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Porphyran, a poly-anionic sulfated polysaccharide isolated from marine red algae Porphyra vietnamensis has been investigated as a reducing agent for size controlled synthesis of gold nanoparticles. The prepared AuNps showed SPR centered at 520 nm with average particle size of 13±3 nm. FTIR spectra suggested that the sulfate moiety is mainly responsible for reduction of aurochloric acid. The capping of the AuNps with porphyran was evident from the negative zeta potential value which also rendered electrostatic stability. Thus, porphyran act as both reducing as well as capping agent. These porphyran capped AuNps are highly stable in a wide range of pH and electrolytic concentration. Porphyran capped AuNps showed enhanced cytotoxicity on human glioma cell lines (LN-229) as compared to native porphyran. Consequently, porphyran capped AuNps have been used for drug delivery application by loading the anticancer drug doxorubicin hydrochloride (DOX). Spectroscopic examinations revealed that DOX conjugated onto porphyran capped AuNps via hydrogen bond between protonated amine group of DOX with porphyran capped AuNps. In acetate buffer (pH 4.5), release rate of DOX was found to be considerably higher as compared to physiological buffer (pH 7.4) from DOX loaded porphyran capped AuNps. Further, these drug loaded porphyran capped AuNps demonstrated higher cytotoxicity on LN-229 cell line as compared with an equal dose of native DOX solution. This established the potential of porphyran capped AuNps as a carrier for anticancer drug delivery.
Keywords: gold nanoparticles, porphyran, cytotoxicity, doxorubicin hydrochloride, and drug delivery
Novel colloidal carriers such as liposomes, polymeric nanoparticles and solid lipid nanoparticles are widely used for delivery of various biomolecules and pharmaceutical actives because they offer lot of advantages like improved efficacy, targeted delivery and reduced toxicity as compared to conventional drug delivery system.1-3 In the present state of affairs, metal nanoparticles are also widely investigated, particularly gold nanoparticles (AuNps) for their unique properties such as comparable size with biomolecules, binding ability to various molecules and optical properties in the visible and NIR regions make them potential candidates for chemical as well as biological applications and these can be synthesized easily.4 Recent burst of research includes biological, diagnostic and therapeutic applications of AuNps.5-11 Several researchers improved the properties of biomolecules and drugs such as amino acids,12 protein,13 DNA,14 insulin,15 ciprofloxacin16 and plasmid17 after conjugation with AuNps without altering their biochemical properties. But, in majority cases chemically synthesis of AuNps18-19 were utilized for conjugation of drug molecules. In this process, chemical reduction of gold salt to AuNps was carried out by using harsh reducing agent such as tri-sodium citrate and sodium borohydrate and organic solvents making them unsuitable for biological applications. To overcome these challenges, the interest in this field has been increased from past few years for utilization of such reducing agent will synthesize size controlled AuNps and give sufficient stability to AuNps under strong electrolytic and pH conditions and making them suitable for therapeutic and drug delivery applications. Recently, Dhar et al., synthesized highly stable AuNps by using gellan gum as a reducing agent for delivery of anticancer drug.20 Also, in another study biodegradable chitosan mediated synthesis of AuNps was improved the transmucosal delivery of insulin.21 In the present study, we have synthesized one pot size controlled AuNps by using naturally occurred reducing agent followed by conjugation of drug molecule on the surface of AuNps.
Porphyran, a poly-anionic sulfated polysaccharide is isolated from marine red algae (Porphyra vietnamensis) found predominantly in rainy season on the west coast of India, especially in the provinces of Maharashtra, Goa and Karnataka.22 Porphyran comprising the hot-water soluble portion of cell wall is the main component of the red algae. Porphyran comprises of disaccharide units consisting of 3-linked-D-galactosyl residues alternating with 4-linked 3, 6- anhydro-l-galactose and the 6-sulfate residues.23 Porphyran is a dietary fiber that contains about 40-50% of seaweed components. Pharmacological functions of isolated porphyran from several porphyra species were demonstrated in different structural and functional studies. Previously few studies have been reported on the antioxidant and anticancer activity of porphyran.23-24
Here, we have explored the use of isolated porphyran for synthesis of stable AuNps, such that it acts as a reducing as well as capping agent. Prepared AuNps were evaluated through suitable techniques to study morphology, surface charge and analyze functional group responsible for reduction of gold ions and binding on AuNps. Effect of pH condition and presence of electrolyte on synthesized AuNps was carried out. Six month stability study of these nanoparticles was performed at ambient temperature. We hypothesize that the porphyran capped AuNps would result in enhanced cytotoxicity as compared to native porphyran through MTT assay method on human glioma cell line (LN-229).26-28 Further to demonstrate applicability for drug delivery we have investigated the loading of anticancer drug doxorubicin hydrochloride (DOX) on porphyran capped AuNps. DOX is a potent anthracycline antibiotic used for various cancer therapies such as malignancies, carcinoma and sarcomas, Lack of tumor targeting capacity of DOX results in narrow biodistribution and undesirable side effects which remain as major problems to be solved.25 This led the company ALZA to develop a product DOXIL® that improved the performance by encapsulating the DOX in a liposome, thereby enhancing the anticancer action of DOX. Here, we have presented the conjugation of DOX via hydrogen bond onto highly stable porphyran capped AuNps may lead to improved cytotoxic effect on human cancer cell line. Also, in-vitro release studies of DOX loaded porphyran capped AuNps was carried out in different pH to demonstrate the effect of pH on the release of DOX.
