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. 2024 Feb 13;7(3):855–862. doi: 10.1021/acsptsci.3c00348

Improved Methodology for Evaluating Nanomedicine Antibacterial Properties in Biological Fluids

Ali Akbar Ashkarran †, Shahriar Sharifi †, Christopher N LaRock ‡, Morteza Mahmoudi †,*
PMCID: PMC10928882  PMID: 38481694

Abstract

graphic file with name pt3c00348_0007.jpg

Accurate assessment of nanomedicines’ antibacterial properties is pivotal for their effective use in both in vitro and in vivo settings. Conventional antibacterial activity assessment methods, involving bacterial coculture with compounds on agar plates, may not fully suit nanomedicines due to their susceptibility to alterations in physicochemical properties induced by biological fluids. Furthermore, these biological fluids might even enhance the bacterial growth. This study introduces a novel, rigorous, and reproducible methodology for evaluating nanomedicine antibacterial properties using cell culture media (i.e., DMEM-FBS10%). To assess the antibacterial activity of the nanoparticles in cell culture media, superparamagnetic iron oxide nanoparticles (SPIONs) were chosen as the model nanomedicine due to their clinical significance. A comparative analysis between the traditional and our proposed methods yielded contrasting outcomes, shedding light on the significant impact of biological fluids on nanoparticle antibacterial activities. While the conventional approach suggested the antibacterial effectiveness of SPIONs against Staphylococcus aureus, our innovative method unveiled a substantial increase in bacterial growth in the presence of biological fluids. More specifically, we found a significant increase in bacterial growth when exposed to bare SPIONs at various concentrations, while the formation of a protein corona on SPION surfaces could markedly reduce the observed bacterial growth compared to the control group. These findings underscore the necessity for more refined evaluation techniques that can better replicate the in vivo environment when studying the nanomedicine’s antibacterial capabilities.

Keywords: nanomedicines, antibacterial properties, antibacterial assessment, biological fluids, protein corona

In recent years, the advent and utilization of nanomedicines have brought about a transformative shift in various aspects of medical treatment and diagnostics, particularly in the context of combating bacterial infections.1 The efficacy of these nanomedicines in addressing bacterial pathogens is pivotal for their therapeutic relevance, thereby demanding precise and reliable methods to assess their antibacterial attributes. Traditional techniques for appraising antibacterial activity, primarily reliant on coculture methodologies on agar plates followed by colony counting,2,3 have long served as the foundation of microbiological research. Nevertheless, these established approaches may not be wholly conducive for assessing nanomedicines, as their interactions with biological fluids have the potential to substantially modify their physicochemical characteristics, subsequently affecting their antibacterial effectiveness.

The dynamic landscape of nanomedicine research necessitates innovative and more precise methodologies capable of mirroring the real-world effectiveness of these agents. In this context, superparamagnetic iron oxide nanoparticles (SPIONs) have garnered considerable attention. SPIONs have exhibited promising antibacterial properties and are increasingly under exploration for their potential clinical applications, especially in combating drug-resistant bacterial strains.4−6 SPIONs have been observed to adhere to the surface of bacterial cells, leading to a reduction in biofilm formation.7 Conversely, they may also facilitate bacterial growth by releasing iron ions, as these ions play a vital role in essential life processes.7−13

In addition to the direct impact of SPIONs on bacteria, the intricate interplay between SPIONs and various nanoparticle types within biological systems compounds the challenge of assessing their antibacterial effectiveness. This complexity arises primarily from the phenomenon of protein/biomolecular corona formation on the surface of nanoparticles. The protein corona represents a layer of biomolecules, predominantly proteins, that spontaneously adhere to the nanoparticle surface upon exposure to biological fluids.14−17 Consequently, what bacteria encounter in vivo are not the pristine nanoparticles but rather the nanoparticles coated with this protein corona.18−20

In this study, we present a novel methodological approach aimed at delivering a more robust, replicable, and real-world assessment of nanomedicines’ antibacterial properties, employing SPIONs as a model nanoparticle. This approach factors in the impact of biological fluids on nanomedicine behavior, providing a more faithful reflection of their efficacy in practical scenarios. Our results underscore the constraints of existing testing methods and emphasize the crucial requirement for enhanced techniques to more accurately evaluate nanomedicines’ antibacterial potential in both in vitro and in vivo contexts.

Results and Discussion

The physicochemical characteristics of the SPIONs employed in this study are summarized in Figure 1. The SPIONs exhibit an average diameter of 13.5 nm and a relatively uniform size distribution, characterized by a surface charge of around −10 mV. Transmission electron microscopy (TEM) images reveal some nanoparticle agglomeration, attributable to the magnetic nature of the SPIONs, yet they corroborate the average size measurements obtained through dynamic light scattering (DLS) analysis.

