AMF-responsive nanobiomagnetite for targeted delivery of amphotericin B against Leishmania amazonensis

Abstract

Cutaneous leishmaniasis remains a neglected tropical disease with limited treatment options. Available therapies include costly and toxic drugs for which recurrent cases of resistance are reported. Drug delivery systems based on the association of approved drugs and nanoparticles have improved pharmacological properties of the drug, such as targeted therapy, enhanced drug solubility, reduced side effects, and potentially lower doses required for effective treatment. In this study, we explored the in vitro potential use of the nanobiomagnetite produced by magnetotactic bacteria functionalized with amphotericin B against promastigotes of Leishmania amazonensis, one of the main pathogens of cutaneous leishmaniasis. Additionally, the antileishmanial activity of the nanoformulation was significantly increased in association with alternating magnetic field (AMF) exposure, indicating an advantage in the therapeutic efficacy of the drug, potentially leading to a combined therapy. In addition, to assess the preliminary safety of the nanoformulation, we assessed its cytotoxicity on HaCaT, hFB, and J774.16 cell lines; none of the tested nanoformulations were cytotoxic toward these cell lines, suggesting their potential for biocompatible therapeutic applications. Moreover, no significant nitric oxide production was detected with the nanoparticle’s interaction on J774.16 macrophages. This finding is vital for further clinical considerations, as it reduces the risk of inflammatory responses. Thus, we demonstrated the biocompatibility and parasitic potential of functionalized nanobiomagnetite as an alternative AMF-responsive therapy in in vitro models. However, in vivo testing is still necessary to assess the nanoformulation activity against Leishmania.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-23720-6.

Introduction

Neglected tropical diseases (NTD) are a group of conditions and infectious diseases defined by the World Health Organization (WHO)^1,2. They are prevalent in tropical and subtropical regions—e.g., Sub-Saharan Africa, Southeast Asia, Latin America, and The Caribbean². NTDs correlate with limited access to healthcare systems, poor sanitation, high poverty rates, and lack of research and investments¹. Therefore, it brings significant health and socio-economic challenges for affected countries, such as productivity loss, that sustains the world’s social, economic, and health inequality^2,3. The impact of NTDs on the lack of the world’s equitability is recognized in the United Nations (UN) 2030 Agenda for Sustainable Development^2,4,5. This agenda encompasses a comprehensive framework to address global challenges, featuring 17 Sustainable Development Goals (SDGs) to foster equitable and sustainable progress. The third SDG, good health and well-being, entails the target to end the epidemics of NTDs by 2030, together with AIDS, tuberculosis, and malaria⁴.

Among NTD total global burden, leishmaniasis affects more than 12 million people worldwide⁶, and it is estimated that 1 billion people are at risk of infection⁷. Leishmaniasis is an NTD caused by protozoans of the Leishmania genus, divided into the subgenus Viannia and Leishmania, that causes different types of the disease^8,9. Leishmaniasis has three main clinical forms: visceral leishmaniasis (VL), cutaneous leishmaniasis (CL), and mucocutaneous leishmaniasis (MCL). The most common form of the disease is CL. CL is characterized by one or multiple lesions on the skin with ulcerative aspects, which can leave disfiguring scars and life-lasting disabilities^7,10. In Brazil, the species Leishmania amazonensis is associated with a rare but severe form of CL, called anergic diffuse cutaneous leishmaniasis (ADCL), characterized by non-ulcerative nodules disseminated through the body, especially the extremities¹¹. Patients usually exhibit a lack of cell-mediated immune response to ADCL, presenting a predominance of Th2 cytokines (such as IL-4) over Th1 response (such as IFN-γ). The poor cell-mediated immune response against L. amazonensis explains the frequent therapeutical failures and relapses observed in ADCL patients, highlighting the importance of L. amazonensis role in the disease’s burden^11,12. The diversity of Leishmania species and their immune-evasion strategies complicates the treatment and prevention approaches¹³. Moreover, leishmaniasis treatment has other challenges to overcome, such as high-costing drugs, long-lasting therapies with high toxicity, and increased resistance to first-choice drugs^14–16. Hence, it is necessary to develop new therapies for CL due to limitations in available treatments. New strategies for leishmaniasis treatment in recent years have focused on therapies that seek to reduce existing drugs’ toxicity and decrease resistance development by Leishmania¹⁴. In this sense, nanoparticles might contribute to creating new delivery systems for approved drugs, allowing improvements in bioavailability, solubility and reducing the toxic effects of currently available drugs^17,18.

Amphotericin B (AmB) is a polyene drug used as a second-choice drug in leishmaniasis treatment⁸. Cure rates of AmB in CL and VL are excellent, being over 97%, depending on the species¹⁹. However, despite the significant activity of this drug, the need for hospitalization, high toxicity, adverse effects, and high cost of AmB, especially in its lipid formulations, means it is not widely used for leishmaniasis treatment⁸. The primary action of AmB is the formation of pores in the membrane, releasing cytoplasmic contents, such as ions and small molecules. AmB also induces alterations in membrane permeability, resulting in the influx of protons, intracellular pH reduction, dissipation of the proton gradient, and ultimately, inhibition of cell growth, resulting in cell death^20–22. The primary targets of AmB are membrane sterols. It has a high affinity for ergosterol due to its efficacy against fungi and parasites compared to the membrane of animal cells, which contains cholesterol^20,23. Despite having a higher affinity for ergosterol, AmB also binds to cholesterol in membranes of mammalian cells^20,23,24. Binding to cholesterol is one of the reasons for AmB’s high nephrotoxicity; as renal tubule cells are rich in cholesterol in their membrane, they are more susceptible to the action of AmB, leading to apoptosis of these cells²³. Within the last years, several studies using a delivery system for AmB were developed trying to minimize its toxicity and solubility in physiological medium while maintaining its efficacy^19,25–27. Therefore, AmB is a great candidate for improving leishmaniasis treatment through a drug delivery system and has been extensively studied for this purpose over the last few years²⁸.