2. Experimental Methods
2.1. Materials and reagents. Doxorubicin hydrochloride was gifted from RPG Life Sciences Limited, Mumbai (India). Hydrochloroauric acid (HAuCl4) was obtained from Sisco India Ltd. Mumbai. The human glioma cell line (LN-229) was porches from American type culture collection (ATCC, USA). The yellow tetrazolium MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) was obtained from Sigma-Aldrich (USA). Analytical grade reagents are from Merck India Ltd., India. All the samples were prepared in deionized water.
2.2. Isolation and characterization of porphyran. Porphyran was isolated from marine red algae (Porphyra vietnamensis).29,30 In brief, dried red algae were soaked in 7.5% formalin for 12 h at ambient temperature. An equal volume of water was added and solution refluxed on boiling water for 8 h. The mixture was centrifuged at 10,000 rpm for 20 min and supernatant was filtered through diatomite. The resulting filtrate was adjusted to pH 7 with sodium hydroxide and 75% volume was evaporated at 65 0C on rota evaporator. Four fold volume of methanol was added to residual solution to precipitate porphyran content. The mixture was centrifuged at 10,000 rpm for 20 min and supernatant was discarded. The precipitated porphyran was washed with 80% aqueous methanol. The residue was freeze dried to yield pale yellow color powdered porphyran. Total sugar and sulfate content in porphyran was determined by the phenol sulfuric acid method31 and Kawai method.32 This freeze dried porphyran was used for further studies.
2.3. Synthesis of gold nanoparticles using porphyran. In a typical experiment, 100 mL an aqueous solution of HAuCl4 (1X10-4 M) containing porphyran (0.01% w/v) was reduced to AuNps by adjusting the pH of solution with sodium hydroxide to 11 followed by heat up to boiling for 10 min yielded dark ruby red colored AuNps. The AuNps dispersion was thoroughly dialyzed for 24 h to remove ionic impurities used during the reduction. After dialysis, the pH of the AuNps dispersion was measured to be 7.
2.4. UV/Visible spectroscopy measurement. The UV/Visible spectra of native porphyran solution (0.1% W/V), porphyran capped AuNps was monitored by UV/Visible spectroscopy (Jasco dual beam UV/Visible spectrophotometer, Model V-570, Japan). Change in surface plasmon resonance (SPR) of porphyran capped AuNps was monitored.
2.5. Loading of DOX on porphyran capped AuNps. A calculated amount of DOX was added to porphyran capped AuNps dispersion (pH 7), obtained as described above, resulting in a final DOX concentration of 10-4 M in solution. The mixture of DOX and AuNps dispersion was incubated for 24 h at room temperature and then centrifuged at 10,000 rpm for 10 min. The obtained pellet after centrifugation was separated from the supernatant solution and redispersed in deionized water prior to further characterization.
