaterials were mixed with yeast the standard Drosophila food at different concentrations, The food was seeded with 50 eggs of Drosophila melanogast‐

**Figure 4.** Biocompatibility of nanoparticles in nonneoplastic (HEK-293) Human Embryonic Kidney cells. Specificity of cellular uptake of different nanoparticles in HEK-293 cells was shown. The cells were incubated in 0.1% DMSO (con‐ trol) and 60mg/ml of each nanoparticle containing culture media separately for 12 hrs prior to process. The cells were counterstained with DAPI. The DIC images and merge figures were shown in the left and right sides of the panel. Scale

40μm

454 Application of Nanotechnology in Drug Delivery

er. After egg hatching (24 h), the larvae were observed to crawl through the food and ingest the suspended nanomaterials. Two of the fluorescent nanotubes were chosen for oral delivery. An important aspect of toxicity and genotoxicity studies is the selection of the assay system. Though In vitro approaches are preferred, the in vivo eukaryotic model, Drosophila appears as an ideal model organism. This has been already used to evaluate the internalization of nanoparticles and to solve open questions concerning cell uptake and live tissue distribution [27, 30]. The organic nanotubes when delivered through the food to the larval stage had no detectable effect on egg to adult survivorship.

propagation and showed more than 90% cell viability even at the higher nanomaterial concentration (120 μg/ml) relative to DMSO treated cells. These results suggest that nanoma‐ terials function as efficient bio-transporters and fail to show any cytotoxicity. These findings were further verified by a parallel study using flow-cytometry measurements. The mitotic cells from confluent cultures incubated with different nanomaterials containing media were assayed. The relative progression of cells from G1 to S phase was also determined. In three separate cultures containing 0 μg/ ml, 30 μg/ml, 60 μg/ml nanoparticles, the phases of cell cycles were progressing normally based on the incubation time, but in higher concentration (120 μg/ml), a fall of G1 number with concurrent increase in G2 and S phase was noticed

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To obtain visual images of assemblies including 3D structure and measurements, many images from fluorescent laser confocal microscope, scanning electron microscope (SEM) were analyzed. The 3D images showed that self-assembled structures, Benzamide microtubes (BAMT-A and BAMT-B) form tube like structure with a hollow space inside. Tubes are nearly 0.178 to 0.207 μm in width. The BAMT-A shows green fluorescence colour that emits from selfassembled 4-alkylamido-N-pyridin-2yl-benzamides coupling with a stearic side chain (C=18). Compound BAMT-B conjugated with a shorter lauric side chain (C=12) does not emit intrinsic fluorescence colour. To add standard fluorescence dye (Rhodamine B, red), BAMT-B organic tubular structure was manufactured by mixing Rhodamine B with tube-forming benzamide during the self-assembly process. The Rhodamine B that is embedded in the surface wall of self-assembled microtubes (BAMT-B) emits red fluorescence as specific signal. Both micro‐ structures with variable side chains are DMSO and ethyl alcohol soluble, but not in water and

Next insect and mammalian cells were used to estimate the efficiency of cellular uptake of two microtubes in vitro. Drosophila S2 cells, nonneoplastic Human Embryonic Kidney (HEK-293) (Figure 2A, B) and neoplastic HeLa cells were grown in small dishes or cover glasses and incubated with the microstructures dissolved in 0.1% DMSO that has no adverse effect on cell physiology (14). After 24 or 48 hrs incubations, cells were fixed in 4% paraformaldehyde, followed by few gentle washes with PBS. The cells were viewed under laser confocal micro‐ scope (Olympus FV1000). The reconstituted images showed that both BAMT-A and BAMT-B was accumulated in the periphery of the nucleus of both insect and human cells incubated with 60mg/ml microtubes containing 0.1% DMSO (Figure 2 a,b). The accumulation is increased proportionately to the concentration of microtubes ingested. In contrast, a negligible amount of fluorescence signals was emitted from the cells incubated in 0.1% DMSO alone for the same period of time. These findings demonstrated that microstructures are accumulated in the cultured cell after penetrating the plasma membrane. Moreover, an identical distribution pattern of BAMT-A and BAMT-B in the insect and human cells further verify that similar to intrinsic green in BAMT-A, embedded red fluorophore remains coupled with BAMT-B tube

Next to test the viability of the culture cells, a colorimetric assay was performed using 3-(4-5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide as described earlier. This assay is based on the reductive capacity to metabolize the tetrazolium salt to blue coloured formazone.

can be stored for prolonged periods without loosing its organic properties.

indicating progression towards asynchrony.

wall after accumulation in the cytosol.