Magnetic nanoparticles (MNPs) are promising tools in developing drug delivery systems due to their features and advantages over other nanoparticles. MNPs are distinguished due to their intrinsic magnetic properties, enabling manipulation through an external magnetic field, allowing guidance and accumulation to specific tissues and organs²⁹. Biological MNPs, called magnetosomes, stand out when compared to synthetic MNPs. Magnetosomes, referred here as bacterial magnetite nanoparticles (BMs) or nanobiomagnetite, are constituted by a magnetic nanocrystal composed of magnetite (Fe₃O₄) or greigite (Fe₃S₄), surrounded by a phospholipid bilayer produced by a high-diverse group called magnetotactic bacteria³⁰. Magnetosomes’ high chemical purity, narrow size range, and permanent magnetization, make them applicable in biotechnology^30–33. The biological membrane of magnetosomes provides oxidation protection, and facilitates molecule anchoring for surface functionalization without the need for a covering step as is necessary for synthetic MNPs^34,35. These properties represent advantages in applying magnetosomes over synthetic MNPs, especially in nanomedicine³⁶. Furthermore, magnetosomes did not present high cytotoxicity when tested against different cell lines^37–40 and in animal models^39,41–43, thus they are considered biocompatible. Moreover, magnetosomes’ biocompatibility, magnetic properties, and potential for functionalization make them versatile platforms for targeted drug delivery in conjunction with magnetic hyperthermia treatment⁴⁴. Magnetic hyperthermia is a therapeutic approach that utilizes magnetic nanoparticles exposed to an alternating magnetic field (AMF) to generate localized heat within a target tissue⁴⁵. This results in controlled hyperthermic conditions that selectively damage or destroy targeted cells, like cancer cells, while minimizing damage to surrounding healthy tissues⁴⁶.

Our research group has previously engineered a nanoformulation of magnetosomes functionalized with amphotericin B⁴⁷. This current investigation assessed the cytotoxicity of the nanoformulation in in vitro models of mammalian cells lines and the antileishmanial activity against promastigotes forms of Leishmania amazonensis IFLA/BR/1967/PH8 strain, a causative agent of CL. Our study proposes a drug delivery system for treating CL, using magnetosomes functionalized with AmB while employing enhanced drug release via AMF application.

Results and discussion

Developing and releasing new drugs involves significant time, investment of resources, and rigorous testing to ensure safety and efficacy. In the context of developing new treatments for CL, researchers often explore innovative strategies, one of which involves new forms of use for drugs that are already known and approved for this disease^48,49. One way to give a new use for known drugs is using nanoparticles for drug delivery systems, which can improve therapeutic efficacy in general⁵⁰. Here, we assessed the in vitro cytotoxicity and the potential of nanobiomagnetite functionalized with AmB against L. amazonensis promastigotes, an effective but expensive and highly nephrotoxic drug for treating CL.

The nanoparticles BMs, BMs-PLL (PLL: poly-L-lysine), and BMs-PLL-AmB were produced following the methodology of our group’s previous work (Fig. 1)⁴⁷. To ensure the preparations were made as described before, all nanoformulations’ membrane thickness was measured, and TEM was conducted to assess the nanocrystals and the membrane integrity. The crystals and membranes were preserved throughout extraction, purification, functionalization, lyophilization, and irradiation steps (Fig. 1). Similarly to what was described previously⁴⁷, the membrane thickness increased in the preparations with PLL and AmB. Average membrane BMs presented 5.16 ± 1.03 nm, BMs-PLL presented 7.58 ± 1.17 nm, and BMs-PLL-AmB, 11.36 ± 1.46 nm.

Fig. 1.

Transmission electron microscopy of the bacterial magnetite nanoparticles. (A) BMs, (B) BMs-PLL, and (C) BMs-PLL-AmB. Black arrowheads indicate the magnetosome’s membrane. BMs: bacterial magnetite nanoparticles; BMs-PLL: bacterial magnetite nanoparticles coated poly-L-lysine; BMs-PLL-AmB: bacterial magnetite nanoparticles coated with PLL and functionalized with AmB.

Aiming to assess if the nanoformulations were active against CL, we tested them on L. amazonensis promastigotes. To calculate the half-maximal inhibitory concentration (IC₅₀), the promastigotes were incubated with BMs-PLL-AmB ranging from 0.00010 to 100 µg/mL and performed the MTT assay after 72 h of incubation. The activity of AmB was evaluated by testing concentrations ranging from 79 pM to 433 µM; the IC₅₀ determined was 0.11 µM (Fig. 2a). The IC₅₀ of BMs-PLL-AmB was 3.13 µg/mL. The same concentration range was tested to access BMs and BMs-PLL activity against the parasite. However, they do not possess anti-leishmania activity in the assessed concentrations, remaining with over 80% cell viability in all tested concentrations (Figure S1). These results indicate the activity of BMs-PLL-AmB against L. amazonensis promastigotes was not intrinsic to BMs or PLL coating but was due to the loaded AmB on the BMs’ surface. The nanoformulation exhibited a loading capacity of 25 µg of AmB per 100 µg of BMs⁴⁷, therefore, the IC₅₀ of 3.13 µg/mL corresponds to an equivalent of 0.85 µM of AmB.

