Padina boryana mediated green synthesis of crystalline palladium nanoparticles as potential nanodrug against multidrug resistant bacteria and cancer cells

Green synthesized nanoparticles (NPs) have emerged as a new and promising alternative to overcome the drug resistance problem. Peculiar nano-specific features of palladium NPs (Pd-NPs) offer invaluable possibilities for clinical treatment. Due to the development of multi-drug resistance (MDR) in pathogenic bacteria and the prevalence of cancers, use of algae-mediated Pd-NPs could be a prospective substitute. Therefore, Pd-NPs were synthesized by a one-step, cost-effective, and environmentally friendly green method using the extract from a brown alga, Padina boryana (PB-extract), and evaluated for their antibacterial, antibiofilm, and anticancer activities. Pd-NPs were physicochemically characterized for size, shape, morphology, surface area, charge, atomic composition, crystal structure, and capping of Pd-NPs by PB-extract biomolecules by various techniques. The data revealed crystalline Pd-NPs with an average diameter of 8.7 nm, crystal size/structure of 11.16 nm/face-centered cubic, lattice d-spacing of 0.226 nm, 28.31% as atomic percentage, surface area of 16.1 m2/g, hydrodynamic size of 48 nm, and zeta-potential of − 28.7 ± 1.6 mV. Fourier-transform infrared spectroscopy (FT-IR) analysis revealed the role of PB-extract in capping of Pd-NPs by various functional groups such as –OH, C=C, C–O, and C–N from phenols, aliphatic hydrocarbons, aromatic rings, and aliphatic amine. Out of 31, 23 compounds were found involved in biosynthesis by Gas chromatography–mass spectrometry (GC–MS) analysis. Isolated strains were identified as MDR Staphylococcus aureus, Escherichia fergusonii, Acinetobacter pittii, Pseudomonas aeruginosa, Aeromonas enteropelogenes, and Proteus mirabilis and Pd-NPs exhibited strong antibacterial/antibiofilm activities against them with minimum inhibitory concentration (MIC) in the range of 62.5–125 μg/mL. Moreover, cell viability assays showed concentration-dependent anti-proliferation of breast cancer MCF-7 cells. Pd-NPs also enhanced mRNA expression of apoptotic marker genes in the order: p53 (5.5-folds) > bax (3.5-folds) > caspase-3 (3-folds) > caspase-9 (2-folds) at 125 μg/mL. This study suggested the possible role of PB-extract capped Pd-NPs for successful clinical management of MDR pathogens and breast cancer cells.

www.nature.com/scientificreports/ Physicochemical characterization of PB extract-capped Pd-NPs. UV-Vis, EDX, and SEM analyses. Liquid samples of PB-extract, Na 2 PdCl 4 , and PB extract-capped Pd-NPs were checked for their absorption in the UV-visible range (280-600 nm) using double beam operation of PerkinElmer Lambda 35 spectrophotometer (Waltman, MA, USA). To measure the surface morphology, the powder of Pd-NPs was put on to a carbon tape and aluminum stub carrying carbon tape was analyzed by a scanning electron microscope attached with EDX at an accelerating voltage of 15 kV (SEM-EDS; JEOL-64000, Tokyo, Japan).
Structure, shape, and size determination of PB extract-capped Pd-NPs. Transmission electron microscope (JEM-1011, JEOL, Tokyo, Japan) was used for the determination of average diameter and shape of Pd-NPs at 200 kV energy. The aqueous suspension (15 μL) of Pd-NPs was put on a Cu-grid followed by drying at 80 °C for 5 h. Prepared grids were analyzed by TEM. To check the crystallinity and phase purity, XRD analysis was performed on Bruker D8 Discover instrument. Cu-Kα radiation (λ = 1.54 Å) was used to obtain the diffraction pattern and data was recorded at 20-80° two-theta (2θ) angle.
