Î±-Amino Acid Rich Photophytonic Nanoparticles of Algal Origin

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#-Amino Acid Rich Photo-phytonic Nanoparticles of Algal Origin Serendipitously Reveals Anti-migratory Property against Cancer Santosh K. Misra, Aaron S. Schwartz-Duval, Fatemeh Ostadhossein, Enrique A. Daza, Zachery M Saldivar, Brajendra K. Sharma, and Dipanjan Pan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 05 Jun 2017 Downloaded from http://pubs.acs.org on June 10, 2017

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ACS Applied Materials & Interfaces

α-Amino Acid Rich Photo-phytonic Nanoparticles of Algal Origin Serendipitously Reveals Anti-migratory Property against Cancer Santosh K. Misra,a Aaron S. Schwartz-Duval,+,a Fatemeh Ostadhossein,+,a Enrique A. Daza,a Zachary M. Saldivar,a Brajendra K. Sharmab and Dipanjan Pan*,a

a

Departments of Bioengineering, Materials Science and Engineering and Beckman Institute,

University of Illinois at Urbana-Champaign, Mills Breast Cancer Institute, and Carle Foundation Hospital, Urbana, Illinois 61801, USA. b

Illinois Sustainability Technology Center, University of Illinois at Urbana-Champaign,

Urbana, IL, 61801, USA *Corresponding author. Email: [email protected]

1 ACS Paragon Plus Environment


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Abstract: Spheroidal nanoparticles of algal (‘phytonic’) origin were synthesized comprising carbonaceous architectures and surface-rich oxygenated functional groups. Nanoparticles were negatively charged and efficiently luminescent post ultraviolet range excitation. A multitude of analytical techniques confirmed the rich profusion of hydroxyl, carboxylate, and amines at the nanoscale while spectroscopic investigation indicated the presence of α-amines, a signature functionality present in amino acids. Confirmed via a series of biological assays i.e. growth regression, anti-migration and protein regression studies, photo-phytonic nanoparticles serendipitously revealed remarkable anti-cancer activity against various stages of breast cancer cells barring the need of an encapsulated drug. We report that nanoparticles derived from algal biomass exhibit intrinsic anti-migratory properties against cancer likely due to the rich abundance of α-amino acids.

Keywords: algae, nanoparticle, breast cancer, metastasis, cell migration


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1. Introduction Green synthesis of nanoparticles for biomedical application has been attractive due to their economical and large-scale productions for feasibility of eventual human use.1-5 A number of synthetic methods has been proposed using plant precursors with or without addition of synthetic molecules.6-11 Plants are generally rich in natural products such as alkaloids, flavonoids, saponins, steroids, tannins and other nutritional compounds. Though rich in various natural products,12-14 feasibility of modulating internal compositions by feeding different nutrients,15-17 requires complex procedure. Algal biomass is an excellent source of nutrients, minerals, proteins and amino acids.18-23 It remained largely unexplored though, if some of the properties of these central biomolecules can be exploited for imaging and therapeutics.24-28 The goal of this work was to investigate if mono dispersed nanoparticles from algae can be obtained in a reproducible manner using a mild, low-destruction protocol. The nanoparticles are expected to preserve some of the fundamental biological functions with the intention that these behaviors can be exploited for bio-imaging and therapeutic applications. Herein, we describe a green microwave-irradiation based synthesis of luminescent (‘photo’) nanoparticles of algal biomass origin (‘phytonic’). Spheroidal photo-phytonic nanoparticles (PPNP) were produced with carbonaceous architectures with traces of Si(O)x and high abundance of oxygenated functional groups including amino acids. Plausible preparation protocol involves ingredients from algae including carbohydrates, proteins and Si(O)x compounds present in various organelles of algae (Fig. 1D). We report that PPNPs are highly luminescent for in vitro cellular imaging and surprisingly exhibit intrinsic antimigratory properties against cancer cells (Fig. 1A).