Fig. 2.

Viability assay of L. amazonensis. (A) IC₅₀ of AmB and BMs-PLL-AmB in L. amazonensis, considering the AmB concentration, both free and loaded in magnetosomes. The IC₅₀ of BMs-PLL-AmB corresponds to 3.13 µg/mL of magnetosomes, which contains 0.85 µM. (B) Assessment of L. amazonensis viability after treatment with AMF. Promastigotes were incubated with BMs (3.13 µg/mL), BMs-PLL (3.13 µg/mL) and BMs-PLL-AmB (¼ IC₅₀ = 0.80 µg/mL, ½ IC₅₀ = 1.60 µg/mL, and IC₅₀ = 3.13 µg/mL) and then submitted to 30 min of AMF (225 kHz, 200 Oe). Untreated cells were exposed to the same system. MTT test was conducted after 72 h of incubation at 28 °C to assess L. amazonensis viability. Results are the mean ± SEM of three independent experiments. The percentage was calculated compared to the control of untreated cells, considered as 100% of viability. There was a statistically significant difference between promastigotes treated with ½ IC₅₀ and IC₅₀ of BMs-PLL-AmB and control cells (**p ≤ 0.01, ***p ≤ 0.001). BMs = magnetosomes; BMs-PLL = poly-l-lysine coated BMs; BMs-PLL-AmB = BMs-PLL loaded with amphotericin B.

The combined treatment of the three nanoparticle types (BMs; BMs-PLL and BMs-PLL-AmB) and magnetic hyperthermia was evaluated against L. amazonensis PH8 promastigotes. MTT assays were conducted (Fig. 2a), and TEM micrographs were taken (Fig. 3) to assess promastigote viability after AMF exposure and to analyze cell morphology before and after magnetic hyperthermia treatment. Notably, AMF application significantly decreased the cell viability of L. amazonensis promastigotes when treated with BMs-PLL-AmB. The concentration of BMs-PLL-AmB, which previously (without AMF application) resulted in a 50% reduction in cell viability (IC₅₀), effectively reduced the viability of the parasites to 11.2 ± 8.6% after AMF exposure (Fig. 2). The AMF application have also enhanced ½ IC₅₀ and ¼ IC₅₀ of BMs-PLL-AmB activities. The former reduced the promastigote viability to 42.45 ± 9.0%, similar to the IC₅₀ activity without AMF exposure, with the latter presenting viability of 62.79 ± 3.2%. Previously, the ¼ IC₅₀ presented 89.02 ± 10.4% viability without magnetic hyperthermia. These findings indicate that AMF application enhances the anti-parasitic activity of the nanoformulation, improving its effectiveness against the parasite. In contrast, no significant reduction in cell viability was observed when BMs and BMs-PLL treated promastigotes were subjected to the AMF at the equivalent concentration of the BMs-PLL-AmB’s IC₅₀ (3.13 µg/mL).

Fig. 3.

Transmission electron microscopy of L. amazonensis before and after treatment with magnetic hyperthermia. Promastigotes were treated with BMs, BMs-PLL, and BMs-PLL-AmB and submitted or not to AMF of 200 Oe, and 225 kHz for 30 min. Untreated cells were used as a control. Black arrowheads = membrane alterations; White arrows = Nucleus; Red arrowheads = electron-dense inclusions; Red asterisk = cytoplasm loss. Scale bars = 1 µm.

Furthermore, morphology changes are visible when cells are treated with ½ IC₅₀ and IC₅₀ of BMs-PLL-AmB even without AMF application (Fig. 3, note the third and fourth rows in the “Without AMF” columns). Cells present alterations in the cell membrane morphology (black arrowheads) and in the nucleus at these concentrations (white arrows). In addition, it was possible to observe the formation of electron-dense structures (red arrowheads) in the cytoplasm of all cells treated with BMs-PLL-AmB, which are probably lipidic inclusions, especially in the IC₅₀-treated cells (Fig. 3). BMs and BMs-PLL did not present robust cell damage in L. amazonensis promastigotes (Fig. 3, fifth and sixth rows).

Notably, there were expressive morphology changes in promastigotes treated with IC₅₀ and ½ IC₅₀ BMs-PLL-AmB when exposed to AMF (Fig. 3, note the third and fourth rows in the “With AMF” columns). At ½ IC₅₀, cells presented diverse inclusions and vesicles, alterations in organelles, and cytoplasm extravasation. This finding is consistent with the cell viability decreasing when exposed to AMF (Fig. 2b). Furthermore, cells treated with IC₅₀ were sorely altered in morphology, presenting cytoplasm loss (note the red asterisks in the fourth row, third and fourth columns). Another observation in BMs-PLL-AmB treated cells was the presence of the nanoparticles around the cells (Fig. 4), and the nanoformulation seemed to stay intact after the treatment, even presenting structures that were similar to the membrane surrounding the nanocrystals (Fig. 5).

Fig. 4.

Transmission electron microscopy of L. amazonensis promastigotes treated with BMs-PLL-AmB and magnetic hyperthermia. (A) Cells treated with IC₅₀ of BMs-PLL-AmB, (B) inset of (A). (C) Cells treated with ¼ IC₅₀ of BMs-PLL-AmB, (D) inset of (C).

Fig. 5.

BMs-PLL-AmB after magnetic hyperthermia in L. amazonensis PH8. (A) and (B), after treatment with IC₅₀ of BMs-PLL-AmB. (C), after treatment with ¼ IC₅₀ of the same preparation. Arrowheads indicate a structure similar to the magnetosome’s membrane.

