Quantification of uptake of NPs in the presence and absence of external magnet

Owing to their magnetic behavior of HER2-CUR-SPION-PLGA NPs, they can

be greatly influenced by an external magnetic field. The presence of an external

magnet enhances the magnetic force and upsurge the cellular uptake of

CUR-SPIONs PLGA NPs. To study the cellular uptake, the cells incubated with

HER2-CUR-SPION-PLGA NPs and one set of cells was exposed to external. Cellular

uptake of NPs was studied based on the quantification of release of curcumin from

the cells by spectrophotometrically and was further confirmed by flow cytometry.

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Figure 10: Magnetic sorting, HER2-CUR-SPION-PLGA NPs with PANC-1 cells (a) Absence of external Magnet (b) Presence of External Magnet and HER2-CUR-SPION-PLGA NPs with MIA PaCa-2 Cells (c) Absence of external Magnet (d) Presence of External Magnet.

Figure 11: VSM Curve of after up take of HER2-CUR-SPION-PLGA NPs (a) PANC-1

and (b) MIA PaCa-2 Cells.

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! The cellular uptake of HER2-CUR-SPION-PLGA NPs by pancreatic cancer cells in the presence and absence of an external magnet exposure for 1 hour was quantified by ethanol extraction method. The released curcumin within the cancer cells was extracted and measured spectrophotometrically. Figure 12 shows the absorbance of ethanolic curcumin extracted from PANC-1 and MIA PaCa-2 in the absence and presence of external magnet. It is clear that there was an increased release of curcumin within PANC-1 and MIA PaCa-2 when the cells were exposed to an external magnet for 1 h. The magnetic exposure resulted in the increased release of curcumin, which may be due to increased cellular internalization of NPs or due to increased release of curcumin within cancer cells. Though we could observe an increase in the absorbance as a function of incubation time in cancer cells, the absorbance values remain almost the same as that after 6 h even after prolonging the incubation time up to 24 h. This may be due to the saturation of NPs binding to HER2 receptors on the cancer cells.

Figure 12: Quantification of uptake of nanoparticles in the presence and absence of

external magnet with PANC-1 and MIA PaCa-2 Cells by UV Spectroscopic analysis.

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The relative extent of cellular uptake of NPs in the absence and presence of external magnet was further confirmed by flow cytometry. In flow cytometry the fluorescence intensity of each cell is quantified and plotted it against the number of cells. Exposure to external magnets for a period of 1 h represented enhanced cellular uptake of HER2-CUR-SPION-PLGA NPs than the cells that was not exposed to an external magnet (Figure 13). Although, the cells that were not exposed to external magnet also showed uptake of NPs owing to receptor mediated endocytosis of the same, the intensity was less when compared to the intensity of entry of NPs in the presence of external magnet. However the intensity of fluorescence from HER2-CUR-SPION-PLGA NPs was more when compared to intensity of fluorescence from CUR-SPION-PLGA NPs. Thus, exposure to external magnets drastically augmented the uptake of HER2-CUR-SPION-PLGA NPs predominantly into cancer cells.

Figure 13: Cellular uptake of NPs in the absence and presence of external magnet by

Flow Cytometric analysis (a, b) uptake of untargeted NPs by PANC-1 and MIA

PaCa-2 cells, (c, d) uptake of HERPaCa-2-CUR-SPION-PLGA NP by PANC-1 and MIA PaCa

cells in the absence of external magnet and (e, f) uptake of HER2-CUR-SPION-PLGA

NP by PANC-1 and MIA PaCa-2 cells in the presence of external magnet.

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! 5.3.7. Therapeutic evaluation of nanoformulation

The in vitro cytotoxic effect of nanoformulation (HER2-CUR-SPION-PLGA NPs) was performed to study the therapeutic efficacy in pancreatic cancer cells by alamar blue assay. The therapeutic effectiveness of various concentrations of HER2-CUR-SPION-PLGA NPs was examined in the presence and absence of external magnet and the results are shown in the Figure 14 (a, b). The cells were exposed to an external magnet for a period of 2 h. The study revealed a distinctive time and dose dependent antiproliferative effect in PANC-1 and MIA PaCa-2 cells. After 96 h of incubation, cells treated with HER2-CUR-SPION-PLGA NPs at a concentration of 1.5 mg/ml showed enhanced toxicity and as a result of which cellular viability was reduced to 14 and 11 % for PANC-1 and MIA PaCa-2 confirming its antiproliferation effects. The results distinctly indicate that curcumin maintains its anticancer activity even after being loaded into PLGA NPs. The results also clearly signified that the enhanced cytotoxicity associated with HER2-CUR-SPION-PLGA NPs in the pancreatic cancer cells could be attributed to HER2 receptor mediated enhanced endocytosis.

To study the effect of external magnet-mediated augmentation in

cytotoxicity, we exposed the cells treated with HER2-CUR-SPION-PLGA NPs to an

external magnet for 2 h. The results demonstrated that the antiproliferative activity

was increased considerably in PANC-1 and MIA PaCa-2 owing to the enhanced

cellular uptake of the HER2-CUR-SPION-PLGA NPs. Upon exposure to external

magnetic field, we noticed enhanced cellular toxicity around 48 h, when compared to

the cellular viability percentage in cells treated with targeted NPs. The onset of

cellular cytotoxicity was quiet early when cell incubated with NPs were exposed to

external magnet. Upon incubation, at 72 h the observed cell viability decreased

further and was 11 and 9 % for PANC-1 and MIA PaCa-2 at the highest

concentration. On the other hand the cell viability was 32 and 35 % for PANC-1 and

MIA PaCa-2 upon incubation with HER2-CUR-SPION-PLGA NPs in the absence of

external magnet. Thus the potential of incorporation of SPIONs into nanoconjugate

was harnessed in the form of increased uptake of NPs and enhanced cytotoxicity of

cancer cells upon exposure to an external magnet.

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Figure 14: Cytotoxicity studies of NPs at different concentration in different cell lines

(PANC-1, MIA PaCa-2) in the absence and presence of external magnet by alamar

blue assay.