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2. Results and Discussion Four different algae species including SCNP (Chlorella 2; single-cell green algae; Table S1),29,30 CHFR (Chlorogloeopsis fritschii; cyanobacterium),31,32 SPR (Spirulina; blue green algae; Table S2)33,34 and CHPT (Chlorella 1; single-cell green algae) were selected for microwave-based syntheses. As-prepared PPNPs, were characterized for their in-aquo size and found to be ca. 100 nm (Fig. 1B). PPNPs (0.1 mg/mL) showed an absorption maximum at 280 nm (Fig. 1C). Additional nanoparticles with added polyethylene glycol (PEG, 20kDa) was prepared as smaller PEG-PPNPs (~30 nm) and found to be very similar in absorption pattern with decrease in absorption efficiency at same concentration of 0.1 mg/mL (Fig. S2). The colloidal stability of PPNPs was tested in various macro-environmental conditions including pH (4.5, 12 and 6.8 (aq. medium)) and 1% fetal bovine serum (Fig. S1). Particles were found to be most stable at pH = 6.8 and lower while at higher pH of 12 a significant aggregation was observed. PPNP (Fig. 1B) and PEG-PPNPs (Fig. S1) showed absorption and emission maxima at 280 and 425 nm (λex= 360nm, 0.1 mg/ml). Zeta potential of PPNPs without (Fig. S3A) and with PEG passivation (Fig. S3B) were found to be changing from highly negative zeta potential to less negative (Table S3). As synthesized, PPNPs and PEG-PPNPs were characterized in anhydrous state by transmission electron microscopy (TEM) showing spheroidal distributions across all the used algal sources. Representative TEM images are shown for PPNPs from CHPT and SPR algal sources (Fig. 2A, B). Anhydrous diameters of PPNPs from CHPT and SPR were found to be of ~5 nm and 30 nm, respectively with a negligible decrease in diameter post PEG passivation (Fig. 2C and D). The topographical height profile from atomic force microscopy (AFM) confirmed their height at z scale (Fig. 2E, F). Surface chemistry and bulk composition of these PPNPs were of high interest due to their unexplored nature in PPNP state. Due to no


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significant difference across algae type, PPNP from SPR was used as representative PPNP for further bio-physical studies. FTIR studies on SPR PPNPs (Fig. 2G) showed the presence of -OH, -NH and -COOH functional groups on particle surface with broad -OH peak in PEG passivated PPNPs (upper panel) with indication of hydrogen bond formation. From comparative studies on all the PPNPs, one of the clear differentiators between algae particles was presence of alpha-amino and alpha-carboxylic acid groups in SPR derived PPNPs. The signature peak at 1650 nm (Fig. 2G, lower panel) was seen, which greatly minimizes when particles were produced with PEG passivation (Fig. 2G, upper panel).35 Raman scattering patterns36 revealed signature G and D bands for SPR (Fig. 2H). A lower G/D intensity (IG/ID) ratio represents loss of graphitic characters of PPNPs post PEG passivation (upper panel). NMR spectrum37,38 of raw SPR in D2O revealed 1H (Fig. 2I, i) and 13C (Fig. 2I, ii) population with chemical shift corresponding to α-amino protons, likely residing in amino acids of algal samples. The peaks ca. 2.8 ppm in the 1H NMR spectrum and the peak ca. 38 ppm in the corresponding 13C NMR spectrum of PPNPs indicate the signals from aspartic acid. It further supports that the α-amino functionalities are arising from the original algae source. Verification of surface chemistries on PPNPs was reinforced by x-ray photoelectron spectra (XPS)39 and energy dispersive x-ray (EDX)40 experiments. The survey spectra revealed the presence of spectral atomic positional peaks for C1s, N1s, O1s, P2p, Si2p and Na1s (Fig. 3). The narrow scan regions from different bond types revealed the specific binding energy confirming the presence of various compositional components of PPNPs. Narrow scan region for N1s indicated the presence of C-N, N-H and NH2 moieties, seemingly the part of peptides or proteins (Fig. 3Ai). Bond properties from narrow scan region for O1s and C1s units were found to be correlated with O=C=O, C-O-C and C-C bonds. P2p narrow scan regions corresponded to elemental phosphorous in different spins of P2p1/2 and P2p3/2 while Si2p in various forms of its oxide.