Figure 1.

Figure 1

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Physicochemical characterizations of SPIONs including TEM (left panels), size distribution, and surface charge (right panels) used in this study.

Our initial investigation into the antibacterial effects of SPIONs against Staphylococcus aureus employed a widely accepted method for assessing antibacterial activity, involving cocultivation of bacteria with SPIONs on solid agar plates, followed by colony counting.21,22 Specifically, 50 μL of bacteria (∼10−6 CFU/mL: colony forming units per milliliter) was mixed with 50 μL of SPIONs at varying concentrations (i.e., 10, 100, and 1000 μg/mL). These mixtures were then spread thoroughly on agar plates and incubated at 37 °C overnight, with colony counting performed on the following day. In line with previous research, our results (Figure 2), based on eight experimental repetitions, confirm the intrinsic antibacterial properties of bare SPIONs against S. aureus.

Figure 2.

Figure 2

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Antibacterial activity of bare SPIONs against S. aureus bacteria at different concentrations (bottom panel) alongside the corresponding optical images (top panel); R1 to R8 denote the eight separate experimental replicates and a P value of <0.05 is considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).

We sought to investigate how SPIONs influence bacterial function and growth within a biologically relevant environment encompassing biological fluids. This exploration is of paramount significance, as it aligns with how biosystems, such as cells, encounter bacteria in a context that mirrors biological relevance, distinct from growth on agar plates. Moreover, biosystems and their associated biological fluids can impact the surface properties of SPIONs23 and, therefore, have the capacity to influence their interactions with bacteria.

To accomplish this, we adapted the conventional approach for probing the antibacterial properties of SPIONs to assess their effects in the presence of cell culture medium. As model cell culture medium, we utilized Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). In order to optimize the experimental conditions and ascertain the reproducibility of this modified approach, we first cultured bacteria (without SPIONs) in the model cell culture medium overnight. Subsequently, we cultured the solution on agar plates to enumerate the colony count, providing a baseline measurement.

To identify the optimal protocol, we initiated the process by inoculating a single bacterial colony in 1 mL of PBS. We then incubated 1 and 5 μL of this suspension with varying concentrations (using serial dilutions of 1 mL of a single bacterial colony) in 1 mL of the model cell culture medium within a 24-well plate overnight. The resultant samples were extracted and centrifuged at 8000g for 5 min to pellet the cultivated bacteria. The pellet was subsequently resuspended in 50 μL of PBS and cultured on agar plates overnight, allowing for the observation of bacterial growth on the subsequent day (refer to Figure S1 of the Supporting Information (SI)). The selection of bacterial concentrations for this experiment (i.e., 10–4, 10–5, and 10–6 CFU/mL) was based on our prior research, which established that culturing a volume of approximately 50 μL with a bacterial concentration of 10–5 CFU/mL yields a substantial number of colonies (∼a few hundred) that can be effectively quantified and counted.

The findings indicated that centrifugation of the culture medium followed by reculturing the pellet on agar plates led to an exceedingly high (almost uncountable) bacterial count. In an effort to quantify bacterial growth, we initially reduced both the concentration and volume of the initial bacteria in the cell culture medium (refer to Figure S2 in the SI for specifics). However, this approach did not resolve the issue of extensive colony formation. Consequently, we proceeded to further dilute the samples, ultimately achieving conditions where isolated colonies were formed and quantifiable (refer to Figure S3 in the SI for details).

Based on the results obtained, the bacterial preparation with a dilution factor of 6 (i.e., 10–6), incubated with cell culture medium and subsequently serially diluted to a factor of 5 (i.e., 10–5), displayed a significant number of observable colonies. To ensure the reproducibility of our findings, this experiment was repeated multiple times, as depicted in Figure S4.

The results of the optimization process led us to employ a dilution factor of 5 for bacteria, ensuring the production of sufficient and reproducible colonies for subsequent experiments (Figure 3). Utilizing these refined experimental conditions, we investigated the antibacterial properties of protein corona-coated SPIONs. It is worth noting that we were unable to assess the antibacterial properties of bare SPIONs under this specific dilution, as the absence of 10% FBS led to the absence of bacterial colonies. This underscores the pivotal role of nutrient supplementation in bacterial growth within culture media (Figures S5 and S6).

Figure 3.

Figure 3

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Experimental design (lower right panel) along with the corresponding optical images of bacteria illustrating the results obtained from technical and experimental replicates employing the refined protocol.

To evaluate the influence of protein corona-coated SPIONs on antibacterial activity against S. aureus bacteria, we initiated the experiment by using a 50 μL sample from an initial colony of bacteria, which was subsequently serially diluted to a concentration of 10–5 CFU/mL. These bacterial samples were then combined with 50 μL of SPIONs at various concentrations (i.e., 10, 100, and 1000 μg/mL) and incubated in cell culture medium (DMEM-FBS10%) overnight within a 24-well plate.

