Polymeric Nanoparticles In Practice For Drug Delivery Applications Biology Essay

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Polymeric nanoparticles are already in practice for drug delivery applications but they have their own limitations [21, 22]. These might be the primary reasons for the design of alternative metal nanoparticles using materials suitable for drug delivery applications. In this work, we have used isolated marine polysaccharide for reduction of HAuCl4, where it acts as a reducing as well as stabilizing agent (scheme 1). The synthesized AuNps were further utilized as a nanocarrier for the delivery of anticancer drug such as doxorubicin hydrochloride.

Scheme 1. Schematic representation showing sulfated polysaccharide reduced gold nanoparticles.

For this application, we have successfully isolated the polysaccharide from marine red algae. This pale yellow colored polysaccharide was dissolved in water on heating. The UV/Visible spectra of isolated polysaccharide (0.1% W/V) in deionised water showed no peak from 260 to 280 nm indicating absence of the protein and nucleic acid like components in the isolated polysaccharide (Fig. 1A).

The evaluation of functional group present in the polysaccharide was carried out by FTIR analysis. The FTIR spectra of the polysaccharide (Fig. 1B) depicted typical bands at 1647, 1417, 1215, 1158, 1019, 933 and 819 cm-1 . The signal at 1215 cm−1 was assigned to the asymmetric stretching vibration of sulfate group, and the signal at 819 cm−1 was indicative of a sulfate group attached to a primary hydroxyl group. Another weak band at 933 cm−1 was due to the 3, 6-anhydro-D-galactose unit in the polysaccharide. From these results and similar finding reported earlier it was confirmed that the isolated polysaccharide is having 3, 6-anhydrogalactose unit and sulfate group [23].

Fig. 1. (A) UV/Visible spectra of 0.1% W/V isolated polysaccharide solution in deionised water and (B) FTIR spectra of isolated polysaccharide.

This sulfate group containing polysaccharide (SP) was used as a reducing and stabilizing agent for synthesis of AuNps. Figure 2A shows the UV/Visible spectra recorded from the dispersion obtained by the reduction of HAuCl4 using 0.01% W/V SP, the band corresponding to the SPR occurred at 520 nm. SPR of AuNps appears in the visible region and can be used to monitor shape, size and aggregation of the nanoparticles. It was observed that HAuCl4 reduction occurs rapidly and the intensity remained unchanged, without any shift in the peak wavelength even after 24 h of reduction time. Inset photographs showed the color of AuNps reduced at 0.01% W/V SP (Fig. 2A).

To evaluate the reduction efficacy of SP, effect of concentration of SP on nanoparticles formation was studied. The information drawn from the UV/Visible spectra of nanoparticles prepared by varying the concentration of SP from 0.01% W/V to 0.05% W/V illustrated that AuNps synthesis was possible even at very low concentration of SP. For further studies, we used 0.01% W/V SP for reduction. Figure 2B depicted the FTIR spectra of SP capped AuNps, where signal of asymmetric stretching vibration of sulfate group shifted to 1283 cm−1 and the signal at 819 cm−1 (sulfate group attached to a primary hydroxyl group) was diminished after synthesis of AuNps indicating the involvement of the sulfur group of polysaccharide in synthesis of AuNps.

Fig. 2. (A) UV/Visible spectra of 0.01 % W/V SP reduced AuNps and inset photograph showed color of AuNps dispersion, (B) FTIR spectra of SP capped AuNps.

HRTEM images (Fig. 3A) revealed that the AuNps appeared to be spherical in shape with narrow size distribution and inset Fig. 3A showed average particle size about 13 ± 3 nm. The selected area of electron diffraction pattern of the AuNps showing the rings designated 1, 2, 3 and 4 arise due to the reflections from (111), (200), (220) and (311) (Fig. 3B). This was further confirmed by the powder X-ray diffractogram recorded from the sample (Inset Fig. 3B), which may be indexed as the band for fcc structures of gold. The XRD pattern thus clearly illustrated that the broadening of Bragg's peaks indicated the formation of nanoparticles [24] and these are in crystalline form.