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Elemental composition of PPNPs were also studied by EDX confirming the presence of C, N, O, P, Si along with Mg and Ca in detectable amounts (Fig. 3B). The peaks assigned as Cu(t) are originating from the Cu tape used for preparing the samples. Interestingly, an in vitro cytotoxicity evaluation of these particles against normal breast cells (MCF-10A) and relative response for cancer cells of same origin at different stages of the disease revealed a serendipitous discovery of anti-cancer property of these PPNPs. Cell growth regression studies were performed on MCF-10A (non-cancerous), MCF-7 (ER (+), early stage), MDA-MB231 (late stage triple negative breast cancer, TNBC) and BT549 (TNBC, metastatic) cells. PPNPs (0.1 mg/mL) could decrease the cell viabilities to a considerably low level of ca. 60 and 50% in MDA-MB231 and BT549 cells, respectively, with low effect in MCF-7 and no difference in MCF-10A cells (Fig. 4A). This observation pointed toward a selective interaction pattern of these PPNPs against late stage breast cancer cells (MDA-MB231 and BT549) compared to early stage (MCF-7) or normal cells. This surprising growth regression pattern could be correlated with some of the surface properties of PPNPs. A closer look at the surface chemistries exposed resemblance with functional moieties of α-amino acids (Fig. 2G-I) and integrated Si(O)x (Fig. 3), which both might be playing a role in blocking cell adhesion and growth.41-43 To study anti-migration properties of PPNPs, cell growth and scratch assay were performed on BT549 cells and compared with MDA-MB231 and MCF-10A cells. A condition that could partially simulate the condition of metastatic cells migrated from tumor site to new host site was adopted.44 It is interesting to report that irrespective of type of PPNP source, cell growth was considerably affected and improved by at least 15-20% more cell death (Fig. 4Biiv) compared to cells treated after being grown for 24h (Fig. 4A). MCF10A cells were unaffected by any of these two conditions of PPNP treatments, indicating negligible interaction between MCF10A surfaces and PPNPs to impact cell adhesion or growth. A


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qualitative analysis for amino acid content on PPNP surface influencing cell migration resulted in high IG values in Raman scattering patterns for PPNP-SPR with high antimigratory property compared to PPNP-CHPT with low IG content and low anti-migratory property (Table S4). In vitro bio-imaging of PPNPs (SPR) also showed strong photoluminescence signals in confocal studies. Distributed PPNPs in extra-nuclear space (green) were found to be colocalized with cytoplasmic dye Qtracker®655 (red) (Fig. 4Ci-iii). The functional ability of PPNPs against metastatic cell growth was confirmed by a scratch assay. Unscratched cells were used as biological controls whereas scratched cells with no PPNP treatments were used as negative controls for cell migration. Untreated cells were found to regain the cell growth density within 24h of experiment (Fig. 5A). However, cells treated with PPNP could not regain its density even after 48h of growth revealing that the PPNPs could reduce the cell migration ability. Mechanistic foundation of this observation was established by studying intrinsic expression of protein typically involved in cell migration and adherence. STAT-3 protein is one of the important factors involved in cell adhesion post metastasis of cancer cells. A decrease in level of STAT-3 could indicate loss in adhesion property of BT549 post PPNP treatments. Indeed, a treatment of BT549 cells with PPNP (0.2 mg/mL) resulted in a significant decrease in the level of STAT-3 protein to ca. 5% with respect to ca. 85% in case of MCF-10A (Fig. 5B, C). 3. Conclusions Nature of metastatic cancer cells are defined as result of cancer cell adaptation to a tissue microenvironment at a distance from the primary tumor. They require properties to allow them not only to adhere to new site but adapt to that microenvironment and continue their proliferation and survival. Host site properties including chemistry of the surfaces and composition of bulk tissue determine the adherence of metastasized cells. Thus, a



