Curcumin Encapsulated Lecithin Nanoemulsions: An Oral Platform for Ultrasound Mediated Spatiotemporal Delivery of Curcumin to the Tumor

Systemic toxicity caused by conventional chemotherapy is often regarded as one of the major challenges in the treatment of cancer. Over years, the trigger-based modality has gained much attention as it holds the spatiotemporal control over release and internalization of the drug. In this article, we are reporting an increase in the anti-tumor efficacy of curcumin due to ultrasound pulses. MDA MB 231 breast cancer and B16F10 melanoma cells were incubated with lecithin-based curcumin encapsulated nanoemulsions and exposed to ultrasound in the presence and absence of microbubble. Ultrasound induced sonoporation enhanced the cytotoxicity of curcumin in MDA MB 231 and B16F10 cancer cells in the presence of microbubble by 100- and 64-fold, respectively. To study the spatiotemporal delivery of curcumin, we developed B16F10 melanoma subcutaneous tumor on both the flanks of C57BL/6 mice but only the right tumor was exposed to ultrasound. Insonation of the right tumor spatially enhanced the cytotoxicity and enabled the substantial regression of the right tumor compared to the unexposed left tumor which grew continuously in size. This study showed that the ultrasound has the potential to target and increase the drug’s throughput to the tumor and enable effective treatment.


Results
physiochemical characterization of cur_ne and MB. The hydrodynamic diameter and zeta potential of the Cur_NE are summarized in Table 1. Cur_NE were found to have a hydrodynamic diameter of 60-120 nm. The Cur_NE were also characterized for its size and morphology through a scanning electron microscope and it had shown to be spherical in size with a diameter varying from 50-120 nm, which is in agreement with the hydrodynamic diameter obtained in dynamic light scattering measurement (Fig. 1I). Moreover, the zeta potential of the Cur_NE with different ratio of lecithin and curcumin did not change substantially and was decreasing from −20 to −45 mV ( www.nature.com/scientificreports www.nature.com/scientificreports/ as the negative zeta potential would substantiate the repulsive forces present between particles and prevent their flocculation 26 . The encapsulation efficiency of the curcumin in Cur_NE increased gradually with the increase in curcumin concentration, as summarized in Table 2. The increase in concentration of curcumin induced a very nominal increase in size of Cur_NE perhaps due to preparation under high pressure homogenization. But the loading capacity of the formulation saturated eventually with the gradual increase in the concentration of the curcumin presumably due to lack of the intra-nanoemulsion space or carrying capacity; and after a specific concentration, encapsulation did not increase further (see Supplementary Fig. S3), rather drug remained free in supernatant and that is why encapsulation efficiency reduced for the ratios 1.5:1 and 1:1 ( Table 2). EE (%) of the NERh6g for the Rh6g was found to be 67.5 ± 3.2%. The hydrodynamic diameter of the MBs was found to be 1200.6 ± 201.8 nm and zeta potential −15.9 ± 4.8 mV, which says that MBs are stable in aqueous solution (Table 1). Electron and light microscopy study revealed the spherical shape of MB in an aqueous medium with 900-1000 nm diameter (Fig. 1II) which is in agreement with the size obtained in DLS. chemical stability and solubility of curcumin. The encapsulated curcumin was characterized for its chemical stability with 1 H NMR spectroscopy and the spectrum of the encapsulated drug was compared with free drug. The chemical shift of the curcumin, both encapsulated and free form is shown in Fig. 2. The existence of the same 1 H spectra for the free drug as well as drug released after lysis of the Cur_NE indicates that curcumin was chemically stable inside Cur_NE and the process of encapsulation was not altering its natural structure. It is evident from the result that the Cur_NE was soluble in PBS while the same concentration of the free drug was lying over the interface between air and liquid ( Supplementary Fig. S3II) whereas the same concentration of encapsulated drug in the form of Cur_NE got easily re-dispersed in PBS without requiring any mechanical agitation or sonication ( Supplementary Fig. S3II).