Concentrations ranging from 0.40 to 25 µg/mL of nanoparticles were used to evaluate cytotoxicity with both lower and higher dosages compared to the calculated IC₅₀ for L. amazonensis. Remarkably, fibroblasts and keratinocytes exhibited robust cell viability, remaining above 80% for all tested concentrations of BMs, BMs-PLL, and BMs-PLL-AmB during the 24–72 h of treatment (Table 1 and Figure S2). Additionally, the impact of the nanoparticles on macrophages was assessed. These cells are involved in the Leishmania life cycle as they participate in the phagocytosis and intracellular survival of the parasite. At 72 h of incubation with the nanoparticles, macrophage viability was slightly affected, remaining above 60% (Table 1), which is not considered highly cytotoxic. However, some concentrations presented statically significant differences when compared to the control of untreated cells (p < 0.05 to p < 0.001) (Figure S2). In 72 h, the negative effect on the number of cells is also observed in control cells since the availability of nutrients and accumulation of cellular residues in the culture medium begin to decrease and increase, respectively, which is expected in cell culture in closed systems.

Table 1.

Cell viability of mammalian cells lines treated with 3.13 µg/ml of BMs-PLL-AmB, the calculated IC50 for L. amazonensis. Data is represented as mean ± SEM, all values were non-significant compared to the untreated cells control.

	HaCaT	hFb	J774.16
24 h	104.24 ± 23.78%	114.96 ± 8.17%	92.59 ± 5.46%
48 h	116.97 ± 3.82%	110.20 ± 15.54%	111.89% ± 4.32%
72 h	90.99 ± 7.65%	103.25 ± 6.37%	63.84 ± 5.6%

In summary, the results of the MTS assay indicated that none of the tested nanoparticles displayed cytotoxic effects on the tested mammalian cells within the evaluated time frame. These findings highlight the in vitro biocompatibility of the nanoformulations with these mammalian cell types, suggesting their potential as biocompatible therapeutic agents for Leishmania amazonensis treatment.

The effects of the three nanoformulations on cell membrane integrity were analyzed using the LDH assay, which quantifies LDH activity in the cell culture supernatant. Two types of control were used: untreated cells and lysed cells, with the latter considered 100% detection of LDH. HaCaT, hFB, and J774.16 cells exposed to nanoparticles for up to 72 h did not demonstrate high levels of LDH enzyme release in the supernatant (Table 2 and Figure S3). None of the tested concentrations showed a significant difference for the control of untreated cells (p > 0.9) This result suggests that the observed LDH release is within the normal range associated with cell death and does not indicate a cytotoxic effect. All concentrations did show significant differences when compared to the control of lysed cells (p ≤ 0.0001).

Table 2.

LDH assay of mammalian cells treated with 3.13 µg/ml of BMs-PLL-AmB, the calculated IC50 for L. amazonensis. Data is represented as mean ± SEM. All values were considered statistically significant (P ≤ 0.0001) when compared to the control of cells lysed with TX-100, considered the 100% release of LDH.

	HaCaT	hFb	J774.16	Control cells
24 h	2.96 ± 2.93%	2.59 ± 0.12%	4.60 ± 0.14%	3.39 ± 3.30%
48 h	1.98 ± 1.73%	1.73 ± 1.07%	3.20 ± 0.24%	6.02 ± 0.68%
72 h	0.25 ± 0.15%	0.50 ± 0.50%	3.79 ± 3.70%	5.17 ± 3.32%

The nitric oxide (NO) dosage test was performed after 24 h of treatment of J774.16 cells (Fig. 6) to assess the effects of BMs, BMs-PLL, and BMs-PLL-AmB on NO production by macrophages. Notably, none of the treatments induced a significant upregulation of NO production by macrophages, as evidenced by the concentration of NO detected in the supernatant of treated cells. NO-induced production concentrations of the nanoformulation were comparable to that found in the control of untreated cells (0.08 ± 0.11 µM of NO). The treatments did not present a statistically significant difference compared to the control of untreated cells (p > 0.9). The positive control of NO production, consisting of cells treated with 1 μg/mL of LPS, demonstrated expressive induction of NO production (2.62 ± 0.05 µM). Macrophages treated with BMs, BMs-PLL, and BMs-PLL-AmB showed a statistically significant difference compared to the LPS-treated cells control (p < 0.0001).

Fig. 6.

Nitric oxide production dosage by J774.16 cells treated with BMs and functionalized BMs. J774.16 cells were treated with BMs, BMs-PLL, BMs-PLL-AmB at various concentrations for 24 h. Control cells (CT) were grown in a medium without treatment. Positive control cells were treated with LPS (1 μg/mL). NO production was measured in the cells’ supernatant. Results are the mean ± SEM of three independent experiments. There was a statistically significant difference by the ANOVA test between the treated cells and the positive control (LPS) (***p < 0.001).

The lack of difference in the control of untreated cells and the significant difference in the positive control of NO production support the observation that none of the treatments led to a robust NO response by the J774.16 macrophages. Excessive induction of NO production can lead to oxidative stress and tissue damage. Moreover, the unaltered NO levels in response to these nanoformulations highlight their suitability for potential nanomedicine applications in combating cutaneous leishmaniasis while avoiding strong inflammatory responses and cytotoxicity.

BMs, BMs-PLL, and BMs-PLL-AmB did not exhibit hemolytic activity when tested from 2 to 250 µg/mL (Fig. 7). These concentrations were approximately 80 times higher than the calculated IC₅₀ for BMs-PLL-AmB (3.13 µg/mL) against the target Leishmania strain. None of the tested concentrations of nanoformulation presented statistical differences from the untreated cells control (p > 0.2), apart from BMs at 250 µg/mL (p = 0.01). In contrast, all treated and untreated cells showed statistical differences compared to the control of lysed cells (p < 0.0001).

