Antimicrobial activities of green synthesized gums-stabilized nanoparticles loaded with flavonoids

Herein, we report green synthesized nanoparticles based on stabilization by plant gums, loaded with citrus fruits flavonoids Hesperidin (HDN) and Naringin (NRG) as novel antimicrobial agents against brain-eating amoebae and multi-drug resistant bacteria. Nanoparticles were thoroughly characterized by using zetasizer, zeta potential, atomic force microscopy, ultravoilet-visible and Fourier transform-infrared spectroscopic techniques. The size of these spherical nanoparticles was found to be in the range of 100–225 nm. The antiamoebic effects of these green synthesized Silver and Gold nanoparticles loaded with HDN and NRG were tested against Acanthamoeba castellanii and Naegleria fowleri, while antibacterial effects were evaluated against methicillin-resistant Staphylococcus aureus (MRSA) and neuropathogenic Escherichia coli K1. Amoebicidal assays revealed that HDN loaded Silver nanoparticles stabilized by gum acacia (GA-AgNPs-HDN) quantitatively abolished amoeba viability by 100%, while NRG loaded Gold nanoparticles stabilized by gum tragacanth (GT-AuNPs-NRG) significantly reduced the viability of A. castellanii and N. fowleri at 50 µg per mL. Furthermore, these nanoparticles inhibited the encystation and excystation by more than 85%, as well as GA-AgNPs-HDN only completely obliterated amoeba-mediated host cells cytopathogenicity. Whereas, GA-AgNPs-HDN exhibited significant bactericidal effects against MRSA and E. coli K1 and reduced bacterial-mediated host cells cytotoxicity. Notably, when tested against human cells, these nanoparticles showed minimal (23%) cytotoxicity at even higher concentration of 100 µg per mL as compared to 50 µg per mL used for antimicrobial assays. Hence, these novel nanoparticles formulations hold potential as therapeutic agents against infections caused by brain-eating amoebae, as well as multi-drug resistant bacteria, and recommend a step forward in drug development.

www.nature.com/scientificreports www.nature.com/scientificreports/ Determination of flavonoids loading efficiency. The loading of HDN/NRG on nanoparticles was determined spectrophotometrically. First the nanoparticles were centrifuged at 12,000 × g for 30 min. Supernatant (containing free drug) was discarded and pellet was collected and re-dispersed in methanol up to the final volume. HDN and NRG were detected and quantified at 285 nm 17,26 . The percentage of encapsulated flavonoids were calculated by using this formula: = × %Flavonoid loaded (Amount of flavonoid loaded/Total flavonoid used) 100 A. castellanii cultures. A. castellanii (ATCC 50492) a clinical strain belonging to the T4 genotype, was routinely cultured in 10 mL growth medium consisting of 0.75% w/v proteose peptone, 0.75% w/v yeast extract, and 1.5% w/v glucose (PYG) in 75-cm 2 tissue culture flasks at 30 °C as described previously 27 . Amoebicidal and encystation assays were performed with healthy A. castellanii trophozoites which are adherent to the surface of tissue culture flask. These active trophozoites were detached by putting culture flasks on ice for 15 min followed by gentle tapping for roughly 5 minutes after changing PYG medium with phosphate buffer saline (PBS) to remove any unhealthy amoeba. Finally, A. castellanii trophozoites suspension was centrifuged at 2500 × g for 10 min to obtain amoeba pellet. The pellet was resuspended in 1 mL PBS, and the population of A. castellanii was determined by cell counting using a hemocytometer. 5 × 10 5 A. castellanii were used for amoebicidal and encystation assays.