**Figure 6.** Biocompatibility and distribution of nanoparticles in four different developmental stages of Drosophila progeny after feeding nanomaterials containing media of the parental population.

Effect of nanoparticles on cell viability and cytotoxicity To address cell viability and cytotoxicity, colorimetric assay was performed using 3-(4-5-dimethylthiazol-2-yl)-2,5-diphenylte‐ trazoliumbromide. The cells incubated in freshly prepared nanoparticles containing media were treated with MTT. Uptake of nanoparticles in all cell types does not disturb normal cell propagation and showed more than 90% cell viability even at the higher nanomaterial concentration (120 μg/ml) relative to DMSO treated cells. These results suggest that nanoma‐ terials function as efficient bio-transporters and fail to show any cytotoxicity. These findings were further verified by a parallel study using flow-cytometry measurements. The mitotic cells from confluent cultures incubated with different nanomaterials containing media were assayed. The relative progression of cells from G1 to S phase was also determined. In three separate cultures containing 0 μg/ ml, 30 μg/ml, 60 μg/ml nanoparticles, the phases of cell cycles were progressing normally based on the incubation time, but in higher concentration (120 μg/ml), a fall of G1 number with concurrent increase in G2 and S phase was noticed indicating progression towards asynchrony.

er. After egg hatching (24 h), the larvae were observed to crawl through the food and ingest the suspended nanomaterials. Two of the fluorescent nanotubes were chosen for oral delivery. An important aspect of toxicity and genotoxicity studies is the selection of the assay system. Though In vitro approaches are preferred, the in vivo eukaryotic model, Drosophila appears as an ideal model organism. This has been already used to evaluate the internalization of nanoparticles and to solve open questions concerning cell uptake and live tissue distribution [27, 30]. The organic nanotubes when delivered through the food to the larval stage had no

**Figure 6.** Biocompatibility and distribution of nanoparticles in four different developmental stages of Drosophila

Effect of nanoparticles on cell viability and cytotoxicity To address cell viability and cytotoxicity, colorimetric assay was performed using 3-(4-5-dimethylthiazol-2-yl)-2,5-diphenylte‐ trazoliumbromide. The cells incubated in freshly prepared nanoparticles containing media were treated with MTT. Uptake of nanoparticles in all cell types does not disturb normal cell

progeny after feeding nanomaterials containing media of the parental population.

detectable effect on egg to adult survivorship.

456 Application of Nanotechnology in Drug Delivery

To obtain visual images of assemblies including 3D structure and measurements, many images from fluorescent laser confocal microscope, scanning electron microscope (SEM) were analyzed. The 3D images showed that self-assembled structures, Benzamide microtubes (BAMT-A and BAMT-B) form tube like structure with a hollow space inside. Tubes are nearly 0.178 to 0.207 μm in width. The BAMT-A shows green fluorescence colour that emits from selfassembled 4-alkylamido-N-pyridin-2yl-benzamides coupling with a stearic side chain (C=18). Compound BAMT-B conjugated with a shorter lauric side chain (C=12) does not emit intrinsic fluorescence colour. To add standard fluorescence dye (Rhodamine B, red), BAMT-B organic tubular structure was manufactured by mixing Rhodamine B with tube-forming benzamide during the self-assembly process. The Rhodamine B that is embedded in the surface wall of self-assembled microtubes (BAMT-B) emits red fluorescence as specific signal. Both micro‐ structures with variable side chains are DMSO and ethyl alcohol soluble, but not in water and can be stored for prolonged periods without loosing its organic properties.

Next insect and mammalian cells were used to estimate the efficiency of cellular uptake of two microtubes in vitro. Drosophila S2 cells, nonneoplastic Human Embryonic Kidney (HEK-293) (Figure 2A, B) and neoplastic HeLa cells were grown in small dishes or cover glasses and incubated with the microstructures dissolved in 0.1% DMSO that has no adverse effect on cell physiology (14). After 24 or 48 hrs incubations, cells were fixed in 4% paraformaldehyde, followed by few gentle washes with PBS. The cells were viewed under laser confocal micro‐ scope (Olympus FV1000). The reconstituted images showed that both BAMT-A and BAMT-B was accumulated in the periphery of the nucleus of both insect and human cells incubated with 60mg/ml microtubes containing 0.1% DMSO (Figure 2 a,b). The accumulation is increased proportionately to the concentration of microtubes ingested. In contrast, a negligible amount of fluorescence signals was emitted from the cells incubated in 0.1% DMSO alone for the same period of time. These findings demonstrated that microstructures are accumulated in the cultured cell after penetrating the plasma membrane. Moreover, an identical distribution pattern of BAMT-A and BAMT-B in the insect and human cells further verify that similar to intrinsic green in BAMT-A, embedded red fluorophore remains coupled with BAMT-B tube wall after accumulation in the cytosol.