Hydrodynamic size, zeta-potential, and surface area measurement. To obtain the hydrodynamic size, 50 µg/mL suspension of Pd-NPs was prepared in DIW and ultrasonicated at 40% amplitude for 15 min. The Pd-NPs suspension was then subjected to analysis by a Zeta Sizer Nano-ZS90, Malvern, UK. The zeta-potential of Pd-NPs was recorded as an average of 20 readings. Specific surface area measurement of PB-capped Pd-NPs was done following Brunauer-Emmett-Teller (BET) analysis using Autosorb-iQ-MP/XR surface area analyzer (Quantachrome Instruments, USA).

Determination of surface functional groups and compounds of PB-extract and Pd-NPs.
To detect the adsorption of functional groups, FT-IR analysis of PB-extract and Pd-NPs was recorded in attenuated total reflectance (ATR) mode on the PerkinElmer system 2000 instrument. The spectra for each sample were scanned three times and average values of percent transmittance were plotted against wavelength (4000-400 cm −1 ). GC-MS analysis of hexane extracts of P. boryana and PB-extract capped Pd-NPs was performed on Shimadzu QP-2010 Plus with Thermal Desorption System TD-20 instrument. Conditions for analysis were kept as follows: He flow at 1.2 mL/min, oven temperature from 80 to 260 °C at 4 °C/min, for 5 min, interface/inlet temperature were set as 280/250 °C. A 0.2 mL solution was injected at a 10:1 split ration at 70 eV. Data for signals obtained for various molecules such as retention time, peak percentage area, molecular mass, etc. was recorded based on the interpretation of the National Institute of Standards and Technology (NIST) library.

Isolation and characterization of clinical bacterial pathogens. Samples for isolation of clinical
bacteria were collected from fluid and sputum of immunocompromised patients diagnosed with respiratory infections following our earlier described method 32 . After biochemical and morphological identification, antibiotic-resistant isolated cultures were molecularly characterized by partially sequencing 16S rRNA gene using universal primers 785F (5ʹ -GGA TTA GAT ACC CTG GTA -3ʹ) and 907R (5ʹ-CCG TCA ATTCMTTT RAG TTT-3ʹ) following our previously demonstrated methods of 16S rDNA amplification and Sanger's dideoxy sequencing. The sequences were processed using BioEdit software 7.2.4. For similarity search, the BLASTn search tool of NCBI was used. The processed sequences for isolated bacterial strains were submitted to the GenBank database and accession numbers were obtained. The strains were stored in Luria Bertani (LB) broth supplemented with glycerol (40%) at − 70 °C in duplicates until further use. Detailed method of phylogenetic analysis can be found in supplementary information.
Antimicrobial drug resistance profiling of bacterial isolates by disc susceptibility test. Drug resistance was checked by Kirby-Bauer's disc diffusion assay of antibiotics with known disc potency on LB agar media following the guidelines of the Clinical and Laboratory Standards Institute (CLSI, 2016) 33   .5 μg/mL was also run in parallel and the results were compared with PB-extract capped Pd-NPs. After incubation, DMEM was gently discarded and cells were rinsed with PBS (1X). MTT at 5 mg/mL rate was added to each well and incubated further for 4 h at 37 °C. MTT retained by cells was solubilized by 0.2 mL dimethyl sulfoxide (DMSO) absorbance at λ max = 550 nm was recorded. Similarly, the NRU assay was performed. After the treatment of MCF-7 cells with Pd-NPs for 24 h, DMEM was supplied afresh carrying 50 μg/mL of neutral red. Further incubation of three hours was given. Microtiter wells containing this mixture were rinsed with a combination of HCHO and CaCl 2 (mixed at a ratio of 0.5:1%). Thereafter, a mix of C 2 H 5 OH (50%) and CH 3 COOH (1%) was added to wells and incubated at 37 °C for 20 min. Absorbance at λ max = 540 nm was recorded. Percent cell viability was plotted over untreated control with an increasing dose rate of PB-extract capped Pd-NPs.