Henrietta lacks cervical adenocarcinoma (HeLa) cells culture. HeLa cells were cultured in Roswell
Park Memorial Institute (RPMI)−1640 supplemented with 10% fetal bovine serum (FBS), 10% Nu-serum, 2 mM glutamine, 1 mM pyruvate, penicillin and streptomycin (100 units/mL and 100 μg/mL respectively), non-essential amino acids, and vitamins to obtain uniform monolayers of cells in 75-cm 2 culture flasks as described previously 28 . Old media was aspirated, and cells were trypsinized with 2 mL trypsin. The cell suspension was centrifuged for 5 min at 2000 × g, and cell pellet was resuspended in 30 mL fresh cell growth media. 200 μL of this cell suspension was seeded in each well of a 96-well plate and the plate was incubated at 37 °C in a 5% CO 2 incubator with 95% humidity for at least 24 h until formation of uniform monolayer of HeLa cells. These were used for N. fowleri cultures, cytotoxicity, and cytopathogenicity assays.
N. fowleri cultures. N. fowleri (ATCC 30174) a clinical isolate from the cerebrospinal fluid of a patient was cultured in 75-cm 2 tissue culture flasks containing HeLa monolayers as feed. N. fowleri was grown at 37 °C in a 5% CO 2 incubator with 95% humidity as described previously 21 .
Amoebicidal assay. Bactericidal assay. Antibacterial potential of nanoparticles and respective controls was determined by using bactericidal assay as described previously 30 . Briefly, bacterial cultures were fixed to an optical density of 0.22 at 595 nm using a spectrophotometer (OD 595 = 0.22) which is equivalent to 10 8 colony-forming units per mL (C.F.U. mL −1 ). An inoculum of 10 μL of above bacteria culture (corresponding approximately 10 6 C.F.U.) was incubated with various concentrations of GA-AgNPs-HDN, GT-AuNPs-NRG and respective controls in 1.5 mL centrifuge tubes at 37 °C for 2 h. For negative controls untreated bacterial culture were incubated with phosphate buffer www.nature.com/scientificreports www.nature.com/scientificreports/ saline (PBS), while 100 μg/mL gentamicin treated bacteria were used as positive control. Next, bacteria were serially diluted and 10 µL of each dilution was plated on nutrient agar plates. These plates were incubated at 37 °C overnight, followed by counting viable bacterial C.F.U.
pathogens-mediated host cells cytotoxicity. The cytopathogenicity assay was carried out as reported previously 31 . 5 × 10 5 A. castellanii, 10 6 C.F.U. of each E. coli K1 and MRSA were incubated with HDN and NRG loaded nanoparticles with respective controls at different concentrations for 2 h at 30 °C. Next, the microbial cultures were centrifuged at 2500 × g for 10 minutes, and supernatants were wasted to remove extracellular materials. The pellet obtained was resuspended in 500 µL of fresh RPMI-1640 which was put on another 24-well plated with HeLa cells monolayer. Cells were incubated at 37 °C in a 5% CO 2 incubator with 95% humidity for 24 h. Finally, supernatants were collected from each well and lactate dehydrogenase (LDH) cytotoxicity assay was performed using LDH kit (Roche) as described previously 23 . The extent of LDH release determines cells damage. Untreated cells were considered as negative control, whereas cells incubated with 0.1% Triton X-100 for 20 min gave maximum LDH release as a result of cell lysis which was taken as positive control. The % cell cytotoxicity was calculated as follows: % cell cytotoxicity = (sample absorbance − negative control absorbance)/(positive control absorbance − negative control absorbance) × 100. The results are representatives of several experiments presented as the mean ± standard error. Cytotoxicity assay. To evaluate the cytotoxic effects of these nanoparticles on human cell, LDH cytotoxicity assay was performed as reported previously 28 . Briefly, 100 µg per mL concentrations of HDN and NRG loaded nanoparticles and respective controls were treated with uniform monolayer of HeLa cells in a 24-well plate, and the cells were incubated for 24 h at 37 °C in a 5% CO 2 incubator. After 24 h, supernatants were collected from each well and cytotoxicity was determined by measuring lactate dehydrogenase (LDH) released by using LDH kit (Roche). Untreated cells were considered as negative control, whereas cells incubated with 0.1% Triton X-100 for 20 min gave maximum LDH release as a result of cell lysis which was taken as positive control. The percentage cell cytotoxicity was calculated as follows: % cell cytotoxicity = (sample absorbance − negative control absorbance)/ (positive control absorbance − negative control absorbance) × 100. The results are representatives of several experiments presented as the mean ± standard error. statistical analysis. Student T test was used to measure statistical correlation and significance. P < 0.05 was the limit for significance using two-sample T test and two-tailed distribution. *Represents P < 0.05, **represents P < 0.01, while ***represents P < 0.001.