2.6. Determination of percent drug loading on porphyran capped AuNps. Drug loading was calculated from the difference between the initial DOX concentration and the superfluous DOX determined in the supernatant liquids. The drug concentration in supernatant was determined by measurements of its UV absorbance at 480 nm using UV/Visible spectroscopy and the percentage loading of DOX on porphyran capped AuNps was estimated by formula (1)
2.7. Spectroscopy, zeta potential measurement, microscopic study and X-ray diffraction. Fourier transform infra red (FTIR) spectra of native porphyran, porphyran capped AuNps, native DOX and DOX loaded porphyran capped AuNps was recorded in KBr pellets using FTIR spectrophotometer (Jasco, Japan). The scan was performed in the range 400 to 4000 cm-1. The zeta potential (ZP) was determined by using the Zetasizer 300 HAS (Malvern, UK). The zeta potential of porphyran capped AuNps and DOX loaded porphyran capped AuNps was determined as such without dilution. Which measured the surface charge on polysaccharide capped AuNps and DOX loaded porphyran capped AuNps. The morphology and size distribution of the porphyran capped AuNps and DOX loaded porphyran capped AuNps dispersion was carried out by high resolution transmission electron microscopy (HRTEM) measurement casting of nanoparticle dispersion on carbon-coated copper grids and allowed to dry at room temperature. Measurements were done on TECHNAI G2 F30 S-TWIN instrument operated at an accelerated voltage of 300 KV with a lattice resolution of 0.14 nm and point image resolution of 0.20 nm. The particle size analysis was carried out using Gattan software (Pleasanton, CA, USA). X-ray diffraction (XRD) measurement of porphyran capped AuNps was carried out by preparing films of nanoparticle dispersion on glass substrates by simple solvent evaporation method at room temperature. The diffraction measurements were carried out on X'pert Pro X-ray diffractometer (Netherlands) instrument operating at 40 kV and a current of 30 mA at a scan rate of 0.388/min.
2.8. pH and electrolytic stability study. In the pH stability study, the pH of porphyran capped AuNps dispersion was adjusted using 0.1N hydrochloric acid (pH 2, 3, 4, 5 and 6) and 0.1M sodium hydroxide (pH 7, 8, 9, 10, 11 and 12) using calibrated pH meter (Delux pH meter 101, India). Change in SPR of porphyran capped AuNps dispersion was recorded after 24 h using a UV/Visible spectrophotometer. Also, the effect of electrolyte was studied by adding varying molar concentration of sodium chloride (101 to 10-6 M) to porphyran capped AuNps dispersion. The change in SPR was recorded after 24 h using UV/Visible spectrophotometer.
2.9. Stability study. The stability study of porphyran capped AuNps complex was carried out at ambient temperature. The change in SPR of the nanoparticle dispersion was recorded up to six month using UV/Visible spectroscopy.
2.10. In-vitro drug release study. The dialysis tube containing DOX loaded porphyran capped AuNps was transferred to a beaker containing 100 mL of phosphate buffer (pH 7.4) by maintain temperature at 37 0C with continuous stirring at 100 rpm. Sink condition was maintained by periodically removing 2 mL sample and replacing equal volume of buffer. The amount of released DOX was analyzed with a spectrophotometer at 485 nm. A similar release study was carried out in acetate buffer (pH 4.5). The experiments were performed in triplicate for each of the samples.
2.11. In-vitro MTT assay. Human glioma cell lines (LN-229) was cultured in Dulbecco's modified eagle's medium (DMEM) supplemented with 1.5 gm/mL sodium bicarbonate, 4 mm glutamine and 5 - 10% fetal bovine serum (Gibco, USA). The cultures were maintained in a humidified atmosphere of 5% CO2 at 37 0C in an incubator. For cytotoxicity testing, the cells were utilized when they reached 70 - 80% confluence. The cells were diluted as needed and seeded as 3 X 103 for LN-229 in 200 µL of media per well, sequentially plated in flat bottom 96 well plates (Becton Dickinson Labwane, USA). This number of cells was selected to avoid potential over confluence of the cells at the end of the four day experiment while still providing enough cells for adequate formazan production. After plating, the 96 well plates were then incubated for 24 h to allow adherence of the cells prior to the administration of various samples for testing. After complete adherence of cells the culture medium was replaced with 200 µL of solution containing mixture of fresh medium and porphyran solution (0.01% w/v), porphyran capped AuNps dispersion, native DOX solution and DOX loaded porphyran capped AuNps in separate wells. Control wells containing cells received only 200 µL of medium. After addition of all the test samples, the plates were incubated up to 72 h for native porphyran and porphyran capped AuNps and 48 h for native DOX and DOX loaded porphyran capped AuNps in CO2 incubator. The cells could be maintained in wells for this period without the need for re-feeding. All experiments were performed in triplicate. MTT assay was based on the measurement of the mitochondrial activity of viable cells by the reduction of the tetrazolium salt MTT (3-(4,5-dimethyathiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) to form a blue water-insoluble product, formazan. MTT (5 mg/mL, 20 µL) was added to respective set of cells and the plates were incubated for an additional 4 h. After 4 h of incubation, the medium was removed and DMSO (200 µL, Sigma-Aldrich, USA) was added to dissolve the formazan crystals resulting from the reduction of the tetrazolium salt only by metabolically active cells. The absorbance of dissolved formazan was measured at 570 nm using a Bio-Rad microplate reader (Model 680, Heraeus, USA). Since the absorbance directly correlated with the number of viable cells, the percent viability was calculated from the absorbance.