Next to test the viability of the culture cells, a colorimetric assay was performed using 3-(4-5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide as described earlier. This assay is based on the reductive capacity to metabolize the tetrazolium salt to blue coloured formazone. Cells were incubated with various concentrations of microtubes. After incubation, cells were added with MTT and the absorbance of coloured product was monitored in a microplate reader at 570nm. In all the cases tested, the cell proliferation estimated by the absorbance and the viability of tumor HeLa and non cancerous HEK-293 cells in the presence of microtubes showed more than 95% viability relative to DMSO treated cells (Figure 2C). These results suggest that microstructures are the efficient molecular transporters for different biologically important cells with no cytotoxicity.

structures in the next successive generations through germ cells. Therefore, lack of heritable transfer of microtubes leads to an ineffective route of microtube spreading in the environment

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In live insects, majority of the internal organs are submerged in haemolymph – a blood equivalent of human. The haemolymph circulate through the open vessel and pump the fluid in a fixed direction at the posterior body cavity by using a series of valves that prevent opposite haemolymph flow. As reported earlier by feeding single walled carbon nanotubes in intact fly postulated that the fluorescent methods are ideal for delivery and diagnostic application. Feeding of microtubes in larvae and adults causes systemic spreading of signals by the gut peristaltic movement to cross the cell membrane barrier. The intensity of the dye associated with the microsized materials is proportionate to the amount of accumulated tubes that were incorporated in the gut cells. Variable intensity of fluorescent dyes in different parts of the body demonstrates different amounts of microtube accumulation in the different organs. Internal organs of larvae and adult tissues were dissected, fixed in 4% paraformaldehyde, processed and scanned under Confocal Fluorescence Microscope. At least five samples of each organ from larvae fed on microtubes were viewed and intensity of fluorescence scored (Supplementary Material). The amount of fluorescence of each tissue of the DMSO and microtube fed larvae and adults was estimated (Gray value/pixel) using Metamorph software. An increased accumulation of fluorescence dye was found in the digestive track (83.06+4.51 in BAMT-A), Malpighian tubules (57.19+3.81), fat bodies (38.41+2.19) compared to the salivary glands (27.73+1.81) and rapidly dividing cells of two imaginal discs (11.52+0.56 in wing discs and 14.32+0.41 in eye discs) (Figure 4). However, variable lengths of the side chains lead to the conspicuous changes in microtubes distribution at the different body parts. Shorter length of lauric side chain exhibited greater accumulation of microstructure. The cells of different discs and larval brain were devoid of any BAMT-A tubes containing longer side chain (C=18) but a considerable amount of BAMT-B (C=12) penetrated the same tissues after feeding equal amount of microtubes (Figure 4). In adults, a distinct pattern of BAMT-A and BAMT-B distribution in different organs was found. BAMT-A mainly accumulated in the abdomen, thorax including haltere and leg assuming their prolonged retention in the body fluid, while BAMT-B conjugated with C-12 side chain widely distributed in eye, antennae, proboscis and adult brain (Figure 4). To examine the preferential penetration of two side chains in adult brains, the amount of dyes in the brain tissue were tested. The BAMT-B with short side chain has a clear advantage in the entry of the brain tissue over BAMT-A in equal concentration. Interestingly, no change in fly behaviour after oral digestion suggest that accumulation of PABA microstructure do not produce any permanent damage in the brain because p-amino‐ benzoic acid functions in the improvement of neuro-degerative damages by inhibiting

Further high concentrations of fluorescence conjugated with BAMR-B were observed in insect cells and in all the organs of Drosophila after feeding. Enlarged view of insect cells and wing disc (Figure 4) verified the accumulation of micro-structure in the cytoplasm of cultured cells and fixed tissues. These finding allowed us to confirm the physical accumu‐ lation of microtubes in both cell types. In contrast, low abundance of the BAMT-A

and their natural entry into the foodchain via participating consumers.

acetylcholinesterase.