Gastric resistance and release kinetics in Sif. The Cur_NE released 0.8 ± 0.08% of encapsulated curcumin within 2 hrs of incubation in SGF and retained >98% of curcumin (Fig. 3E). It showed that the Cur_NEs were resistant to the gastric fluid and therefore encapsulated curcumin would be available for release and absorption in the small intestine. After 48 hrs incubation in SGF, Cur_NEs were still intact and supported the data that showed only 0.8 ± 0.08% of drug released from nanoemulsions within 2 hrs as Cur_NEs were still intact and  Table 2. Encapsulation efficiency of the Cur_NE with varying ratio of lecithin and curcumin. www.nature.com/scientificreports www.nature.com/scientificreports/ morphologically stable (Fig. 3B). In SIF the Cur_NE released 30 ± 3.2% of the encapsulated content in 4 hrs (Fig. 3E) and it was evident from the TEM image of the SIF digested Cur_NEs that have diffused core and periphery (Fig. 3C,D). Diffused core and periphery obtained due to the presence of pancreatin and bile salt in SIF that digested the lecithin. cellular internalization of cur_ne without ultrasound. Cellular internalization study showed that free curcumin was internalized meagrely by cells with an internalized amount of just 0.15 ± 0.05 µg whereas curcumin in the form of Cur_NE was getting internalized in a substantially higher amount of 7.6 ± 1.32 µg within 1.5 hrs of incubation. As we increased the concentration of curcumin in lecithin: drug ratio, internalization was increasing with the increase in the concentration of curcumin to the extent where ratio reached 2:1 but after that, it decreased (see Supplementary Fig. S6). Out of all the ratios, Cur_NE synthesized from 2:1 ratio of lecithin and curcumin showed the highest internalization and it was 50 times higher to free curcumin. Therefore, this ratio was considered for in vitro cytotoxicity and in vivo anti-tumor efficacy study.

MB induced sonoporation in MDA MB 231 and B16F10 cells. SEM analysis of the cells treated with
ultrasound in presence of MB revealed that insonation led to the opening of the cell membrane and also the addition of MB further amplified the process of pore generation in terms of both number/cell as well as the diameter. The number of pores/cell was limited to 2-3 pores (Fig. 4B,F) in case of only ultrasound treatment (in ≈ 85% of cells, a total of 100 cells were analyzed) but in presence of MB same number increased up to 8-12 pores/cell (Fig. 4C,D,G,H) in ≈90% of the cells. As far as the diameter of the sonopores is concerned, the diameter of pores generated through ultrasound solely was restricted to 100-300 nm (Fig. 4B,F) whereas in the presence of MB, the width of the pores increased up to 500-700 nm, as shown in Fig. 5C,D,G,H. The observation was similar in both B16F10 and MDA MB 231 cells. Moreover, another interesting finding was breaching (≈1 µm wide) of the cell membrane in few cells due to severe disintegration of membrane following insonation in presence of MB (see Supplementary Fig. S8). As per the literature, opening of membrane due to insonation is transient in nature, so the limitation of our experiment is that it can give a comparative aspect of the effect of ultrasound exposure over cells in presence and absence of MB as by the time we fixed the cells some of the pores would have resealed themselves.

Cellular internalization of NERh6g with ultrasound. Cellular internalization study of NERh6g indi-
cates that the exposure of ultrasound in the presence of MB increased the cellular uptake as we observed in CLSM analysis of MDA MB 231 and B16F10 cells (Fig. 5I). Moreover, the gradual increase in the intensity of ultrasound viz. 0.5, 0.7 and 0.9 W/cm 2 increased the fluorescence intensity inside cells which could have been possible due to enhancement in the internalization of NERh6g (Fig. 5I). The addition of MB further enhanced the process of internalization as it provided a higher intracellular intensity of the rhodamine 6g (Rh6g) inside the cells at above-mentioned ultrasound intensity (Fig. 5I). The fluorescence intensity of the internalized amount of NERh6g www.nature.com/scientificreports www.nature.com/scientificreports/ was quantified by flow cytometry also and found to be increased 3-fold in case of ultrasound treatment as compared to control where only NERh6g was incubated with cells ( Fig. 5IIA-C). Ultrasound application not only enhanced the cellular uptake but also the number of cells involved in internalization. In the case of only NERh6g cytotoxicity and apoptosis assay. Cytotoxicity of the Cur_NE was determined both with and without the assistance of MB in MDA MB 231 and B16F10 cells. As was observed in the CLSM study, NERh6g internalization was enhanced by ultrasound with and without MB and so did the cytotoxicity, as summarized in Table 3. Mere encapsulation of the curcumin inside nanoemulsions reduced the IC 50 of the drug by 21-fold and further application of ultrasound over MDA MB 231 cells at intensity 0.9, 1.5 and 2 W/cm 2 reduced the IC 50 by 36-, with NERh6g and exposed to ultrasound at 0.9 W/cm 2 intensity and 50% duty cycle for 30 sec.