To assess the apoptosis induced by Pd-NPs, MCF-7 cells were treated with 62.5 and 125 μg/mL concentration of Pd-NPs. Total RNA was extracted from cells and purified by a commercially available RNA purification kit procured from Roche, Mannheim, Germany as per manufacturer's instructions. Extracted RNA was visualized following agarose gel electrophoresis (1%) and quantification of RNA was done by a NanoDrop spectrophotometer. The cDNA from RNAs was synthesized by using the Fermentas cDNA synthesis kit (Burlington, ON, Canada). cDNA synthesis was performed as per the protocol provided by the manufacturer. Primer sequences for housekeeping GAPDH gene (for normalization of gene expression) and four apoptotic genes namely bax, p53, caspase-9, and caspase-3 are provided in the supplementary information (Table S1). PCR amplification of genes was performed in 35 cycles following the program: first cycle 95 °C for ten minutes; 35 cycles at 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. DIW was used as a template for negative control. Gene expression data were analyzed by 2-△△ Ct method fold changes in gene expression were compared with control. Data analysis. Each experiment was performed three times with triplicate samples for each test concentration. Data shows mean values and error bars represent standard deviation (S.D.). Significant differences between the values were calculated by student's t-test at 95% confidence limit using Sigma Plot 14.0. All methods were carried out in accordance with relevant guidelines and regulations. Clinical cultures of bacteria were isolated from pus/wound samples of the registered patients and informed consent was obtained. Experimental protocols were approved by institutional committee of the University as and when required. No consent from ethical committee was required for sample collection for the isolation of bacteria.

Results and discussion
Many physiological or structural changes occur alone or simultaneously in bacteria when it is encountered by an antibiotic. The following are major antibiotic resistance mechanisms reported in clinical bacteria: (i) occurrence of bacterial mutations, (ii) horizontal gene transfer, (iii) destruction/modification of antibiotic molecules, (iv) decrease in cell membrane permeability to inhibit antibiotic penetration, (v) higher expression of efflux pumps in the membrane, and (vi) alteration of antibiotic target sites 34 . Moreover, bacterial species can form biofilms that is highly resistant towards antibiotics than planktonic cells, sometimes > 1000 folds 35 . On the other hand, the development of cancers and inefficient cure by available anti-cancer drugs further complicates their clinical treatments. Based on the drug sensitivity/resistance profile, cancers could be also be categorized as MDR cancer 36 . Therefore, environmentally sustainable, non-toxic, and cost-effective nano-therapeutics are in trend to treat such resistance in clinical bacteria and cancer cells.
So far, few studies report the utilization of brown seaweed (marine algae) for Pd-NPs production 16,22 even though the marine algal population is chemically rich possesses a wide range of compounds with promising anti-oxidant, anti-cancer, anti-inflammatory, and anti-microbial activities. The rich biomolecular composition of PB-extract and previous evidences on Pd-NPs biological activity prompted us to investigate the role of PBextract in capping of Pd-NPs and interactions with six clinical bacteria and human breast cancer (MCF-7) cell line. The capping of NPs (i.e. adsorption of molecules on the surface of NPs) decides its final morphology and thus prevents the overgrowth of NPs. The method for synthesis and capping of Pd-NPs by PB-extract is detailed in Fig. 1 depicting (i) bio-reduction of Pd 2+ ions to Pd 0 seeds and (ii) capping/stabilization of Pd-NPs growth by molecules of PB-extract via surface adsorption. Production of Pd-NPs started following the reduction of Pd 2+ to Pd 0 from electrons liberated mainly from the reducing sugars and polyphenols containing biomolecules of P. boryana extract. As a result, the color of solution changed drastically from pale yellow to dark brown. This could be well corroborated with earlier observations 37 , where leaf biomolecules of Solanum trilobatum while interacting with Pd 2+ ions gave dark precipitation from the reaction mixture which could be due to surface plasmon resonance (SPR) owing to the collective oscillation of electrons. The biomolecules of PB-extract when donating electrons for Pd 2+ ions reduction are oxidized and are expected to form intermediate Pd-organic complexes. Ions are then converted to Pd 0 by free electrons generated in the medium 38 . Frequent collisions among Pd 0 atoms lead to the production and growth of Pd-NPs that are meanwhile capped by other organics of PB-extract giving specific size and shape to growing Pd-NPs seeds.