Further, these microsized structures were scaled up for *in vivo* use precisely to facilitate organ distribution and to combat different physiological hurdles in live organisms. To test overall viability and growth, after feeding the PABA containing microtubes to larvae, pupae and adult Drosophila were estimated. After hatching, larvae undergo an intense 4-5 day feeding, when they increase weight by 200-folds. In subsequent immobile pupal stage, they stop feeding, therefore no marked gain in adult fly weight from the larval stage is noticed. To feed larvae with highest possible doses of BAMT-A and BAMT-B, dry Baker's yeast was mixed with concentrated suspensions of microstructures (60μg in 100μl) in 0.1 % DMSO solutions, followed by centrifugation and decantation. The resulting pastes were used in equal amount as the sole food source for various batches of equal number of larvae. Indeed viability of the larvae, pupae and adult was marginally higher in sole DMSO (0.1%) fed flies relative to the flies fed on yeast paste containing microtubes (Figure 3). The feeding of low percentage DMSO (0.1%) has no apparent effect on fly physiology and viability. To investigate the effect of the microtubes on overall growth, behaviour and physiology of the flies, the size of the adults and the sexual behaviour of newly emerged flies were estimat‐ ed. No size difference between DMSO and BAMT-A and BAMT-B fed male and female flies and abnormalities in their sexual behaviour were marked (Figure 3). The egg laying capacity of adult females for six consecutive days after hatching and the sex of eclosed flies were counted. No significant difference was detected in egg laying capacity and male/ female ratios when compared to the wild type, DMSO or microtubes fed females (Figure 3). Taken together organic microtubes and their side alkyl chain modifications have no adverse effect on cellular physiology, behaviour, and sensitivity of fly sex and other pharmacokinetics parameters of live cells in the insects.(same as publication)

Organic nanotubes claimed a greater stability and better self-life and biocompatibility. To verify such claim of orally ingested microtube in live organisms, we reared the newly hatched adult flies, generated from microtubes or DMSO fed larvae, in the normal culture media for 7 consecutive days and examined under fluorescence microscope. Nearly equal level of fluo‐ rescence intensity was found from body parts of the adult flies reared in normal food media for 0 to 7 days (Figure 3d). Culture in food media without microtubes does not reduce the stability and self-life of the fluorescence organic microtubes for a limited period. The fluores‐ cence intensity was reduced conspicuously after extending the culture on an average of 23-25 days and was eliminated within 47 days from the organs of the adult flies reared in the normal media. We further examined fluorescence intensity of the mature fertilized eggs laid by adult females reared in normal food for 7 days after hatching. No fluorescence signal was emitted from the eggs (Figure 3d). It eliminates the rare possibility of genetic inheritance of micro‐ structures in the next successive generations through germ cells. Therefore, lack of heritable transfer of microtubes leads to an ineffective route of microtube spreading in the environment and their natural entry into the foodchain via participating consumers.

Cells were incubated with various concentrations of microtubes. After incubation, cells were added with MTT and the absorbance of coloured product was monitored in a microplate reader at 570nm. In all the cases tested, the cell proliferation estimated by the absorbance and the viability of tumor HeLa and non cancerous HEK-293 cells in the presence of microtubes showed more than 95% viability relative to DMSO treated cells (Figure 2C). These results suggest that microstructures are the efficient molecular transporters for different biologically

Further, these microsized structures were scaled up for *in vivo* use precisely to facilitate organ distribution and to combat different physiological hurdles in live organisms. To test overall viability and growth, after feeding the PABA containing microtubes to larvae, pupae and adult Drosophila were estimated. After hatching, larvae undergo an intense 4-5 day feeding, when they increase weight by 200-folds. In subsequent immobile pupal stage, they stop feeding, therefore no marked gain in adult fly weight from the larval stage is noticed. To feed larvae with highest possible doses of BAMT-A and BAMT-B, dry Baker's yeast was mixed with concentrated suspensions of microstructures (60μg in 100μl) in 0.1 % DMSO solutions, followed by centrifugation and decantation. The resulting pastes were used in equal amount as the sole food source for various batches of equal number of larvae. Indeed viability of the larvae, pupae and adult was marginally higher in sole DMSO (0.1%) fed flies relative to the flies fed on yeast paste containing microtubes (Figure 3). The feeding of low percentage DMSO (0.1%) has no apparent effect on fly physiology and viability. To investigate the effect of the microtubes on overall growth, behaviour and physiology of the flies, the size of the adults and the sexual behaviour of newly emerged flies were estimat‐ ed. No size difference between DMSO and BAMT-A and BAMT-B fed male and female flies and abnormalities in their sexual behaviour were marked (Figure 3). The egg laying capacity of adult females for six consecutive days after hatching and the sex of eclosed flies were counted. No significant difference was detected in egg laying capacity and male/ female ratios when compared to the wild type, DMSO or microtubes fed females (Figure 3). Taken together organic microtubes and their side alkyl chain modifications have no adverse effect on cellular physiology, behaviour, and sensitivity of fly sex and other

pharmacokinetics parameters of live cells in the insects.(same as publication)