www.nature.com/scientificreports www.nature.com/scientificreports/ 46-, and 51-fold, respectively, as compared to Cur, as shown in Fig. 6A. Cur_NE + US 0.9, Cur_NE + US 1.5, and Cur_NE + US 2 refers to the ultrasound treatment in the absence of MB at intensity 0.9, 1.5 and 2 W/cm 2 , respectively, for 30 sec at 50% duty cycle. Similarly, in B16F10 cells, the IC 50 value of Cur_NE, Cur_NE + US 0.9, Cur_NE + US 1.5 and Cur_NE + US 2 was found to be 14-, 19-, 23-, and 33-fold, respectively, lower to the Cur (Fig. 6C). In another set of experiment, at the same concentration of Cur_NE, 0.2 mg and 0.3 mg/ml of MB was added to the solution followed by ultrasound treatment at 2 W/cm 2 for 30 sec with 50% duty cycle. IC 50 value reduced by 1.6-and 2-fold after treatments with Cur_NE + US2 + MB (0.2) and Cur_NE + US2 + MB (0.3) respectively as compared to Cur_NE + US2 in MDA MB 231 cells, as shown in Fig. 6B. In B16F10 cells, the IC 50 value of Cur_NE + US 2 reduced by 1.4-and 2-fold due to the addition of MB at concentration 0.2 and 0.3 mg/ml,  Table 3. IC50 of the Cur_NE in cancer cells. www.nature.com/scientificreports www.nature.com/scientificreports/ respectively (Fig. 6D). Apoptosis assay showed that the presence of MB and ultrasound increased the population of early and late apoptotic MDA MB 231 cells (see Supplementary Fig. S9).
Anti-tumor efficacy. The anti-tumor efficacy study illustrated that the application of ultrasound reduced the tumor volume and relative growth rate of the tumor. In most of the groups, the volume of the right tumor (insonated) was significantly (P ≤ 0.01, P ≤ 0.001) smaller than the left tumor which was not exposed to   www.nature.com/scientificreports www.nature.com/scientificreports/ ultrasound, as shown in Fig. 8IIIA,D, except the group treated with PBS and free curcumin (P = 0.2, P = 0.06). When mice were given oral administration of only Cur_NE and exposed with ultrasound, the right tumor showed 1.6-fold higher growth inhibition as compared to the left tumor on the 15 th day after treatment started (Fig. 8C). The oral administration of Cur_NE followed by I.V. injection of MB after 2 hrs and ultrasound exposure over right tumor reduced the relative growth rate of the tumor by 11-fold (P ≤ 0.001) compared to the group treated with free curcumin at the end of 15 th day after treatment started (Fig. 8C). Ultrasound exposed right tumor had shown a substantial reduction in growth rate as compared to the left tumor of the same mice. It was also proved in another experiment where mice were given an oral dose of NERh6g nanoemulsions and after 2 hrs, MBs were injected and the right tumor was exposed with ultrasound whereas the left tumor was kept unexposed. The CLSM analysis of the thin section of tumors showed that the intensity of Rh6g in the right tumor was substantially higher than the left tumor (Fig. 8I,II). Although, the distribution of Rh6g was decreasing from periphery to center still the decrease in intensity was not substantial (Fig. 8I,II). In the peripheral region, the Rh6g intensity of the right tumor was 4-fold higher than the corresponding region of the left tumor (Fig. 8I,II). In the central region also, the intensity of Rh6g was 4.5-fold higher in the right tumor as compared to the corresponding region in the left tumor (Fig. 8I,II). Moreover, in Kaplan-Meier survival study it was found that during treatment the maximum percentage (90.9%) of animal survived in a group treated with Cur_NE + MB + US whereas the least survival percentage (51.9%) was observed in a group treated with PBS (Fig. 8III,E). The group treated with free curcumin, 75.6% (Fig. 8IIIE) of animals survived at the end of treatment schedule whereas the group Cur_NE + US showed a survival percentage of 80%. Kaplan-Meier study showed a significant (log-rank test, P ≤ 0.04) trend of increase in survival percentage. As far as the decrease in body weight of mice is concerned, in all the groups, relative body weight decreased initially post cells injection but soon it increased gradually (Fig. 8IIIF). Once treatment started, the decrease in body weight followed the trend of PBS > Cur free > Cur_NE > Cur_NE + MB. It suggests that the decrease in body weight occurred due to the decrease in tumor volume as a decrease in both, body weight as well as tumor volume, followed the same pattern.