UV-vis spectroscopic analysis. NPs have optical features which give a preliminary idea about their shape, size, and SPR 39 . The comparative UV-vis spectra (Fig. 2) showed a broad absorption of PB-extract with a peak overlapping the UV and visible region. The precursor salt (Na 2 PdCl 4 ) showed two characteristics peaks at 311 nm and 402 nm, while Pd-NPs exhibited a sharp peak at 293 nm. The absorption near to 293 nm by Pd-NPs has also been observed in other studies such as 268 nm 22 . In Na 2 PdCl 4 , two signals can be assigned to the transition of ligand to metal charge transfer between Pd 2+ and Cl -. Absence of these two signals in the Pd-NPs spectrum advocates reduction of Pd 2+ ions to NPs 40 as also visually observed (Fig. 2 inset).

Surface morphology and elemental composition of PB-capped Pd-NPs. The morphological
analysis of dehydrated powder of PB-capped Pd-NPs through SEM is shown in Fig. 3A,B at two different magnifications. Aggregates of variable sizes were recorded, however, the shape was pleomorphic. The elemental composition of Pd-NPs revealed the presence of Pd with carbon, oxygen, and chlorine (Fig. 3C). The Pd-NPs after synthesis were washed many times to remove the ions and unbound PB-extract, therefore, C, O, and Cl could appear in EDX spectra from the algal extract. The percentage of Pd was high in the EDX spectrum as 28.31% (Fig. 3C inset) and the peak clarity of EDX confirmed the purity of synthesized Pd-NPs. The peak at 3.1 keV in the EDX spectrum represents Pd as reported earlier 41 . Average Diameter, crystalline size, and structure of NPs. The TEM micrographs showed spherical shape Pd-NPs (Fig. 4A,B) with a narrow range of particle size distribution from 5 to 20 nm (Fig. 4C). The average particle diameter calculated from the TEM size distribution was 8.7 nm. Pd-NPs were found well dispersed and agglomeration was absent. There was least direct particle-to-particle adherence and nil fusion of Pd-NPs during TEM analysis that could be due to the corona formation by biomolecules of PB-extract during capping. In Fig. 4B, the lattice d-spacing of 0.226 nm is shown while the distance between planes was measured. This d-spacing confirmed the crystallinity of PB-capped Pd-NPs. The XRD pattern of Pd-NPs (Fig. 4D) revealed one major (111) and three minor signals (200), (220), and (311) which were sharp and intense. The crystalline plane of (111) was well-matched with Pd (d-spacing of 0.23 nm) and revealed an FCC structure. The XRD derived average particle size was calculated as 11.16 nm by using Debye-Scherrer's equation which is good agreement with TEM size. Peaks could corroborate with the standard JCPDS file of crystalline Pd (file no. 05-0681) 42 . Thus XRD and TEM data confirmed the purity and crystalline nature which is consistent with other plant and green algae mediated fabrication studies of Pd-NPs 16,43 , however, the antibiofilm and anti-cancer activities were lacking.
Surface area, hydrodynamic size, and zeta-potential. The  www.nature.com/scientificreports/ was found as − 28.7 ± 1.6 mV (Fig. 5A,B). The increase in size compared to the primary size measured by TEM and XRD shows some sort of particle aggregation in the aqueous solution which depends on the frequency of NPs collisions as inter-particular interactions increases. Due to these collisions, the average path length covered by NPs decreases thereby increasing the hydrodynamic size 44 . A similar kind of variation has been seen in another study where size of Pd-NPs measured by DLS increased up to 24.20 nm as compared to size (4 nm) measured by TEM 38 which was suggested due to the presence of biomolecules from Delonix regia. The stability of Pd-NPs was assessed by determining the zeta-potential that gives information about surface electrostatic potential and movement of NPs in the suspension. The zeta-potential of − 28.7 ± 1.6 mV denotes sufficient stability of Pd-NPs for effective biological applications. This could probably be due to the efficient capping of Pd-NPs by biomolecules of algal extract producing repulsion among Pd-NPs in solution 45 .