Results
Characterization of GA-AgNPs and GA-AgNPs-HDN for determination of size, PDI, zeta potential and surface morphology. Size and shape of the nanoparticles have a key importance in the design of drug delivery systems. Smaller the size of the nanoparticles greater will be the surface to volume ratio, therefore the chances of interactions between the bioactive drug molecules and the nanoparticles increases which ultimately enhances the therapeutic efficacy of the drug 32 . GA-AgNPs possess 107.1 ± 2.56 nm mean size with PDI 0.270 ± 0.03. Similarly, GA-AgNPs-HDN exhibit 182.8 ± 1.02 nm and PDI 0.422 ± 0.01 results are shown in Fig. 1A,B. Larger mean size and PDI of the HDN loaded nanoparticles than that of their unloaded analogue is attributed to unequal scattering of the drug moieties over the surface of GA-AgNPs. Zeta potential of GA-AgNPs and GA-AgNPs-HDN was −18.6 ± 0.54 mV and −19.1 ± 1.34 mV respectively as shown in Fig. 1C,D. Zeta potential is another important parameter for the determination of nano-carrier stability. AFM and TEM of GA-Ag-NPs and GA-Ag-NPs-HD were investigated to find their morphology. Both nanoparticles found to be spherical in  Fig. 2E. The FT-IR spectrum of HDN revealed its distinctive peaks at 3466.1and 2925.50 cm −1 assign to OH and C-H groups of HDN. The peak for C=O, C = C and aromatic C=C appeared at 1645.27, 1515.9 and 1443.0 cm −1 respectively (Fig. 2F). The FT-IR spectrum of GA-AgNPs-HDN shows all the representative peaks at their respective places with slight changes in absorbance. FT-IR analysis confirms the chemical stability of HDN as all peaks are present on their respective positions in the spectrum shown in Fig. 2F. The drug loading % was found to be 73.66%.
Characterization of GT-AuNPs and GT-AuNPs-NRG for determination of size, PDI, zeta potential and surface morphology. GT-AuNPs and GT-AuNPs-NRG exhibit 183 ± 1.04 and 221 ± 1.08 nm mean size with PDI 0.351 ± 0.02 and 0.410 ± 0.03 respectively which are shown in Table 1. The size of the GT-AuNPs as compare to NRG loaded counterpart is relatively larger which confirms the NRG loading on the surface of GT-AuNPs. Zeta potential of GT-AuNPs is −34.1 ± 0.1 and for GT-AuNPs-NRG is −27.6 ± 0.5 mV respectively. The drug loading % was found to be 72%. Surface morphology of both GT-AuNPs and GT-AuNPs-NRG were   Table 1. The average size, polydispersity index (PDI), and zeta potential of GT-AuNPs and GT-AuNPs-NRG with the % NRG loading efficiency.
www.nature.com/scientificreports www.nature.com/scientificreports/ investigated through AFM and TEM, and they were found to be spherical in shape as shown in Fig. 3A-D. shows a representative surface plasmon resonance band of GT-AuNPs-NRG with maximum absorbance at 540 nm.  -HDN abolished viability of A. castellanii and N. fowleri. Amoebicidal assay revealed that GA-AgNPs-HDN killed all the A. castellanii trophozoites at 50 µg per mL, and significantly reduced the number of cells by 90% at 25 µg per mL as compared to GA alone, HDN alone, and GA-AgNPs (Fig. 4A). Notably GA-AgNPs-HDN was found to be more effective than positive control Chlorhexidine. Whereas, GT-AuNPs-NRG did not produce cidal effects when statistically compared with GT alone, NRG alone and GT-AuNPs. On the other hand, both GA-AgNPs-HDN and GT-AuNPs-NRG exhibited significant amoebicidal effects against N. fowleri as compared to gums alone, drugs alone and gums stabilized nanoparticles (Fig. 4B). GA-AgNPs-HDN caused 99% reduction in N. fowleri viability at 25 µg per mL which is also significantly more effective than Amphotericin B alone. These results suggest that GA-AgNPs-HDN is an exceptional formulation which hold potential for further studies.