On the following day, we again serially diluted the samples to a concentration of 10–5 CFU/mL. Subsequently, 50 μL of these diluted samples was spread onto agar plates and incubated once more overnight. The colonies on the agar plates were then counted on the following day (as shown in Figures 4 and S7 in the SI). This entire process was repeated a total of six times for distinct experimental repetitions.

Figure 4.

Figure 4

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Experimental design and photographs showing the effect of protein corona-coated SPIONs on bacterial growth (three repeats) at various concentrations of SPIONs.

The results of our study indicated an increase in the number of colonies in the presence of protein corona-coated SPIONs, in contrast to the control group of bacteria.

To investigate the influence of bare SPIONs on bacterial growth, we employed a lower bacterial dilution in DMEM medium devoid of FBS (Figure 5), as the absence of FBS results in a lower bacterial growth, and therefore, a higher number of initial colonies (lower bacterial dilution) are required to achieve a meaningful quantification. Specifically, for this experiment, the initial bacterial concentration used in the cell culture medium was 10–1 CFU/mL. To achieve this concentration, a single colony was inoculated into a 1 mL vial, and then, it was diluted to 10–1 CFU/mL by adding 9 mL of PBS. This initial concentration was utilized in bare DMEM medium. Subsequently, there were two sequential serial dilutions, resulting in a concentration of 1 × 10–2 CFU/mL in the second round of dilution in the cell culture medium. After this, 50 μL of the 10–2 CFU/mL sample was recultured on agar plates for colony counting (as depicted in Figure 5, top panel).

Figure 5.

Figure 5

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Experimental design and images demonstrating the impact of bare SPIONs on bacterial growth (three repetitions) at different SPIONs’ concentrations in DMEM culture medium in the absence of FBS.

It is important to note that FBS serves as a crucial source of nutrition for bacterial growth. Therefore, in the absence of FBS, we opted for a lower number of dilutions to increase the likelihood of identifying isolated colonies.

The average colony count results (Figure 6) indicate a significant increase in bacterial viability in the presence of bare SPIONs within DMEM lacking FBS. This increase in viability may be attributed to the release of iron ions, which is typically limiting within humans due to sequestration and positively impacts the growth of bacterial pathogens.13,24−26 While most pathogens use this iron as cofactors or incorporate it into biomolecules, recent research demonstrated that some even have organelles dedicated to storing iron in the particle form intracellularly for future utilization.27

Figure 6.

Figure 6

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Viability of the bacteria in the presence of bare SPIONs and protein corona-coated SPIONs at various concentrations in DMEM cell culture medium (*P < 0.05, **P < 0.01, and ***P < 0.001).

This result contradicts the well-established antibacterial properties of SPIONs observed in conventional antibacterial assays, largely owing to the fundamental disparities between these two culture media. In fact, SPIONs have greater physical interactions with bacteria on agar plates compared to liquid culture media, resulting in stronger bacterial inhibition.28 In other words, the interaction between bacteria and nanoparticles is less pronounced in a liquid environment with a greater molecular spacing. Moreover, liquid environments offer a more nutrient-rich milieu for bacterial growth during agitation, thereby promoting increased growth.29

Another noteworthy observation we made was that the formation of a protein corona on the surface of SPIONs resulted in a slower rate of bacterial growth when compared with bare SPIONs (Figure 6). Several factors may contribute to this reduction: (i) reduced interactions: protein corona-coated SPIONs may exhibit fewer interactions with bacteria due to their lower surface energy in comparison to bare SPIONs and (ii) iron release: the release of iron from the surface of protein corona-coated SPIONs may be reduced, impacting bacterial growth. Additionally, liquid environments, as previously mentioned, provide a more nutrient-rich medium for bacterial growth during agitation, potentially leading to enhanced growth.29 Our observations have unveiled a noteworthy trend in bacterial growth. When subjected to bare SPIONs at various concentrations, we observed a substantial increase in the bacterial proliferation. However, the presence of a protein corona formed around SPIONs due to the presence of FBS significantly curtailed bacterial growth. These proof-of-concept findings underscore the influence of the protein source and the bioavailable iron supplied through SPIONs on enhancing bacterial proliferation. Consequently, overlooking the pivotal role of biological fluids in conventional antibacterial approaches may lead to an incomplete understanding of the true antibacterial potential of NPs in vivo.