Discussion
Cur_NE from different ratios of lecithin and curcumin were developed and characterized. It was found that at all the ratios provided spherical shaped stable Cur_NE with diameter 60-120 nm and optimum negative zeta potential. Moreover, the 1 H NMR spectrum revealed that the process of encapsulation was not altering the chemical structure of the curcumin and was stable inside the Cur_NE. An increase in the concentration of curcumin at a fixed concentration of lecithin eventually saturated the loading capacity. The ratio 2:1 showed higher internalization as compared to 1.5:1 and 1:1, as the hydrophilicity of latter nanoemulsions (1.5 & 1:1) would have decreased compared to the former one (2:1) due to the gradual increase in filling of curcumin. Hence, ratio 2:1 was preferred over all the other ratios for further studies. The developed nanoemulsions was biocompatible with normal fibroblast cell line (see Supplementary Fig. S12). Solubility assay revealed that the encapsulation of the curcumin inside lecithin nanoemulsions turned the whole nanostructure hydrophilic and that is why all the ratios got easily resuspended in PBS but the same amount of free drug was highly hydrophobic and could not be dispersed in PBS.
The developed MBs were proved to be echogenic i.e. capable of generating ultrasound contrast (see Supplementary Fig. S5). Application of ultrasound over cells in presence of MB lead to the cavitation of latter which in turn exerted shear stress over the cells situated nearby and generated small pores in the plasma membrane as observed in Fig. 4 in both MDA MB 231 and B16F10 cells. Cells treated with ultrasound developed small pores in the plasma membrane but pores generated after ultrasound treatment in presence of MB were higher in count/cell and also larger in diameter compared to ultrasound treatment in absence of MB. Moreover, it was found that if MBs cavitated and burst near the cell, it created the pore with a wide diameter (≈1 µm) due to an extreme shear force and jet propulsion around cells (see Supplementary Fig. S8). These pores facilitated the intracellular delivery of therapeutics.
That is why the addition of MB further enhanced the Rh6g intensity inside the cells as compared to the ultrasound alone and hence the ultrasound application will be very crucial in increasing cytotoxicity of a drug. The insonation also increased the population of cells involved in internalization and hence the application of ultrasound would ensure the maximum participation of the constituent cells of tumor in the uptake of drug and therefore maximum regression of the tumor. Through the same mechanism, the internalization of Cur_NE would have enhanced in the presence of ultrasound and MB, which resulted in a substantial reduction in IC 50 of curcumin. These sonopores greater in number and diameter would have ensured the higher uptake of curcumin to reduce the IC 50 and even after the expulsion of the drug through pgp receptor, enough amount of drug would have been inside the cells to stop the cellular proliferation (see Supplementary Fig. S10). Moreover, the insonation of cells; in the presence as well as absence of MB, increased the percentage of apoptotic cells. It is already reported that the application of low intensity pulsed ultrasound (>0.5 W/cm 2 ) substantially increased the intracellular Ca 2+ concentrations and expression of caspase 3, Bcl-2 and Bax which in turn increased the apoptosis of hepatocellular carcinoma cells. Ca 2+ dependent expression of apoptotic inducer and curcumin would have synergistically increased the population of late apoptotic cells 27 . Moreover, the increased uptake of curcumin inside cells due to presence MB and ultrasound would have enhanced the signal cascade responsible for the apoptosis of cells. Park et al. 2013 reported that curcumin enhanced the TNF related ligand-induced apoptosis in breast cancer cells MCF-7, T47D and SK-BR-3 28 .