Fig. 7.

Hemolytic activity assessment of BMs, BMs-PLL, and BMs-PLL-AmB. The nanoformulations exhibited statistically significant differences from the positive hemolysis control (100% hemolysis), indicating their reduced hemolytic activity. Notably, only the nanoparticle BMs at its highest tested concentration showed a statistical difference compared to the control of untreated cells (CT). This observation highlights the specificity of the nanoparticle’s response under these conditions, suggesting their potential for biomedical applications with minimal impact on cellular integrity. Results are the mean ± SEM of three independent experiments (*p < 0.05, (****p < 0.0001).

The absence of hemolytic effects across this broad concentration range indicates the nanoparticles’ biocompatibility with human erythrocytes. Furthermore, the observed absence of hemolysis at such high concentrations highlights these nanoparticles’ potential biosafety and non-toxicity when exposed to blood components, making them suitable for prospects of intravenous administration, especially when considering their demonstrated leishmanicidal activity against L. amazonensis.

BMs have great potential in drug delivery systems due to their unique features, intrinsic magnetic properties, and reported biocompatibility⁵¹. As the target disease is a cutaneous disease, we aimed for cells that are close to the infection site of CL, such as fibroblasts, keratinocytes, and macrophages since they are targeted cells by Leishmania species. Different tests were conducted to evaluate different types of toxicity in these three cell lines. MTS assay was carried out to evaluate mitochondrial activity, thus indirectly measuring apoptosis. BMs, BMs-PLL, and BMs-PLL-AmB were non-toxic to HaCaT, hFB, and J774.16 cells up to 25 µg/mL until 72 h of incubation. BMs’ biocompatibility is well reported in diverse mammalian cell lines, and although their sensibility to BMs changes, overall, the results demonstrate its safety. It has been reported that DNA, cell size, and membrane integrity of H22, HL60, and EMT-6 cell lines were not affected by 9 µg/mL BMs of Ms. gryphiswaldense MRS-1⁴¹. The IC₅₀ of BMs were 25.41 ± 3.83 µg/mL and 4.73 ± 0.78 µg/mL for H22 and HL60 cells, respectively. EMT-6 cells were less sensitive to magnetosomes, as they could not estimate its IC₅₀⁴¹. HT-29 cells were tested with up to 1 mg/mL of BMs, and cells remained viable for 24 h⁵². MSR-1 BMs were also compatible with BeWo, FaDu, HCC78, and hPC-PL cells up to 194.4 µg Fe/mL in 24 h⁵³. Additionally, LDH enzyme is only released from cells when there is membrane damage, its detection indirectly measures necrosis. Membrane integrity of ARPE-19 cells was not altered as well when treated with MSR-1 BMs up to 72 h³⁸, likewise our findings.

However, most studies were made with magnetosomes of Magnetospirillum species and not with magnetosomes produced by Mv. blakemorei strain MV-1 ^T, which are less studied for biomedical applications. So far, internalization and toxicity of Mv. blakemorei strain MV-1 ^T magnetosomes were assessed in HeLa cells³⁷. It was observed that the nanoparticles persisted up to 120 h within the cells and no cytotoxicity or morphological alterations were observed in this period when cells were treated with 740 µg/mL³⁷.

Magnetotactic bacteria are Gram-negative microorganisms, so the possible presence of LPS in BM nanoformulations raises concerns about their pyrogenic potential. Thus, assessing NO production in J774.16 macrophages treated with these nanoformulations serves as an indicator of LPS or other endotoxins presence⁵⁴. Our investigation demonstrated that BMs, BMs-PLL, and BMs-PLL-AmB, even at a maximum concentration of 25 µg/mL, did not induce NO production. These results corroborate other biocompatibility assessments presented in this study and others³⁷, including cell viability and membrane integrity evaluations, confirming the nanoparticles’ overall biocompatible profile.

Revathy and colleagues³⁹ conducted a study to evaluate the hemolytic activity of the BMs produced by Ms. gryphiswaldense MSR-1, revealing maximum hemolysis of 3.5% at 150 µg/mL. Our investigation demonstrated that BMs, BMs-PLL, and BMs-PLL-AmB showed no hemolytic activity even at higher concentrations, specifically up to 250 µg/mL. The absence of hemolytic effects observed in our study supports the biocompatibility and safety profile of the functionalized BMs, suggesting their potential suitability for biomedical applications. Especially when there was no hemolysis even at concentrations almost 80 times higher (250 µg/mL) than the IC₅₀ for BMs-PLL-AmB (3.13 µg/mL), corroborating the safety potential of this new anti-Leishmania nanoparticle. The difference in the hemolytic activity of BMs between our study and Revathy and colleagues³⁹ could be related to the BMs shape (prismatic or cuboctahedral) or extraction and purification process, that included an SDS washing purification step.