GA-AgNPs-HDN and GT-AuNPs-NRG inhibited encystment and excystation of A. castellanii.
As encystment of A. castellanii is responsible for the resistance against drugs, these nanoparticles were tested for inhibition of encystation. GA-AgNPs-HDN and GT-AuNPs-NRG significantly inhibited the encystation of A. castellanii at 100 µg per mL as compared to respective controls (Fig. 5A). GA-AgNPs-HDN caused 95% inhibition and GT-AuNPs-NRG inhibited the encystation by 85%. The de-differentiation of cysts into trophozoites causes recurrence of infection in most of the cases. Therefore, the effects of GA-AgNPs-HDN and GT-AuNPs-NRG were also evaluated against excystation. While treated with pre-formed mature cysts of A. castellanii, GA-AgNPs-HDN inhibited excystation by 84% at 100 µg per mL (Fig. 5B). Contrary, GT-AuNPs-NRG did not exhibit significant excystation when compared with GT-AuNPs. Since most of the lead compounds and drugs have limited effects against cysts of A. castellanii, these nanoparticles showed consistent effects against trophozoite as well as resistant cyst stage. www.nature.com/scientificreports www.nature.com/scientificreports/ GA-AgNPs-HDN exhibited significant bactericidal effects. Figure 6 represents the bactericidal effects of GA-AgNPs-HDN and GT-AuNPs-NRG tested at 50 and 0.5 µg per mL against MRSA and E. coli K1. GA-AgNPs-HDN showed significant bactericidal activity at 50 µg per mL against MRSA (Fig. 6B), and 0.5 µg per mL against E. coli K1 (Fig. 6D). GT-AuNPs-NRG did not exhibit bactericidal effects at 50 µg per mL against both tested bacteria (Fig. 6C,F). Figure 7 presents the corresponding field emission scanning electron microscopic (FE-SEM) analysis of bacteria before and after treatment with GA-AgNPs-HDN.

GA-AgNPs-HDN reduced the pathogens-mediated host cell cytotoxicity. The pretreatment of
A. castellanii and E. coli K1 with GA-AgNPs-HDN resulted in significant reduction of their cytopathogenicity against human cells. Figure 8A describes that untreated A. castellanii caused more than 80% cell cytotoxicity against HeLa cells, contrary, GA-AgNPs-HDN (50 µg per mL) completely obliterated the host cells cytotoxicity as compared to relative controls. Similarly, the pretreatment of 0.5 µg per mL GA-AgNPs-HDN with E. coli K1 abolished the cytotoxicity of bacterium against HeLa cells as compared to untreated E. coli K1 which exhibited 74% cytotoxicity (Fig. 8B).

GA-AgNPs-HDN and GT-AuNPs-NRG showed minimal cytotoxicity against human cells. When
tested against human cells, all test samples showed minimal cytotoxic effects (Fig. 9). GA-AgNPs-HDN exhibited only 11% cytotoxicity while GT-AuNPs-NRG caused 23% cytotoxicity against HeLa at a higher concentration of 100 µg per mL as compared to amoebicidal effects which were recorded at 50 and 25 µg per mL. The cytotoxicity profile against human cells suggest that these nanoparticles are biosafe and can further be evaluated for potential in in vivo studies.  50 . castellanii and (B) N. fowleri. The viability of amoebae was determined after amoebicidal assay as described in the materials and methods section. Briefly, A. castellanii or N. fowleri trophozoites were incubated with GA and GT alone, AgNPs alone, AuNPs alone, HDN alone, NRG alone, GA-AgNPs, GT-AuNPs, GA-AgNPs-HDN, and GT-AuNPs-NRG and negative and positive controls at 50 and 25 µg per mL at 30 °C for 24 h. Next, the viability was measured by Trypan blue exclusion assay. The results are presented as the mean ± standard error of various experiments performed in duplicate. *Represents P < 0.05, **represents P < 0.01, while ***represents P < 0.001. P values were obtained using two-sample T test and two-tailed distribution.