Surface functional groups/biomolecules analyzed by FT-IR and GC-MS. The IR signals detected
for extract and NPs (Fig. 5C) are assigned to various functional groups in Table 1. All the major signals detected for extract were also found in Pd-NPs except one which was for C-H stretch at 2086 cm −1 . The FT-IR spectrum of Pd-NPs suggests the possible role of -OH functional groups having compounds such as polyols including terpenoids, tannins, saponins, etc. 46 forming complex with Pd-NPs as revealed by narrowing of the peak. The shift in transmittance of FT-IR signals of Pd-NPs as compared to extract could be due to possible interactions of functional groups with metal ions (during reduction) and atoms or smaller NPs (during capping) as observed in the current study 47 . The signals at 2986, 1643, and 1062 cm −1 of extract were shifted to 2974, 1637, and 1059 cm −1 in Pd-NPs after bio-reduction suggesting the involvement of aliphatic hydrocarbons, aromatic rings, aliphatic amines 48,49 .   (Table S2) which were identified as long-chain hydrocarbon acids, esters, and acid chlorides. The major compounds of extract were: 2-palmitoylglycerol (peak area 25.14%), tricosanoic acid, 2-methoxy-, methyl ester (peak area 19.46%), oleic acid glycidyl ester (peak area 6.78 + 4.91 = 11.78%), cinnamyl linoleate (peak area 4.87%), 9,12-Octadecadienoyl chloride (peak area 3.24%), oleic acid chloride (peak area 2.89%), methyl oleate (peak area 2.78%) and monoolein (1.49%). The variation in the structure of long-chain hydrocarbons is evident from linear molecules 50 to cyclic hydrocarbons 51 with various functional moieties such as -OH, > C=O, C-O-C, and -COOH as detected in the present study. Moreover, the single compound could appear as two or more individual peaks. This difference is due to the presence of stereoisomers, identical mass, and functional moieties. Under similar experimental conditions, the GC-MS analysis of Pd-NPs reflected 23 compounds similar to those in the extract ( Table 2). The major among them were tricosanoic acid, 2-methoxy-, methyl ester (peak area 31.89%), 2-palmitoylglycerol (peak area 18.41%), oleic acid chloride (peak area 14.52%), oleic acid glycidyl ester (peak area 6.95%), glycol stearate (peak area 4.14%), monoolein (peak area 4%), 9,12-octadecadienoyl chloride (peak area 3.42%), and oleic acid, 3-hydroxypropyl ester (peak area 2.76%). These compounds were involved in the surface capping and stabilization of Pd-NPs. Similar to our study, long-chain aldehydes of various fatty acids such as palmitic, oleic, linoleic, and linolenic acids have been detected in red, green, and brown marine algae 52 . Likewise, an array of long-chain hydrocarbon fatty acids including oleic and palmitic acids were reported as abundant molecules in some species of Chlorophyta and Rhodophyta 53 . In another study, palmitic acid was also reported in brown algae by GC-MS analysis 54 . These type of compounds such as palmitic acid, stearic acid, linolenic acid, tetracosane, tetradecanoic acid, sitosterol etc. have also been detected in plants such as Triticum aestivum 55 , Catharanthus roseus 56 and Moringa oleifera 56 by GC-MS.

Zone of inhibition and MIC of PB-extract capped Pd-NPs.
Bacteria were not found sensitive to PBextract, however, the zone of inhibition produced by gentamicin and Pd-NPs were variable among test strains (    www.nature.com/scientificreports/

Pd-NPs concentration-dependent growth of isolates and membrane destruction.