Fig. 3. (A) HRTEM images and inset (A) showed particle size distribution graph. (B) electron diffraction pattern of SP capped AuNps and inset (B) XRD patterns of SP capped AuNps respectively.

Zeta potential of AuNps was found to be - 31.05 mV. The negative charge indicated that the AuNps were wrapped with the SP. In general, particle aggregation is less likely to occur for charged particles with optimum zeta potential (~ ±30 mV) due to electrostatic repulsions [10].

Stability of AuNps at different pH and electrolytic conditions is a very important requirement for varied biomedical applications. In this regard, we studied the stability of AuNps by monitoring the SPR over reasonable period of time at different pH and electrolytic conditions. It should be noted that a red shift in UV/Visible spectra is associated with either an increase in the mean size of the particles or aggregation of nanoparticles or a combination of both [25]. In case of pH study, the pH of AuNps dispersion was adjusted from pH 2-12. The sample was incubated overnight and analyzed for any change in the SPR. Figure 4A depicted that insignificant change in peak intensity and SPR shift was observed in pH range of 3 to 12. Inset photographs suggested that the color of AuNps turned to blue indicating the aggregation of nanoparticles at pH 2. Also, addition of electrolyte (NaCl) up to 0.01 M caused no major aggregation (Fig. 4B), thus meeting the stipulations laid out for the utility of these particles for drug delivery applications. Nanoparticles obtained by borohydrate or citrate reduction routes aggregate at slight change in their pH and electrolytic condition [26]. This evaluation was easily confirmed by the SPR position of AuNps and its aggregates, the insignificant change in its position under change in the pH and electrolytic conditions indicating the excessive stability of SP capped AuNps. Also, in long term stability study, AuNps did not show shift in SPR (Fig. 4C) and inset HRTEM image of SP capped AuNps revealed that no change was observed in particle size and shape over six month stability period.

Fig. 4. UV/Visible spectra of SP capped AuNps (A) at varied pH, (B) electrolytic condition and (C) UV/Visible spectra of room temperature stability study samples of AuNps. Inset (A) and (C) showed the color change of AuNps at varied pH conditions and HRTEM images of six months stability sample of AuNps respectively.

Several researchers have made attempts to investigate the macrophage-stimulating activity of the SP by means of in-vitro and in-vivo study. Yamamoto et al. reported that the oral administration of several seaweeds could cause a significant decrease in the incidence of carcinogenesis [27]. In recent years, algal polysaccharides have been reported to have free-radical scavenging activity and act as an antioxidant for the prevention of oxidative damage in living organisms [28, 29]. Previously, Kwon et al. reported the anti-proliferative effect of SP (Porphyran) on human gastric carcinoma cell line (AGS), where 0.25 and 0.5% of SP showed significant inhibition of cell growth as compared to control [14].

In order to demonstrate the effect of the nanocarrier such as AuNps on the cytotoxic activity of SP, we have carried out cytotoxicity study of native SP and SP capped AuNps on human glioma cell lines (LN-229) and human gastric carcinoma cell line (AGS) using in-vitro MTT assay. It was observed that native SP and SP capped AuNps revealed cytotoxic effect on both the cell line. However enhancement in cell cytotoxicity was observed in case of SP capped AuNps (cell viability: 10% for LN-229 and 50% for AGS) as compared to native SP (cell viability: 40% for LN-229 and 87% for AGS) after 72 h incubation (Fig. 5). The higher cytotoxicity in case of SP capped AuNps can be attributed to the greater uptake potential of the AuNps as compared to native SP [11]. These results clearly revealed that the AuNps helped as a nanocarrier for enhancement of cytotoxic effect of SP on human carcinoma cell lines after capping on AuNps as compared to native SP.