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serendipitous finding related to PPNPs as anti-metastatic agents opened the possibility of artificially modifying the surface of host tissue as a strategy to counter metastasis and turn functioning as anti-cancer therapy for later stage cancer with higher possibilities of metastasis. Among all the used PPNPs, particles from SPR were found better anti-migratory properties compared to particles obtained from SCNP, CHFR and CHPT, probably due to higher αamino acid and Si(O)x content. Our results indicated

45

for the first time that carbonaceous

nanoparticles can be derived from algal sources having functional richness with α-amino acid and Si(O)x which could be responsible for this therapeutic potential and can further be explored for improving such therapeutic regimes. Cell migration and protein expression studies concluded for the first-time role of PPNPs in STAT-3 modulation and decrease in cell migration and adhesion, primarily in case of metastatic cancer cells. 4. Experimental Section 4.1. Synthesis and Physiochemical Characterizations The spray dried food grade algae, Spirulina (SPR; Blue green algae), Chlorella 1 (CHPT; Single-cell green algae) and Chlorella 2 (SCNP; Single-cell green algae) were obtained from Illinois Sustainable Technology Center as was procured from buyalgae and Chlorogloeopsis fritschii (CHFR; cyanobacterium) was obtained from ISTC center. A dispersion of 100 mg.ml-1 of algae was made in water and then subjected to kitchen microwave at P10 for 45 min with intermittent stops. Prepared dry mass was hydrated in 5 mL of water for 30 min before performing probe sonication at Amp 1, temp 40 ̊C, with pulse rate of 2 sec ON and 1 sec OFF for 30 min. Sonicated suspension was filtered through 0.45 and 0.22 µm syringe filters, sequentially. Prepared suspensions were checked for physiochemical characteristics and found to be of nanoparticle with photo responsive in nature of phytonic origin, hence called photophytonic nanoparticles (PPNPs). The hydrodynamic diameter and zeta potential of the NPs (1 mg/mL) were measured on a Malvern Zetasizer ZS90 instrument (Malvern


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Instruments Ltd, United Kingdom) at fixed angle of 90º. The morphology of the samples were observed under the transmission electron microscopy (TEM) on a JEOL 2100 Cryo TEM machine (Tokyo, Japan) equipped with Gatan UltraScan 2k × 2k CCD camera. Holey carbon coated copper grids were used for TEM sample preparation. Atomic force microscopy (AFM) was performed on samples deposited on a freshly cleaved mica sheet secured on a glass slide in the tapping mode (Asylum Cypher (Asylum Research, Santa Barbara, CA)). The absorbance of the PPNP suspensions (0.1 mg/mL) in the UV and visible spectrum range was recorded on a GENESYS 10 UV–vis spectrometer (Thermo Scientific, MA, USA). Elemental composition of PPNPs was investigated using SEM/EDS on Hitachi (Schaumburg, Illinois) S-4700 SEM with Oxford Instruments (Abingdon, Oxford shire) ISIS EDS X-ray Microanalysis System and Centaurus BSE detector. The chemical characteristics of the NPs was inferred from 1H NMR analysis using a 500 MHz machine (Varian VXR 500 (Varian, Inc., Palo Alto, CA)) equipped with a 5-mm Nalorac QUAD probe in deuterium oxide (D2O) (Cambridge Isotope Laboratories, Inc., MA, USA). The data was analyzed using MestRenova™ 8.1 software (Mestrelab Research SL; Santiago de Compostela, Spain). X-ray photoluminescent spectrum (XPS) was obtained on a thick vacuum dried layer of the NPs applied on the glass surface using Physical Electronics PHI 5400 spectrometer with Al Kα (1486.6 eV) radiation. The spectrum was referenced to the adventitious C 1s feature at 284.8 eV.