3. Results and discussion
In this work, we have used porphyran isolated from marine red algae (Porphyra vietnamensis) for reduction of HAuCl4, where it acts as a reducing and stabilizing agent (scheme 1). The synthesized AuNps were further utilized as a nanocarrier for the delivery of anticancer drug such as DOX.
3.1. Chemical analysis of porphyran. This pale yellow colored isolated porphyran was dissolved in water on heating. It contained 78.92 % of total sugar and 9.62 % of sulfate. Further, UV/Visible spectra of isolated porphyran (0.1% w/v) in deionized water showed no peak from 260 to 280 nm indicating absence of the protein and nucleic acid like components in the isolated porphyran (Figure 1A). The evaluation of functional group present in the porphyran was carried out by FTIR analysis. The FTIR spectra of the porphyran (Figure 1B) depicted typical bands at 1645, 1417, 1230, 1158, 1019, 933 and 819 cm-1. The signal at 1230 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 we confirmed that the isolated porphyran contained 3, 6-anhydrogalactose unit and sulfate group.33
3.2. Synthesis and evaluation of AuNps. This isolated porphyran was used as a reducing agent and stabilizing for synthesis of AuNps. Preliminary study revealed that the reduction of gold ions in presence of porphyran did not occur up to pH 7, but as pH increased up to 11 reduction occured as indicated by the color change. At pH 11 the color of solution changed from colorless to dark ruby red on heating for 10 min. Figure 2A shows the UV/Visible spectra recorded from the dispersion obtained by the reduction of HAuCl4 using 0.01% w/v of porphyran at pH 11, 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 porphyran (Figure 2A). Figure 2B depicted the FTIR spectra of porphyran reduced 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 porphyran in synthesis of AuNps.
Zeta potential of porphyran reduced AuNps was found to be - 31.05 mV. The negative charge indicated that the AuNps were properly wrapped with poly-anionic porphyran. In general, particle aggregation is less likely to occur for charged particles with optimum zeta potential (~ ±30 mV) due to electrostatic repulsions.20 HRTEM images (Figure 3A) revealed that the porphyran capped AuNps appeared to be spherical in shape with narrow size distribution and inset Figure 3A showed average particle size about 13 ± 3 nm. The selected area of electron diffraction pattern of the porphyran capped AuNps showing the rings designated 1, 2, 3 and 4 arise due to the reflections from (111), (200), (220) and (311) (Figure 3B). This was further confirmed by the powder X-ray diffractogram recorded from the sample (Inset Figure 3B), which may be indexed as the band for face centered cubic (fcc) structures of gold. The XRD pattern thus clearly illustrated that the broadening of Bragg's peaks indicated the formation of nanoparticles34 and these are in crystalline form.
3.3. Stability study at different pH condition and electrolytic concentration. For varied drug delivery applications,35 we studied the stability of porphyran capped 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.36 In case of pH study, the pH of porphyran capped 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 change in peak intensity and SPR shift was not observed in pH range of 3 to 12. Inset photographs suggested that the color of porphyran capped AuNps turned to blue indicating the aggregation of nanoparticles at pH 2. Also, addition of electrolyte (NaCl) up to 1 X 10-2 M caused no major aggregation (Figure 4B). Borohydrate or citrate reduced AuNps aggregate at slight change in their pH and electrolytic condition.35 This evaluation was easily confirmed by the SPR position of porphyran capped AuNps and its aggregates, the insignificant change in its position under change in the pH and electrolytic conditions indicating the excessive stability of porphyran capped AuNps. Also, in long term stability study, AuNps did not show shift in SPR (Figure 4C) and inset HRTEM image of porphyran capped AuNps revealed that no change was observed in particle size and shape over six month stability period.