A. castellanii (1 × 10 5 ) were inoculated in PBS in the presence of GA-AgNPs-HDN and GT-AuNPs-NRG and respective controls at 100 µg per mL with encystation media and incubated at 30 °C for 72 h. Next, 0.25% sodium dodecyl sulfate (SDS) was added and incubated at room temperature for 10 min to lyse A. castellanii trophozoites followed by enumeration of amoebae cysts using a hemocytometer. (B) Excystation assays was performed by incubating GA-AgNPs, GT-AuNPs, GA-AgNPs-HDN, GT-AuNPs-NRG and respective controls (100 µg per mL) with A. castellanii cysts (1 × 10 5 ) in growth medium, PYG at 30 °C for 72 h. After this period, amoebae were counted using a hemocytometer. The results are presented as the mean ± standard error of various experiments performed in duplicate. *Represents P < 0.05, **represents P < 0.01, while ***represents P < 0.001. P values were obtained using two-sample T test and two-tailed distribution.  GT-AuNPs-NRG shows no antibacterial activity (F). The results are presented as the mean ± standard error of various experiments performed in duplicate. *Represents P < 0.05, **represents P < 0.01, while ***represents P < 0.001. P values were obtained using two-sample T test and twotailed distribution. www.nature.com/scientificreports www.nature.com/scientificreports/

Discussion
Brain-eating amoebae are opportunistic protist pathogens associated with diseases of fatal severity. The molecular pathways to target these microbes are limited which results in challenges in development of effective therapeutics 4 . Current management and treatment are unspecific and ineffective due to which the CNS infections caused by brain-eating amoebae almost always proved to be deadly 33 . Furthermore, the clinical procedures suffer from limitations including long term use of medications (a mixture of drugs including biguanides, azoles, amidines, antibiotics) and still the chances of recurrence are high 34 . On the other hand, ever growing drug resistance in most commonly present bacteria, and lack of newer and improved antimicrobial agents pose serious challenges to healthcare systems 35 . Therefore, there is an urgent need to develop novel, sustainable, and effective modalities of chemotherapeutics against infectious diseases. Nanotechnology has proved to be a model alternative to target infectious diseases 36 . Due to small size of nanomaterials, these are efficient drug delivery carriers for minimizing the pharmacokinetics and pharmacodynamics limitations of compounds and drugs known to have medicinal values 37 . Flavonoids are important nutraceutical and biologically active class of secondary metabolites natural products. Flavonoids obtained from citrus fruit plants are rich of drug candidates against a variety of diseases such as; infectious diseases, cancer, neurodegenerative etc. [38][39][40][41] . However, their clinical applications have some common shortcomings, and their poor bioavailability is one of the major factors 14 . In this study, we synthesized silver and gold nanoparticles stabilized with plant gums and loaded them with two most common citrus fruits flavonoids HDN and NRG to utilize their antimicrobial activity against brain-eating parasites A. castellanii and N. fowleri and multi-drug resistant bacteria MRSA and neuropathogenic E. coli K1. °C in a 5% CO 2 incubator as described in materials and methods section. Next, cell-free supernatant was collected, and cytotoxicity was determined using Lactate dehydrogenase (LDH) assay kit (Roche). (B) E. coli K1 caused 74% cytotoxicity to HeLa cells. Upon pretreatment with 0.5 µg per mL GA-AgNPs-HDN, the host cells cytotoxicity was reduced to 1%. The results are presented as the mean ± standard error of various experiments performed in duplicate. *Represents P < 0.05, **represents P < 0.01, while ***represents P < 0.001. P values were obtained using two-sample T test and two-tailed distribution.