Release kinetics study in SGF and SIF revealed that the formulation is resistant towards the gastric juice of stomach but sensitive to the intestinal fluid. Hence, it will not release the curcumin in the extreme acidic condition of gastric juice but will be easily digested in the small intestine and enhance the absorption of the curcumin. It was observed that the Cur_NE got agglomerated in SGF but the addition of NaOH (increased the solution's pH 6.8-7.4) re-dispersed the Cur_NE completely and intact Cur_NEs were present in solution (Fig. 3F,G) 29 . Naturally, the bicarbonate released from the pancreas will play a role in this neutralization of HCl. Hence, when which was not exposed to ultrasound (D) peripheral region of the right tumor which was exposed to ultrasound for 60 sec at intensity 2 W/cm 2 , duty cycle of 50%, (E) central region of the left tumor, (F) central region of the right tumor. II. The average pixel intensity of the red fluorescence from each image was calculated and plotted. Ten points were considered from each image for calculating pixel intensity. P value was calculated by Student t-test, and values are presented in the form of mean ± SD. ****P ≤ 0.0001 and **P ≤ 0.01. III. Antitumor efficacy of the Cur_NE in the presence and absence of MB injection. The right tumor was exposed to ultrasound whereas the left tumor was kept unexposed. www.nature.com/scientificreports www.nature.com/scientificreports/ Cur_NE will cross the stomach, the pH of the small intestine would re-disperse the Cur_NE again in colloidal form (Fig. 3F). Moreover, the re-dispersion would expose the individual particles to pancreatin and facilitate the enzymatic digestion of Cur_NE to release free fatty acid, monoglycerides, and curcumin. Thereafter, this free curcumin would be absorbed either alone or in combination with monoglycerides and fatty acid in the form of chylomicrons. Curcumin released from Cur_NE would have entered the circulation presumably through two pathways: (i) transcytosis of the curcumin through intestinal epithelial cells and, (ii) chylomicrons encapsulated with curcumin diffuses to the lymphatic system and then to blood circulation (Fig. 7I) 30,31 . Moreover, the higher concentration of curcumin in Peyer's Patch suggests that the Cur_NE got absorbed through the route of the first-pass metabolism and entered the circulation through the lymphatic capillary in the form of chylomicrons. Consequently, T 1/2 , C max , and AUC 0-∞ as well as biodistribution of the curcumin in vital organs increased several-fold.
Effect of ultrasound intensities on fibroblast normal cells showed that intensity of 2 W/cm 2 did not induce any cell death and more than 90% cell viability was maintained. Also, the histopathology examination of the insonated skin revealed that the ultrasound stimulus, at 2 W/cm 2 and 50% duty cycle for 60 sec, was safe for topical application as not induced inflammation or ulceration in the skin (see Supplementary Fig. S11). Furthermore, one obvious observation was that in every group right tumor was smaller in size as compared to the left tumor which is attributed to the exposure of ultrasound that increased the uptake of curcumin from extracellular region to inside the cell due to sonoporation. Although the reduction in the tumor volume was not found to be substantial in left tumors in all the treatment. This might have happened due to less uptake of curcumin by the tumor in absence of ultrasound; on the contrary, the exposure of ultrasound over the right tumor enhanced the intracellular delivery of the drug to the intratumoral space. This was evident from a result where we demonstrated that the exposure of ultrasound stimulus could increase the delivery of dye in the region of interest. That is why a substantial reduction in volume observed in the tumor exposed to ultrasound.
Moreover, the repeated use of ultrasound in the presence of MB created sheer stress that led to the death of the cells and that is why even in the absence of any drug, at the end of 15 days, reduction in right tumor occurred in a group treated with PBS, although reduction in tumor was not statistically significant (P = 0.2). Hence, simple ultrasound exposure may not be able to regress the tumor effectively. As per the literature, ultrasound exposure also generates ROS, and its repeated exposure might have produced enough amounts of ROS which led to the death of cancer cells 32 . Hence, the oral delivery of the formulation followed by MB injection and ultrasound treatment provides a unique modality of oral chemotherapy for the treatment of cancer. www.nature.com/scientificreports www.nature.com/scientificreports/ Release kinetics in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). The drug retention ability of the Cur_NE in the stomach was evaluated by monitoring the release profile over 48 hrs in SGF. SGF (pH 1.5-2) was prepared as reported previously after making desired changes in the protocol 33 . In short, 0.2% NaCl and 0.32% pepsin were dissolved in 7 ml HCl and finally, volume was adjusted to 1000 ml. The release medium comprised 0.5% (v/v) tween 80 in SGF. Cur_NE, with concentration 1 mg/ml curcumin, was packed inside dialysis bag (HIMEDIA LAB 387 Dialysis Membrane-50, molecular wt. cut-off 12,000 to 14,000) and kept in sink condition with the required volume of release medium in USP dissolution apparatus type-II (ELECTROLAB, TDT-08L). The amount of released curcumin was estimated at different time points 0, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 36 and 48 hrs using HPLC at 420 nm after making appropriate dilutions.