Organic nanotubes claimed a greater stability and better self-life and biocompatibility. To verify such claim of orally ingested microtube in live organisms, we reared the newly hatched adult flies, generated from microtubes or DMSO fed larvae, in the normal culture media for 7 consecutive days and examined under fluorescence microscope. Nearly equal level of fluo‐ rescence intensity was found from body parts of the adult flies reared in normal food media for 0 to 7 days (Figure 3d). Culture in food media without microtubes does not reduce the stability and self-life of the fluorescence organic microtubes for a limited period. The fluores‐ cence intensity was reduced conspicuously after extending the culture on an average of 23-25 days and was eliminated within 47 days from the organs of the adult flies reared in the normal media. We further examined fluorescence intensity of the mature fertilized eggs laid by adult females reared in normal food for 7 days after hatching. No fluorescence signal was emitted from the eggs (Figure 3d). It eliminates the rare possibility of genetic inheritance of micro‐

important cells with no cytotoxicity.

458 Application of Nanotechnology in Drug Delivery

In live insects, majority of the internal organs are submerged in haemolymph – a blood equivalent of human. The haemolymph circulate through the open vessel and pump the fluid in a fixed direction at the posterior body cavity by using a series of valves that prevent opposite haemolymph flow. As reported earlier by feeding single walled carbon nanotubes in intact fly postulated that the fluorescent methods are ideal for delivery and diagnostic application. Feeding of microtubes in larvae and adults causes systemic spreading of signals by the gut peristaltic movement to cross the cell membrane barrier. The intensity of the dye associated with the microsized materials is proportionate to the amount of accumulated tubes that were incorporated in the gut cells. Variable intensity of fluorescent dyes in different parts of the body demonstrates different amounts of microtube accumulation in the different organs. Internal organs of larvae and adult tissues were dissected, fixed in 4% paraformaldehyde, processed and scanned under Confocal Fluorescence Microscope. At least five samples of each organ from larvae fed on microtubes were viewed and intensity of fluorescence scored (Supplementary Material). The amount of fluorescence of each tissue of the DMSO and microtube fed larvae and adults was estimated (Gray value/pixel) using Metamorph software. An increased accumulation of fluorescence dye was found in the digestive track (83.06+4.51 in BAMT-A), Malpighian tubules (57.19+3.81), fat bodies (38.41+2.19) compared to the salivary glands (27.73+1.81) and rapidly dividing cells of two imaginal discs (11.52+0.56 in wing discs and 14.32+0.41 in eye discs) (Figure 4). However, variable lengths of the side chains lead to the conspicuous changes in microtubes distribution at the different body parts. Shorter length of lauric side chain exhibited greater accumulation of microstructure. The cells of different discs and larval brain were devoid of any BAMT-A tubes containing longer side chain (C=18) but a considerable amount of BAMT-B (C=12) penetrated the same tissues after feeding equal amount of microtubes (Figure 4). In adults, a distinct pattern of BAMT-A and BAMT-B distribution in different organs was found. BAMT-A mainly accumulated in the abdomen, thorax including haltere and leg assuming their prolonged retention in the body fluid, while BAMT-B conjugated with C-12 side chain widely distributed in eye, antennae, proboscis and adult brain (Figure 4). To examine the preferential penetration of two side chains in adult brains, the amount of dyes in the brain tissue were tested. The BAMT-B with short side chain has a clear advantage in the entry of the brain tissue over BAMT-A in equal concentration. Interestingly, no change in fly behaviour after oral digestion suggest that accumulation of PABA microstructure do not produce any permanent damage in the brain because p-amino‐ benzoic acid functions in the improvement of neuro-degerative damages by inhibiting acetylcholinesterase.

Further high concentrations of fluorescence conjugated with BAMR-B were observed in insect cells and in all the organs of Drosophila after feeding. Enlarged view of insect cells and wing disc (Figure 4) verified the accumulation of micro-structure in the cytoplasm of cultured cells and fixed tissues. These finding allowed us to confirm the physical accumu‐ lation of microtubes in both cell types. In contrast, low abundance of the BAMT-A microtubes in the salivary glands and rapidly dividing cells of imaginal discs probably represent the secondary uptake after the microtubes enter into the haemolymph. These findings demonstrate that differential uptake and specificity of live cell targeting by the microtubes depend on the cellular physiology, chemical modification of carrier to travel and enter live cells.