0% 25% 50% 75% 100% Cytotoxicity against human cells Figure 9. GA-AgNPs-HDN and GT-AuNPs-NRG did not exhibit cytotoxicity against HeLa cells at 100 µg per mL. These nanoparticles and the respective controls were incubated at 30 °C with HeLa cells monolayer for 24 h at 37 °C in a 5% CO 2 incubator. Following this incubation, cell-free supernatant was collected, and cytotoxicity was determined using Lactate dehydrogenase (LDH) assay kit (Roche www.nature.com/scientificreports www.nature.com/scientificreports/ Green synthesis of nanoparticles involves the reduction of metal ions by using environmentally and eco-friendly materials which act as reducing and stabilizing agents. Microorganisms and plant materials have been widely used for the biosynthesis of nanoparticles 42 . Green synthesized nanoparticles have been extensively used against microbial diseases, however, only few results are reported against parasitic diseases 43 . Besides metallic nanoparticles, green polymers including cellulose and starch have also been used for clinical and biomedical applications including bones healing and substitution [44][45][46] . The synthesis and stabilization of nanoparticles is dependent on the reducing and capping ability of the material used. In this study, the reduction of silver and gold is accomplished by using biocompatible natural gums; GA and GT. This green approach is being exploited for the formation of nanoparticles by avoiding any toxic reducing agent and harsh temperature conditions 17 . These nanoparticles were thoroughly characterized by various instrumental techniques before subjected to biological evaluation against brain-eating amoebae. The role of Toll-like receptors (TLRs) in innate immune responses to pathogens is well recognized 47,48 . Our previous study showed that, HDN is known to reduce the expression of mRNA in TLRs which as a result reduce inflammation 17 . As the TLRs can influence the immunopathogenesis of CNS parasitic infections, we proposed that using TLRs targeting compounds can decrease the activity of inflammatory cytokines which may affect the parasite clearance and host survival. However, upon loading of HDN on GA-AgNPs caused surprisingly drastic amoebicidal effects, the mechanism of which is yet unknown. On the other hand, NRG acts as inhibitor of cytochrome P450 49 , which is known to be a common pathway associated with antimicrobial mode of action against brain-eating amoebae 50 . In our previous report, we showed the antibacterial effects of GT-AuNPs-NRG against a variety of bacteria, however their IC 50 values were high (in the range of 250-300 µg per mL) 26 . GA-AgNPs-HDN are found to be more potent which showed significant bactericidal effects at 50 and 0.5 µg per mL against MRSA and E. coli K1 respectively. Interestingly, Gram-negative E. coli K1 which have additional peptidoglycan cell wall as compared to Gram-positive bacteria is found to be more susceptible to GA-AgNPs-HDN. The mode of action of such potent antimicrobial effects of these nanoparticles however is still to be determined.

Conclusions
The green synthesis of silver and gold nanoparticles stabilized with natural glycosidic polymers of plant gums (gum acacia and gum tragacanth) was achieved. These nanoparticles were further loaded with citrus fruits flavonoids HDN and NRG to obtain GA-AgNPs-HDN and GT-AuNPs-NRG. The nanoparticles were characterized by zetasizer, zetapotential, AFM, UV-vis spectrophotometric, and FT-IR analyses. GA-AgNPs-HDN and GT-AuNPs-NRG were tested against brain-eating amoebae A. castellanii and N. fowleri, as well as multi-drug resistant bacteria MRSA and neuropathogenic E. coli K1. These nanoparticles exhibited potent amoebicidal and bactericidal effects, and also inhibited the encystation and excystation processes of A. castellanii. Furthermore, these nanoparticles significantly reduced the pathogens-mediated host cells cytotoxicity. Interestingly, these nanocarriers did not show cytotoxicity against human cells even at higher concentration as compared to their concentration used for antimicrobial effects. This study demonstrates a potential development of effective antimicrobial nano-formulations based on naturally occurring flavonoids. These results are anticipated to be a major step forward in developing efficient nanomedicine against pathogenic microbes including brain-eating amoebae and bacterial infections. The mechanism of action and in vivo studies are part of our future research.

Data Availability
Data will be provided upon request on case to case basis.