Release kinetics was also studied in SIF with pH 6.8-7.4 and was performed similar to the release kinetics in SGF. The composition of the SIF was preferred as reported by USP and changes were made in the protocol as per requirement. Briefly, 0.05 M monobasic potassium phosphate, 0.15 M NaOH, 3 mM sodium taurocholate and 1% w/v of pancreatin at pH 6.8 33 . The release medium comprised 0.5% (v/v) of tween 80 in SIF.

MB induced sonoporation in
Briefly, 14 µg NERh6g was added and the cells were treated with ultrasound at a 50% duty cycle for 30 sec with varying intensity viz. 0.5, 0.7, 0.9 W/cm 2 . In another set of experiment, cells were supplemented with 14 µg of NERh6g and 50 µg of MB before exposing with ultrasound at 50% duty cycle for 30 sec at an intensity of 0.9 W/ cm 2 . The cells were incubated for 1.5 hrs. After incubation, to stain the nucleus, 0.02 nM of Hoechst 33342 (ANASPEC, Inc.) dye was added and left for 10 min. Likewise, at the same concentration of NERh6g, cellular uptake study was performed by flow cytometry 25 .

Cytotoxicity in MDA MB 231 breast cancer and B16F10 melanoma cancer cells. MDA MB 231
and B16F10 cells were grown overnight in 96 well plates and fresh medium containing serial dilutions of Cur_NE with curcumin concentration 100, 25, 6.3, 1.6, 0.4, 0.1, 0.02 µM was added. After the addition of Cur_NE, cells were treated with ultrasound at a 50% duty cycle at intensities 0.9, 1.5 and 2 W/cm 2 for 30 sec. In another set of experiments, the effect of MB on cytotoxicity was observed and in addition to Cur_NE, 0.2 and 0.3 mg/ml of MB was also added and serially diluted. At the end of 72 hrs, MTT assay was performed to determine the cell viability.
Apoptosis assay. The flow cytometry assay was performed to determine the apoptotic induction ability of the Cur_NE in presence of MB and ultrasound 25 . Cells were treated with Cur, Cur_NE, Cur_NE + US, and Cur_NE + MB + US; the US stands for the ultrasound treatment at intensity 2 W/cm 2 , duty cycle 50% for 30 sec. The concentration of Cur, Cur_NE, Cur_NE + US used for the treatment was equal to the IC 50 concentration of Cur_NE + MB + US (0.3) and following ultrasound exposure entire tests and control were incubated further for 72 hrs. In vivo pharmacokinetic study. The pharmacokinetic study was performed in 6-8 weeks old albino Wistar rats (n = 6) with body weight 200-250 g. Ten mg/kg body weight single dose (oral or I.V.) was administered and after administration, 0.5 ml blood sample was collected through retro-orbital puncture at intervals 0, 1, 2, 6, 8, 10, 24 hrs. Plasma was processed and curcumin was extracted as reported previously 25 . To study biodistribution, the entire organ was weighed and then 0.5 g of organs viz. liver, kidney, spleen, lung, brain, Peyer's patch and heart were homogenized in 2 ml acetate buffer (pH 5) and the rest of the process of curcumin extraction was similar to the previously reported article 25 . In vivo anti-tumor efficacy. To study the in vivo anti-tumor efficacy, tumors were generated on both the flanks of the mice (C57BL/6, n = 6, 6-8 weeks old) as reported by us 25 . Thereafter, Forty mg/kg body weight of free Cur, Cur_NE was administered orally to the mice of all groups and then after 2 hrs of dosing, 250 µg of MB was injected through the tail vein. Just after injection of MB, ultrasound treatment was given at parameters 2 W/cm 2 , 50% duty cycle for 60 sec to the right tumor whereas the left tumor was kept unexposed. A total of 5 treatments were given, first three on alternate days and last two after a gap of two days. To determine the tumor uptake, 0.5 mg (20 mg/kg body weight) of NERh6g was administered orally to the tumor bearing mice. Then after 2 hrs, 250 µg of MB was injected through the tail vein and just after that right tumor was exposed with ultrasound. Tumors were excised and then analyzed by CLSM 25 .

Statistical analysis.
All the experiments were conducted in triplicates unless stated otherwise. The graphs are showing data in the form of mean ± SD. The statistical significance of the results was calculated by the Student t-test (two-tailed model).