An interesting aspect we observed is that the efficacy of the AmB loaded into the BMs-PLL-AmB (IC₅₀ = 0.85 µM of AmB in 3.13 µg/mL of Fe₃O₄) nanoformulation is comparatively lower than that of free AmB (IC₅₀ = 0.11 µM). Our group’s previous description of BMs-PLL-AmB showed that the AmB release of the nanoparticles increased four times with the application of an AMF⁴⁷. In standard conditions (37 °C), 15.0 ± 1.2% of drug release was observed in this nanoformulation, while with AMF application, this percentual increased to 53.8 ± 6.2%⁶⁶. The release occurs probably due to the residual presence of AmB still anchored in the BMs’ membrane. The enhanced controlled release was attributed to the nanoparticle’s rotation under an AMF (e.g., Brown relaxation) instead of the hyperthermia effect⁴⁷. Furthermore, as experiments with promastigotes of Leishmania are conducted at 28 °C, AmB release may be lower at this temperature than the previous tests conducted at 37 °C⁴⁷. Therefore, the loaded AmB might not be fully released in the medium, not achieving the same activity level as the free AmB, explaining the lower activity of BMs-PLL-AmB.

Nevertheless, our primary objective was combining nanoparticle treatment with magnetic hyperthermia, creating a drug delivery system with controlled release of AmB at the infection site, and evaluating the preliminary cytotoxicity and parasitic activity. A substantial increase in temperature during experiments was not observed. However, AMF application indeed enhanced the activity of BMs-PLL-AmB nanoformulation, especially when applied to the IC₅₀ and ½ IC₅₀. In only one application of AMF, the IC₅₀ reduced the viability to 11.2 ± 8.6%, while the ½ IC₅₀ reduced the viability to half. So far, the use of AMF or magnetic hyperthermia for treating Leishmania parasites is not extensive in the literature. Iron oxide MNPs were used to kill axenic amastigotes of Leishmania mexicana in vivo⁵⁵. This study has shown that 200 µg/mL of MNPs under an AMF of 452 kHz frequency and 30 mT intensity for 40 min reduced the viability of axenic amastigotes by 70%. Similar to our results (Fig. 3), the authors observed ultrastructure and morphology alterations in the cells⁵⁵. The concentration of synthetic MNPs used by Berry et al. (200 µg/mL) was approximately 64 times greater than the highest concentration used in this study (3.13 µg/mL). In addition, the exposure time, frequency, and intensity of the applied AMF in our study were also lower. Here, we applied an AMF of 200 kHz and 20 mT for 30 min, in contrast to 452 kHz, 30 mT for 40 min of the mentioned study. Therefore, with lower AMF and minor concentration, we found similar results with BMs-PLL-AmB, reducing about 90% viability of cells with only one magnetic hyperthermia cycle at 3.13 µg/mL. However, it is worth mentioning that the studies were conducted with different Leishmania species and different forms of the parasite (e.g., promastigotes and axenic amastigotes). Thus, further studies are necessary to compare the nanoparticles’ activities properly, particularly studies in animal models, in which the hyperthermia effect could be more accurately evaluated.

Other works focused on applying MNPs to treat Leishmania but did not use magnetic hyperthermia. Fe₃O₄ MNPs functionalized with piroctane olamine (Fe₃O₄@PO NPs) or without functionalization (Fe₃O₄ NPs) were tested against amastigotes of L. major presenting an IC₅₀ of 31.3 ± 2.26 µg/mL and 62.3 ± 2.15 µg/mL⁵⁶. It was also observed that the lesion size in infected mice decreased by 8.1 and 9 mm when treated with Fe₃O₄@PO NPs 1 or 2 mg/mL, respectively. Lesions were reduced by 4.8 mm and 6.1 mm when applying 1 or 2 mg/mL of Fe₃O₄ NPs while increasing by 8.2 mm in untreated animals⁵⁶. Paramomycin-loaded cobalt ferrite (CoFe₂O₄) core–shell nanospheres coated by PEG-PLGA (NP@PEG-PLGA/PM) presented activity in concentrations from 5 to 60 µg/mL, reducing over 50% of promastigotes viability after 72h⁵⁷. However, none of the works used magnetic hyperthermia to enhance/control the drug release of their nanoparticle’s activity. Nevertheless, our paper presents the potential of AmB-loaded BMs in inhibiting L. amazonensis, especially when associated with AMF exposure, using smaller quantities of nanoparticles. AMF’s ability to enhance the therapeutic efficacy of the nanoformulation further suggests that this combined approach could lead to more efficient and targeted treatments for CL.

As biological-origin magnetic nanoparticles produced by magnetotactic bacteria, BMs offer a notable advantage over their synthetic counterparts regarding sustainability and green processes. The inherent biological origin of BMs aligns with green nanotechnology principles, as their production occurs via biologically controlled processes without the need for aggressive chemicals⁵⁸. The eco-friendly nature of these magnetic structures significantly reduces the environmental footprint associated with BMs manufacturing, emphasizing their potential as sustainable alternatives to synthetic MNPs^59,60.

The SDG for eliminating the epidemics of NTDs is a prominent target of the 2030 Agenda⁴. Integrating green-synthesized magnetic nanoparticles, such as magnetosomes, in treating CL presents a promising stride toward this aim. These nanoparticles can offer targeted and efficient treatment modalities, minimizing adverse environmental⁶¹ and human health effects⁵¹, aligning with eco-friendly considerations by reducing the impacts on biological systems and the environment. By offering cost-effective and innovative treatments for NTDs, particularly those affecting marginalized populations, nanotechnology-driven solutions can significantly improve access to healthcare, thereby reducing disparities in disease management and enhancing social equity.

Conclusion

In conclusion, this study demonstrates the significant potential of magnetosomes to develop innovative and effective therapies for CL. By coupling magnetosomes with AmB and integrating AMF application, we have achieved remarkable results in Leishmania amazonensis promastigotes. Furthermore, adding AMF exposure as a combined therapy significantly enhanced the leishmanicidal effect, based on the improved antiparasitic effect of BMs-PLL-AmB. However, the most significant limitation of this work was testing the nanoparticles only in the promastigote forms of L. amazonensis, which are not the intracellular replicative form found in humans. Therefore, the results obtained here are promising but require further investigation.