Fig. 5. Percent cell viability of LN-229 and AGS cancer cell line after exposure to 0.01 % SP and 0.01% SP capped AuNps at the end of 72 h.

After the successful synthesis of stable SP capped AuNps, we have envisaged this system for drug delivery application through the subsequent attachment of a bioactive molecule. Therefore, we have selected a low bioavailable anticancer drug such as DOX (pKa=8.2) for loading on SP capped AuNps. The loading efficiency of DOX loaded AuNps was found to be 60% after 24 h incubation at room temperature (Fig. 6A) and inset photograph represented HRTEM image of DOX loaded AuNps revealed insignificant change in particle size (14±3 nm) (Fig. 6A). The decrease in the zeta potential (from -31.05 mV to -19 mV) of DOX loaded AuNps was ascribed to the presence of positively charged DOX on the surface of AuNps. Thus even after DOX loading, SP capped AuNps remained as a stable dispersion owing to the electrostatic repulsion through the negative surface charge. It was thought that along with the electrostatic interaction other attractive forces including hydrogen bond could be playing a major role facilitating the drug loading process. The hydrogen bonding hypothesis between protonated amine groups of the DOX molecule with SP capped AuNps is also supported by FTIR, where NH stretching band of native DOX at 3314 cm-1 shifted to 3413 cm-1 in case of DOX loaded AuNps suggested the formation of hydrogen bond between protonated amine group of DOX with SP capped AuNps (Fig. 6B).

Fig. 6. (A) UV/Visible spectra of native DOX solution, supernatant and inset photograph represented HRTEM image of DOX loaded AuNps and (B) FTIR spectra of native DOX and DOX loaded AuNps.

To establish the capabilities of the AuNps drug carrying technology, we determined the cytotoxicity of native DOX solution and DOX loaded AuNps on human glioma cell lines (LN-229) using in-vitro MTT assay. The wells that received only media were regarded as control showing 100 % cell viability at the end of 48 h. Figure 7 illustrated dose dependant cytotoxic effect of DOX in the form of either DOX loaded AuNps or native DOX on the LN-229 cells after 48 h exposure. DOX loaded AuNps exerts a higher cytotoxic effect than native DOX on LN-229 cells at the same dose. At the end of 48 h, the decrease in cell viability with native DOX and DOX loaded AuNps in the concentration range studied (1.0-20 µg/mL) was found to be between 60-35% and 48-20%, respectively.

Fig. 7. Percent cell viability of human glioma cell line (LN-229) after exposure to control media, native DOX solution and DOX loaded AuNps (48 h).

Previously, L. Serpe et al. reported increased cytotoxicity of doxorubicin when incorporated in solid lipid nanoparticles due to the fast internalization of doxorubicin loaded solid lipid nanoparticles followed by the drug's release inside the cells [30]. In our study, we observed a significant increase in the cytotoxicity of DOX on LN-229 when loaded on AuNps compared to native DOX. The increase in cytotoxicity of DOX loaded AuNps may be due to the enrichment in internalization of DOX loaded AuNps by an endocytosis mechanism as compared to the passive diffusion mechanism of native DOX into cells [31].

4. Conclusion

In conclusion, we have reported size controlled synthesis of gold nanoparticles by using isolated marine sulfated polysaccharide from red algae. These nanoparticles exhibited stability in a wide range of pH and electrolyte concentration. Further, applicability of these nanoparticles as carriers for the delivery of the cationic anticancer drug was demonstrated by successful loading of doxorubicin hydrochloride (DOX) onto AuNps. In-vitro cell line study revealed higher cytotoxicity of sulfated polysaccharide capped AuNps and DOX loaded AuNps in human carcinoma cell lines as compared to native polysaccharide and native DOX solution. As per these results, further in vivo anti-tumor activity of DOX loaded AuNps and toxicity of sulfated polysaccharide capped AuNps following chronic use are under investigation in our laboratory.

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