**6. Mode of uptake of PABA nanomaterials**

through the clathrin-dependent endocytosis pathway.

mechanism.

Broadly, there are two modes of entries, either PABA nano-materials transverse the cell membrane via endocytosis orenergy independent nonendocytotic mechanism. We have carried out a series of investigations on uptake mechanism and cellular internalization for PABA conjugates. Endocytosis is an energy dependent mechanism. The process is hindered at a low temperature (at 4°C instead of 37°C) or in ATP deficient environment. Cells incubated in media containing nanoparticles were either cultured at 4°C or pretreated with NaN3 for inhibiting the production of ATP, thereby hampering the endocytosis process. The level of fluorescent intensity in the cytosol of each cultured cells was reduced dramatically relative to cells cultured in regular standard conditions (Figure 3). This reduction determines that PABA conjugates enter in the sub-cellular compartment of cultured cells via endocytosis. We also sub-categorized the endocytosis pathway including phagocytosis, pinocytosis, clathrin dependent receptor mediated and clathrin independent mechanisms.Internalization often occurs when the clathrin coat on the plasma membrane forms conspicuous invagination in the cell membrane leading to the budding of clathrin-coated vesicles. As a result, extracellular species located on the cell membrane are trapped within the vesicles and invaginated inside the cells. To disrupt the formation of clathrin coated vesicles on the cell membranes, cells were preincubated in sucrose (hypertonic) soluton or K+-depleted media before treatment with all seven nano-particles. Data showed a drastic reduction in PABA nano-particle uptake (Figure 3C), which suggests that a clathrin dependent endocytosis process is involved in entry

Organic Nanotubes: Promising Vehicles for Drug Delivery

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

461

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

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

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

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

Here, several critical questions relevant to biocompatibility and application of p-aminozben‐ zoic containing microstructures are addressed. (1) It determines the parameters of chemi‐ cal and biological modifications suitable for oral administration. However, the impact on specific cellular physiology and efficient uptake of microstructures by carrier molecules based on their chemical modifications cannot be ignored. It is evident that distribution and accumulation is prone to shorter (C=12) alkyl side chain. (2) These organic microtubes overcome cell physiological, pharmacokinetic barriers and show an efficient cellular uptake in animal cultured cells (5,19). (3) It has no adverse effects in physiology, behaviour and growth of insects that shows strong similaries with human at molecular level (3) Finally, modifications in alkyl side chain play an important role in biodistribution. The side chain modifications in BAMT-A and BAMT-B alter the shape of microstructures by changing self assembly properties and discriminate tissue specific distribution specifically in adult eye, its precursor cells, wing imaginal discs and neuronal tissues in larval and adult brain but do not show any conspicuous changes of their biodistribution in cultured insect and human cells. The chemical modifications of self assembled p-aminobenzoic moiety also show a better stability and long self-life before degradation but no short-term toxicity, impaired growth of Drosophila larvae and adults after feeding solely on microtubes containing media. Moreover, no obvious effect on fecundity or impairment of fertility was noticed in the adult female flies. During self assembly, chemical modifications of PABA, and their longer stability in the internal organs provides the microtubes as better-suited and sustainable cargo in live organisms, unlike inorganic nanomaterials that neither accumu‐ late in the live cells nor produce cytotoxicity in the cellular environment. Most logical extension of this work would be the cell specific target delivery in which coupled small molecule on the surface wall have define properties for attachment of specific cell surface and/or stringent proteins for facilitating antigen-antibody reactions. This approach could be used not only to deliver small regulatory RNA and DNA as therapeutic materials but also to optimize pill like properties for oral ingestion with no pharmacokinetic barriers. Though this study used only one structure moiety of drug molecules, it is likely that the method would work with other types organic microstructure as long as three major criteria of orally administered molecule biocompatibility, pharmacokinetics and a capacity for multivalency are considered. Therefore chemical modulations on the surface should be incorporated in the design to attach the multivalency of small ligands. These changes in organic microstructure were described recently. Such attempts for synthesizing tissue or cell guided orally ingested microstructure may favour to make the next generation micropill to deliver biomaterials for effective gene therapy and novel cargo useful for molecular diagnostics.