The antibacterial activity of silver NPs has been a well-documented and recognized phenomenon in the field of nanomedicine for many decades against a broad spectrum of bacteria including S. aureus.18,22 We have performed a control experiment using commercially available silver NPs with an average size of 25 nm and tested their antibacterial activities in similar experimental conditions to bare and protein corona-coated SPIONs in cell culture medium supplemented with 10% FBS (Figure S8). The results revealed that at the same concentrations used for bare SPIONs (i.e., 10, 100, and 1000 μg/mL), silver NPs completely inhibited the growth of S. aureus bacteria. These findings reveal that in the same experimental conditions, some nanomaterials (i.e., silver) may act as an antibacterial agent and some (i.e., bare/protein corona-coated SPIONs) may enhance the bacterial growth due to possible reasons discussed earlier.

Conclusions

Traditional methods for evaluating the antibacterial properties of nanomedicines, such as coculturing bacteria with nanomaterials on agar plates, may not adequately account for the influence of biological fluids. In this study, we explored the antibacterial effects of bare and protein corona-coated SPIONs—a nanoparticle of clinical significance in nanomedicine—against S. aureus. Our findings indicate a significant increase in bacterial growth when exposed to bare SPIONs at various concentrations. Interestingly, the formation of a protein corona around SPIONs, triggered by FBS, markedly reduces but does not completely inhibit bacterial growth compared with the control group. These initial results suggest that the synergistic roles of the protein source and bioavailable iron (through SPIONs) improve bacterial proliferation. Neglecting the crucial role of biological fluids in traditional antibacterial approaches may lead to an incomplete understanding of the true antibacterial efficacies of nanoparticles in vivo. These findings point to the necessity for more advanced and accurate evaluation methods that can mimic in vivo conditions when assessing the antibacterial potential of nanomedicines.

Experimental Details

Materials

SPIONs (30 mg/mL, commercially known as Ferumoxytol) was purchased from Feraheme (www.feraheme.com) and diluted with phosphate-buffered saline (PBS 1×, HyClone) solution to the desired concentration for all experiments. DMEM and FBS were ordered from Fisher Scientific, mixed, and prepared freshly with the desired concentrations. S. aureus Rosenbach 25923 was ordered from ATCC. Tryptic soy agar (TSA) was ordered from BD Bioscience and prepared by dissolving 40 g of agar in 1 L of pure water and autoclaving at 120 °C for 45 min. After cooling, the agar was poured out into the sterile plates and allowed to reach room temperature and stored at 4 °C for further use. Commercially available powder silver nanoparticles (∼25 nm) were ordered from Nanocomposix, diluted with PBS 1× to desired concentrations, and used as control NPs compared with bare SPIONs.

Bacterial Culture

Before the microbiological experiments, all glasswares were sterilized by autoclaving at 120 °C for 20 min. The antibacterial activity of SPIONs was tested against Gram-positive S. aureus bacteria as a representative. For the preparation of bacterial solution, a single colony of bacteria was taken from an agar plate that was already cultured overnight and inoculated into a 1 mL tube. The 1 mL solution containing bacteria was then serially diluted to a factor of 5 to reach approximately 10−6 colony forming units per milliliter (CFU/mL) concentration of bacteria. For basic control experiments, 50 μL of SPIONs with desired concentrations was separately mixed with 50 μL of diluted bacterial suspension (10−6 CFU/mL) and spread on the surface of nutrient agar plates and incubated at 37 °C overnight. All experiments are performed at least in triplicate followed by colony counting on the next day. All bacterial culture experiments in the presence of cell culture medium are carried out in a 24-well plate containing 1 mL of cell culture medium (i.e., bare DMEM or DMEM-FBS10%) in each well plate. The plate was then placed in a shaker incubator overnight at 37 °C to allow the bacteria to grow. On the following day, the plate was taken out and each individual well (as a representative of each sample) was diluted serially and cultured again on agar plates overnight at 37 °C followed by colony counting on the next day to access the antibacterial activities of each sample.

Characterization

DLS and ζ-potential analyses were performed to measure the size distribution and surface charge of the nanoparticles using a Zetasizer Nano series DLS instrument (Malvern company). A helium–neon laser with a wavelength of 632 nm was used for size distribution measurement at room temperature. Transmission election microscopy (TEM) images were acquired by a JEOL JEM-1400 Plus transmission electron microscope (Peabody, MA) operating at 120 kV. Formvar-coated carbon TEM grids were prepared by drop-casting nanoparticles suspended in 95% ethanol and dried at room temperature.

Acknowledgments

The authors acknowledge the support of the US National Institute of Diabetes and Digestive and Kidney Diseases (grant DK131417).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.3c00348.

  • Experimental designs and optical images of grown bacteria (PDF)

The authors declare no competing financial interest.

Supplementary Material

pt3c00348_si_001.pdf (1MB, pdf)

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Associated Data

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Supplementary Materials

pt3c00348_si_001.pdf (1MB, pdf)

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