4.2. Cell viability studies Cells of human breast cells of cancer origin MCF-7, MDA-MB231, BT549 and MCF-10A of non-cancerous origin were used for cell viability, selectivity and migration studies as described. Cells were obtained from American Type Culture Collection (ATCC, VA, USA). MCF-7 and MDA-MB231 cells were cultured in Dulbecco’s Modified Eagle's Medium



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(DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% pen-strep under 99% humidity and 5% CO2. BT549 cells were grown in RPMI-1640 Medium (ATCC® 302001™) medium supplemented with 10% fetal bovine serum (FBS) and 1% pen-strep under 99% humidity and 5% CO2. Non-cancerous MCF-10A cells were cultured using base medium (MEBM) along with the additives from Lonza/Clonetics Corporation as a kit: MEGM, Kit Catalog No. CC-3150 supplemented with 5% fetal bovine serum (FBS) and 1% pen-strep under 99% humidity and 5% CO2. The cells were passaged via trypsinization (0.1%) containing 0.02% ethylenediaminetetraacetic acid (EDTA) after being washed with Dulbecco’s phosphate-buffered saline (DPBS, pH 7.4). For cell viability and effect of PEG10K passivation on PPNPs evaluations, MTT assays were performed on various cell types. A population of 10,000 cells suspended in 200 µl of appropriate medium type were seeded in each well of a 96- well plate and were allowed to grow for ~24h to reach ~80% confluence. The cells were incubated with NPs at concentration of 0.025, 0.05 and 0.1 mg.ml1

for 48h.The cells without PPNPs were considered as control. After 44 h of incubation, the

cells were further incubated with 20 µl of MTT solution (5 mg.ml-1) for an additional 4 h. The old media was replaced with 200 µl of DMSO to solubilize the formed formazan crystals and the absorbance at 592 nm was measured on multi-well plate reader (BioTek Synergy HT, USA). Absorption intensity was correlated to the cell viability.

4.3. Cell internalization using confocal microscopy BT549 cells were cultured as described above. BT549 cells (400,000) were suspended in 500 µl of media and were grown on sterilized 22 mm diameter microscope cover slips (Fisher Scientific, Hampton, NH, USA) for 24 h to reach ~80% confluence. Grown cells were treated with 0.1 mg.ml-1 of the PPNPs for 4h while the cells without treatment were chosen as control. The Qtracker® 655 Cell Labeling Kit (ThermoFisher Scientific, Grand Island, NY,


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USA) was utilized for staining the cytoplasmic content at a concentration of 1 nM. Treated cells were washed with chilled DPBS twice and then was incubated with the chilled paraformaldehyde (4% wt/vol in PBS) for 10 min before being washed by DPBS twice again. Finally, the coverslips were mounted on microscope slides with DAPI-containing mounting medium (VECTASHIELD mounting medium with DAPI). The slides were imaged using a Leica SP8 UV/Visible Laser Confocal Microscope (Leica Micosystems, Germany) under 405 nm laser.

4.4. Scratch wound healing and Cell migration assay Scratch or cell migration assays refer to the measurement of 2D cell migration into a cell free area. These are generally created by a central linear scratch across the surface of a tissue culture well after growing a confluent monolayer of cells.1-3 Migration may be quantitated manually by standard microscope or by using quantitation software. BT549 cells (400,000) were plated 6 well plate using DMEM supplemented with 10% FBS for 24 h to reach ~80-90% confluence as a monolayer. Without changing the medium, gently and slowly a scratch using 1 ml pipette tip across the center of the well was made. This gave a resulting gap distance approximately equal to the outer diameter of the end of the tip. After creating scratch, cell monolayer was gently washed twice with culture medium to remove the detached cells. Cells were grown untreated (negative control) or treated with 0.1 mg/mL of PPNP for next 24 and 48h of time point. Cells without scratch were used as biological controls. At the end of incubation time points cells were again imaged under bright field to measure change in scratch gaps and compared with untreated scratched cells. The gap distances were quantitatively evaluated using software ImageJ.

4.5. Statistical analysis



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The tests were done in triplicate and the results were expressed as the mean±standard deviation. The data were analyzed on the GraphPad Prism 6.0 software using analysis of variance (ANOVA) Bonferroni correction for post hoc. The p

Î±-Amino Acid Rich Photophytonic Nanoparticles of Algal Origin

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