When the exposure of Pd-NPs was increased from 7.81 μg/mL at a geometrical progression with a common ratio of 3 up to 250 μg/mL, the number of viable cells and thus CFU/mL at logarithmic scale was reduced (Fig. 7A). A ≤ 50% reduction in cell population was observed at 31.25 μg/mL for E. fergusonii, S. aureus, and A. pitti, 62.5 μg/mL for A. enteropelogenes and P. aeruginosa, and 125 μg/mL for P. mirabilis. Regression analysis between concentrations of Pd-NPs versus average Log 10 CFU/mL resulted in an R 2 value of 0.70 which shows a negative correlation (Fig. 7B). So far no study has reported the CFU-based bacterial inhibition by algal mediated Pd-NPs. Progressive damage to the bacterial cell membrane permeability was also noticed (Fig. 7C). PI clearly distinguishes between live and dead cell due to its ability to permeate only the membrane damaged cells followed by binding to DNA and fluorescence emission (λexc = 532 nm). Cells exposed to low concentration of Pd-NPs (7.81-15.62 μg/mL) showed less number of membrane altered cells. However, a significant (P ≤ 0.05 or 0.01) number of membrane compromised cells at a higher dose rate suggests substantial interaction of Pd-NPs with the bacterial cell membrane.

Biofilm inhibition of isolates by PB-capped Pd-NPs. Biofilm formation of test strains was first com-
pared with biofilm positive strains and it was found that all the MDR strains could form the biofilm ( Figure S1). PB-extract capped Pd-NPs significantly reduced the biofilm formation in a dose-related fashion as compared to control (Fig. 8) 65 . Besides, extra polymeric substance (EPS), biofilms also contain lipids, proteins, and DNA that resist higher concentrations of antibiotics and compromise the host immune system. The smaller size Pd-NPs synthesized in our study with a large surface area and biologically active capping material could serve as an alternative or supplement to antibiotics effectively inhibiting the growth of pathogens. While interacting with bacterial cells, PB-extract capped Pd-NPs can exert toxic impacts on bacterial growth and metabolism by direct reactions including the following: (i) attachment to peptidoglycan (PG; a polymer of sugars and amino acids around cell membrane) layer due to the linkage between free amino groups (-NH 2 ) and hydroxyl (-OH), carbonyl (> C=O), epoxide or ester groups of biomolecules present in PB-extract. This Table 2. GC-MS analysis of PB extract mediated Pd-NPs. www.nature.com/scientificreports/ binding can also facilitate the inside entry of Pd-NPs into periplasmic space, (ii) creation of new pores in the cell membrane by physical interaction with phospholipids and membrane lipid peroxidation and thus altering the membrane permeability, (iii) inactivation of cellular enzymes, and (iv) destruction of biofilm formation by negatively charged EPS mediated mobilization of positively charged Pd-NPs or Pd 2+ ions (released from Pd-NPs) to biofilms as depicted in (Fig. 9). Indirectly, encounter and binding of -SH groups of proteins with Pd-NPs can trigger a modification of PO 4 efflux system leading to cell membrane exfoliation from cytoplasm, intracellular oxidative stress, dysfunction DNA replication system, and leakage of cell content.