Additionally, magnetosomes from Magnetovibrio blakemorei strain MV-1^T are rarely studied for biomedical applications in literature. Previously, these magnetosomes had only been tested in HeLa cells³⁷. Thus, the present work provides further information on the biocompatibility and safety assessment of MV-1 magnetosomes with three additional mammalian cell lines (HaCaT, hFB, and J774.16). Overall, the main goal of this work was to validate the safety of the nanoformulation in in vitro models and evaluate the potential anti-Leishmania activity by preliminary tests in promastigotes. Nonetheless, in vitro tests with infected macrophages and, mostly, in vivo tests are necessary to further investigate the biocompatibility and activity of the nanoformulations against the parasite, validating the results observed in this work and confirming its potential as a therapeutic agent.

Methods

Cells and cell culture

L. amazonensis IFLA/BR/1967/PH8 was maintained in PBHIL medium (11.1 mM Glucose, 6.85 mM NaCl, 53.7 mM KCl, 95.9 mM NaH₂PO₄, NaOH 75 mM, 2 g/L Peptone, 2 g/L Brain–Heart infusion, 0.25 g/L Liver broth, 10 µg/ml Hemin, pH 7.2)⁶² supplemented with 10% (v/v) inactivated Fetal Bovine Serum (FBS) (Thermo Fisher Scientific) and incubated at 28 °C. Human keratinocyte cell line (HaCaT), human fibroblast (hFB), and mouse macrophage cell lines (J774.16) were kindly donated by Prof. Dr. Leonardo Nimritcher (Instituto de Microbiologia Paulo de Góes, UFRJ, Brazil). The cells were maintained in Dulbecco’s Modified Eagle medium (DMEM) High glucose (Sigma-Aldrich) supplemented with 10% (v/v) FBS (Thermo Fisher Scientific), and 1% (v/v) PenStrep (Gibco) at 37 °C and 5% CO₂.

Cultivation of magnetotactic bacteria and magnetosome purification

Magnetovibrio blakemorei strain MV-1 ^T strain was cultivated according to a previously published method^63,64. Cells were harvested and magnetosomes were purified as described before⁴⁷.

Functionalized BM production

Purified magnetosomes (BMs), poly-L-lysine (PLL) coated magnetosomes (BMs-PLL), and PLL-coated magnetosomes functionalized with AmB (BMs-PLL-AmB) were produced as described before by Correa and colleagues⁴⁷. Purified BMs, BMs-PLL and BMs-PLL-AmB were frozen overnight at − 20 °C, lyophilized for 2 h, and then irradiated by Co₆₀ (15 kGy) for sterilization⁶⁵. All nanoparticles were tested regarding LPS presence by the LAL endotoxin detection kit (Nova Biotecnologia LTDA, Brazil), and were negative to the presence of this endotoxin.

Cell treatment for cytotoxicity assays

HaCaT, hFB, and J774.16 cells were seeded at a density of 1.25 × 10⁵ cells/mL, 3.75 × 10⁵ cells/mL, and 10⁶ cells/mL, respectively, into 96-well microtiter plates for 24 h at 37 °C and 5% CO₂. The medium was removed, and cells were treated with medium containing BMs, BMs-PLL, or BMs-PLL-AmB at concentrations ranging from 0.4 to 25 μg/mL of nanoparticles for 24 h, 48 h, and 72 h. A non-treated cell control was performed in all cytotoxicity assays.

Cell viability determination

Cell viability was measured after incubation with the produced nanoparticles by CellTiter® 96 AQueous One Solution Reagent (Promega) assay according to the manufacturer’s instructions. Briefly, after 72 h of treatment, 10% of the MTS reagent was added and incubated at 37 °C for 3 h until reading at 490 nm in an EL808 microplate reader (BioTek). Cells treated with 1% formaldehyde (v/v) were used as the positive death control.

Lactate dehydrogenase assay

Cell membrane integrity was evaluated by lactate dehydrogenase (LDH) leakage assay. After cell treatment with nanoparticles for 24, 48, and 72 h, 80 μL of cell supernatant was removed and centrifuged. The supernatant was transferred into a new 96-well plate, and 120 μL a solution containing 0.3 mM B-nicotinamide adenine dinucleotide reduced form (NADH) (Sigma-Aldrich), and 4.7 mM pyruvic acid (Sigma-Aldrich) in phosphate-buffered saline (PBS) 1 × buffer was added. The plate was immediately read at 340 nm⁶⁶ in a 2 h kinetic performed at an EL808 microplate reader (BioTek) to determine absorbance decrease in the reaction medium. Cells disrupted with 10% Triton X-100 (TX-100) were used as the LDH maximum leakage control.

Determination of NO production by macrophages

J774.16 macrophages were also tested regarding nitric oxide (NO) production. After J774.16 cells were treated with BMs, BMs-PLL, and BMs-PLL-AmB for 24 h, the cells’ supernatants were removed and transferred into a new 96-well plate. NO production was measured in the supernatant according to the Griess method⁶⁷. Cells treated with 1 μg/mL of LPS were the positive control for NO production.