3.4. In-vitro cytotoxicity of porphyran capped AuNps. Several researchers have made attempts to investigate the macrophage-stimulating activity of the porphyran 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.37 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.38, 39 Previously, Kwon et al. reported the anti-proliferative effect of porphyran on human gastric carcinoma cell line, where 0.25% and 0.5% of porphyran showed significant inhibition of cell growth as compared to control.24
In order to demonstrate the effect of the nanocarrier such as AuNps on the cytotoxic activity of porphyran, we have carried out cytotoxicity study of equal concentration of native porphyran and porphyran capped AuNps on LN-229 cell line using in-vitro MTT assay method. Both native porphyran and porphyran capped AuNps revealed cytotoxic effect on LN-229 cell line. It was interesting to note a fourfold increase in cytotoxicity of porphyran capped AuNps as compared to native porphyran (Figure 5). This clearly demonstrated the role of AuNps for the enhancement of cytotoxic effect of porphyran on human glioma cell lines. Previous report justified that porphyran exhibited cytotoxicity on human gastric carcinoma cell line via induction of apoptosis related signaling through activation of proapoptotic molecules (Bax and caspase-3), suppression of anti-apoptotic molecule (Bcl-2), inhibition of IGF-I receptor and decrease in the level of Akt activation.24 In our case, improvement in cytotoxicity of porphyran capped AuNps can be attributed to the greater uptake potential by endocytosis of the AuNps as compared to native porphyran.21,40
3.5. Evaluation of DOX loaded porphyran capped AuNps. After the successful synthesis of stable porphyran capped AuNps, we have envisaged this system for drug delivery application through subsequent loading of a bioactive molecule. Therefore, we have selected a low anthracycline ring containing anticancer drug DOX (pKa=8.2) for loading on porphyran capped AuNps. Loading efficiency of DOX on porphyran capped AuNps was found to be 60% after 24 h incubation at room temperature. Figure 6A represented HRTEM image of DOX loaded porphyran capped AuNps revealed insignificant change in particle size (14±3 nm) and inset image represented uniformly redispersed DOX loaded porphyran capped AuNps dispersion. The decrease in the zeta potential (from -31.05 mV to -19 mV) of DOX loaded porphyran capped AuNps was ascribed to the presence of positively charged DOX on the surface of porphyran capped AuNps. Thus even after DOX loading, porphyran 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 porphyran 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 porphyran capped AuNps suggested the formation of hydrogen bond between protonated amine group of DOX with porphyran capped AuNps (Figure 6B).
3.6. In-vitro drug release study. Figure 7A depicted the release of DOX from DOX loaded porphyran capped AuNps in acetate buffer (pH 4.5) and phosphate buffer (pH 7.4). At the end of 7, 98% and 15 % of DOX was released in acetate and phosphate buffer respectively. This result revealed that release of DOX was found to be higher in acidic pH as compared to basic pH. This pH dependant release may help to improve efficacy of DOX because uptake of drug loaded nanoparticle through endocytosis process leads to exposure to an acidic environment,41 which may initiate rapid release of DOX from DOX loaded porphyran capped AuNps. Such efficient release would ultimately result in improved cytotoxic efficacy against tumor cells. Also, very low release of DOX in basic pH will help to reduce toxicity of DOX to the normal tissue because physiological pH of body is maintained at pH 7.4, such pH condition may inhibit the release of DOX from DOX loaded porphyran capped AuNps.42
3.6. In-vitro cytotoxicity of DOX loaded porphyran capped AuNps. To establish the capabilities of the porphyran capped AuNps drug carrying technology, we determined the cytotoxicity of native DOX solution and DOX loaded porphyran capped AuNps on LN-229 cell line using in-vitro MTT assay method. Figure 7B illustrated dose dependant cytotoxic effect of DOX in the form of either DOX loaded porphyran capped AuNps or native DOX on the LN-229 cells after 48 h exposure. DOX loaded porphyran capped 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 porphyran capped AuNps in the concentration range studied (1.0-20 µg/mL) was found to be between 60-35% and 48-20%, respectively.
Previously, Serpe et al. reported higher 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.43 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 porphyran capped AuNps may be due to the enrichment in internalization of DOX loaded porphyran capped AuNps by an endocytosis mechanism as compared to the passive diffusion mechanism of native DOX into cells.44 In an earlier cytotoxicity study of anticancer drug loaded AuNps, Chen et al. have reported the intracellular accumulation of methotrexate conjugated AuNps owing to AuNps mediated endocytosis.40
In conclusion, we have reported size controlled synthesis of AuNps by using isolated marine porphyran 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 DOX onto porphyran capped AuNps. In-vitro cell line study revealed higher cytotoxicity of porphyran capped AuNps and DOX loaded porphyran capped AuNps in human glioma cell lines as compared to native porphyran and native DOX solution. Further, in-vivo toxicity study of porphyran capped AuNps and in-vivo anti-tumor activity of DOX loaded porphyran capped AuNps are under investigation in our laboratory.