In vitro cancer cell cytotoxicity and apoptosis. MTT assay quantifies the production of cellular oxidoreductase enzymes (NADPH mediated) following the reduction of MTT stain and thus reveals the metabolic activity of cells. These enzymes reduce the MTT to an insoluble form of formazan. Whereas, NRU uptake assay measures the influx of neutral red into lysosomes of metabolically active cells. The data exhibited a dose-dependent significant (P ≤ 0.05 and 0.01) reduction of MCF-7 cellular activity and cell viability was found decreased as compared to control cells (untreated) (Fig. 10A). At 62.5 and 125 μg/mL of Pd-NPs, the percent reduction in viable cells was found as 38% and 53% by MTT assay, and 32% and 45% by NRU assay. This data clearly shows lysosomal toxicity and the destruction of cellular metabolism by Pd-NPs. The cytotoxic activity of green Pd-NPs to human leukemia cancer  www.nature.com/scientificreports/  www.nature.com/scientificreports/ cell lines has been suggested due to the physicochemical interaction of Pd-NPs with DNA, proteins, phosphate groups, cell cycle arrest, free radical formation, and leakage of lactate dehydrogenase 66 . Under NPs stress, cancer cells regulate gene expression to circumvent cellular disruption thereby restoring signaling and cell cycle. In current study, two concentrations (62.5 and 125 μg/mL) of Pd-NPs induced expression of apoptotic marker genes in folds in the following order: p53 (4.5-folds) > caspase-3 (2.5-folds) > bax (2-folds) > caspase-9 (1.5-fold) at 62.5 μg Pd-NPs/mL and p53 (5.5-folds) > bax (3.5-folds) > caspase-3 (3-folds) > caspase-9 (2-folds) at 125 μg Pd-NPs/mL ( Fig. 10 B-E). The enhanced expression of p53 mRNA transcripts suggests multiple targets of Pd-NPs in MCF-7 cells including generation of oxidative stress, dysfunction of mitochondria, aberration on cell cycle, and apopto- www.nature.com/scientificreports/ sis. Similarly, bax is a well-known apoptosis inducer. Higher expression of two of the major caspases (caspase-9 and caspase-3) emphasizes the fragmentation of nuclear material and suggests the role of mitochondria in p53 apoptosis. Moreover, higher expression of p53 enhances the transcription of bax, caspase-9, and caspase-3 as proapoptotic genes 67 . Cancer cell death mediated by PB-extract capped Pd-NPs is schematically presented in Fig. 9.
In a comparative analysis, PB-extract capped Pd-NPs were found more active against pathogenic biofilms and breast cancer cells as compared to bare-PdNPs Fig. 11. The enhanced in vitro clinical performance of PB-extract capped Pd-NPs is possibly due to the capping of P. boryana biomolecules. The apparent difference in antibiofilm and anticancer potential of two species of Pd-NPs probably arises as a result of bioactive corona of PB extract biomolecules and functional groups around Pd-NPs which might help in the enhanced uptake of PB-capped Pd-NPs by bacterial and cancer cells (primary factor) which in turn results in Pd-NPs toxicity (secondary factor). Similar kind of results have been reported in two other studies where green synthesized α-Fe 2 O 3 and CuO NPs were compared with uncapped α-Fe 2 O 3 68 and CuO NPs 69 . Results showed substantial reduction of cell viability and biofilm formation by E. coli. S. aureus, and P. aeruginosa.

Conclusion
The current study is perhaps the first study which explored the constituents of marine brown seaweed P. boryana by FT-IR and GC-MS analysis and proved their role in bio-reduction and bio-capping of Pd-NPs. The green synthesized Pd-NPs were fairly small in size, spherical and crystalline which were biologically effective in the range of 31.25-125 μg/mL against six MDR bacteria and human breast cancer MCF-7 cell line. The antibiofilm and anticancer efficiency of PB-extract capped Pd-NPs was higher than the uncapped or bare-PdNPs. A very few algae have been used for Pd-NPs fabrication but lack the detailed exploration of capping and assessment of biomedical potential. The developed synthesis method is cost-effective, green, and can be easily scaled up. Our green Pd-NPs are further warranted for in vivo research in an animal model to determine their safety to humans. However, the PB-extract capped Pd-NPs can be applied for the coating of medical appliances to control the resistant bacterial infections thus safeguarding the health of patients.  . Panels B-E shows fold changes in gene expression of four apoptotic genes (bax, p53, caspase-9, and caspase-3). Data represents mean values of three independent replicates and error bars represent standard deviation (S.D.). *P ≤ 0.05 and **P ≤ 0.01 as calculated by student's t-test.