Hemolysis detection

Human blood was collected, and erythrocytes were isolated via centrifugation. Subsequently, the erythrocytes were washed and resuspended in 1 × PBS to achieve a 4% (v/v) erythrocyte suspension. BMs, BMs-PLL, and BMs-PLL-AmB were resuspended in 1 × PBS (pH = 7.4) and added to the erythrocyte suspension at increasing concentrations, ranging from 2 µg/mL to 250 µg/mL. The suspensions were then incubated at 37 °C for 1 h for nanoparticle interactions with erythrocytes. Then, the suspensions were centrifuged, and the supernatants were collected to measure the released hemoglobin. The absorbance of the supernatants was spectrophotometrically assessed at a wavelength of 540 nm. As a positive control, erythrocytes were treated with deionized water to induce complete hemolysis. Control samples containing erythrocytes suspended in PBS without nanoparticles were included as the negative control. The hemolytic activity of each nanoparticle formulation was calculated as a percentage of hemolysis relative to the positive control, considered as 100% hemolysis.

Antiparasitic assay

MTT assay evaluated L. amazonensis viability after treatment with the nanoformulations. L. amazonensis promastigotes were resuspended to a final concentration of 10⁵ cells per well. BMs, BMs-PLL, and BMs-PLL-AmB were added to the culture in 96-well plates at final concentrations ranging from 0.00010 to 100 μg/mL, and AmB was added at final concentrations ranging from 7.87·10^–11 to 432.86 µM for 72 h at 28 °C (final working volume of 100 μL). MTT reagent (5 mg/ml) was added, and the plate was incubated at 28 °C for 4 h in the absence of light. 100 μL of DMSO were added, and the plate read at 570 nm in a SpectraMax 190 microplate reader (Molecular Devices).

Magnetic hyperthermia application

L. amazonensis promastigotes cell suspensions (10⁶ cells/mL) in the presence or not of BMs, BMs-PLL, and BMs-PLL-AmB were submitted to an alternating magnetic field of 200 Oe (20 mT) with 225 kHz of frequency for 30 min using an EasyHeat magnetic hyperthermia device (Ambrell Induction Heating Solutions). AMF was applied about 1 h after incubation of L. amazonensis with nanoformulations. After AMF exposure, promastigotes were incubated for 72 h at 28 °C, and MTT assay was performed as described in the antiparasitic assay topic.

Transmission electron microscopy (TEM)

Promastigotes non-treated and treated with BMs, BMs-PLL, and BMs-PLL-AmB with or without magnetic hyperthermia application were harvested after 72 h of incubation through centrifugation at 2000 rpm for 10 min, 4 °C. After, cells were washed in PBS buffer, and fixed overnight at 4 °C using 2.5% glutaraldehyde and 0.1 M sodium cacodylate buffer. Cells were washed with 1 M sodium cacodylate buffer with 3.7% sucrose and 5 mM CaCl₂. The samples were post-fixed for 1 h at room temperature with osmium tetroxide 1% with ferricyanide with 0.1 M sodium cacodylate buffer with 3.7% sucrose and 5 mM CaCl₂. Then, post-fixed samples were washed with sodium 0.1 M cacodylate buffer. Serial dehydration with acetone was carried out with 30–100% acetone, each concentration for 15 min, repeating the last 3 times. Afterward, samples were infiltrated on the epoxy resin (Epon) with acetone, gradually inverting the proportion of solvent: resin (2:1; 1:1; 1:2) each step for 24 h at room temperature. Finally, samples were infiltrated with only resin 2 times for 24 h at 4 °C. Infiltrated samples were transferred to silicon molds and included in Epon resin for 72 h at 68 °C. Ultrathin sections were made by ultramicrotomy; they were obtained with Leica EM U6 (Leica Microsystems, Bannockburn, IL, USA). The ultrathin sections with a thickness of 60 nm were collected with formvar/carbon-coated 300 mesh copper grids (Electron Microscopy Sciences, Hatfield, USA) and stained for TEM with uranyl acetate and lead citrate.

All nanoformulations were verified by TEM to analyze the magnetosomes and functionalized magnetosome’s membrane and crystal integrity by depositing the suspension on 300 mesh copper grids coated with Formvar/carbon. The grids were air-dried, and membrane integrity and thickening after functionalization were analyzed by measuring with the software ImageJ⁶⁸. All samples were analyzed in a Fei Morgagni transmission electron microscope (FEI Company, Hillsboro, OR) with an accelerating voltage of 80 kV.

Statistical analysis

Statistical analysis was evaluated with two-way ANOVA and Tukey multiple comparison test using Prism 6 software (GraphPad Software Inc.); p ≤ 0.05 was considered statistically significant. Data were shown as mean ± standard error (SEM) of at least 3 independent experiments.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

M.V., D.N., and L.S. conducted the experiments. M.V. wrote the first draft of the manuscript, analyzed and interpreted the data, and produced the figures. F.G. provided the facilities for magnetic hyperthermia experiments. M.V., D.N., L.S., F.G. A.S., and F.A. conceptualized and designed the study. M.V., D.N., and F.A. revised the manuscript. F.A. supervised, administered the project, and acquired funding. All authors read and approved the final manuscript.

Funding

This work was supported in part by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), grant reference number 001. The Brazilian agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) provided financial support.

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Declarations

Competing interests

The authors declare that they have no competing interests.

Ethics approval and consent to participate

This study was reviewed and approved by the Research Ethics Committee of the Clementino Fraga Filho University Hospital, from the Federal University of Rio de Janeiro, Rio de Janeiro, Brazil (process number 6.094.729). Written informed consent was obtained from the subjects prior to blood collection. The consent process included a detailed explanation of the study objectives, the potential risks, the assurance of confidentiality, and the voluntary nature of participation. All experiments were performed in accordance with relevant guidelines and regulations provided by the Research Ethics Committee, which follows the Declaration of Helsinki.

Consent for publication

Not applicable.