Gold-Conjugated Curcumin as a Novel Therapeutic Agent against Brain-Eating Amoebae.

Balamuthia mandrillaris and Naegleria fowleri are free-living amoebae that cause infection of the central nervous system, granulomatous amoebic encephalitis (GAE) and primary amoebic meningoencephalitis (PAM), respectively. The fact that mortality rates for cases of GAE and PAM are more than 95% indicates the need for new therapeutic agents against those amoebae. Considering that curcumin exhibits a wide range of biological properties and has shown efficacy against Acanthamoeba castellanii, we evaluated the amoebicidal properties of curcumin against N. fowleri and B. mandrillaris. Curcumin showed significant amoebicidal activities with an AC50 of 172 and 74 μM against B. mandrillaris and N. fowleri, respectively. Moreover, these compounds were also conjugated with gold nanoparticles to further increase their amoebicidal activities. After conjugation with gold nanoparticles, amoebicidal activities of the drugs were increased by up to 56 and 37% against B. mandrillaris and N. fowleri, respectively. These findings are remarkable and suggest that clinically available curcumin and our gold-conjugated curcumin nanoparticles hold promise in the improved treatment of fatal infections caused by brain-eating amoebae and should serve as a model in the rationale development of therapeutic interventions against other infections.

Introduction

Pathogenic free-living amoebae, including Naegleria fowleri and Balamuthia mandrillaris, are opportunistic protists that cause infection of the central nervous system (CNS).[1−3] Infections with free-living amoebae almost always lead to death, indicating the lack of effective treatments against those virulent pathogens.[4−6]N. fowleri is known to infect the CNS of young children and adults, causing primary amoebic meningoencephalitis (PAM), leading to a rapid onset of the disease and death within days.[7] Amphotericin B, used in combination with rifampin and azole compounds, is the main treatment option but results in severe side effects, including nephrotoxicity.[8−10] Like N. fowleri, B. mandrillaris also targets the CNS, causing the fatal disease termed granulomatous amoebic encephalitis (GAE).[11] The mortality rate of patients suffering from GAE due to B. mandrillaris infections is very high, resulting in death in over 95% of the reported cases.[11−14] Patients suffering from GAE caused by B. mandrillaris are usually treated with multidrug regimens, usually including miltefosine and pentamidine isethionate.[13,14]

Curcumin is a hydrophobic polyphenol derived from the rhizome of the herb Curcuma longa. Curcumin, the active component of turmeric, has been traditionally used as an inflammatory agent in the treatment of several diseases.[15] Human models and animal studies have demonstrated the extreme safety of curcumin.[16] Curcumin expressed antioxidant activities in the rat brain as it inhibits lipid peroxidation at low concentrations.[17] Micromolar concentrations of curcumin also resulted in the inhibition of superoxides and hydroxyl radicals, further confirming its antioxidant activities.[18] Anti-inflammatory activities of curcumin in both chronic and acute models of inflammation have been reported.[19] Antiviral activity of curcumin has been demonstrated in vivo in humans.[20] Curcumin also possess antibacterial activities as demonstrated in a study where curcumin inhibited the growth of Helicobacter pylori.[21] Antifungal and antiparasitic activities of curcumin have also been demonstrated, such as its inhibitory effects against Plasmodium falciparum at a low IC50 of 5 μM and its activities against Trichomonas vaginalis, Giardia lambelia, Toxoplasma gondii, and Cryptosporidium parvum.(22−24) Anticancer activities of curcumin at low micromolar concentrations have also been reported.[25] Also clearly established are the myocardial infarction protective, hepatoprotective, nephroprotective, thrombosis suppressing, and antirheumatic effects of curcumin.[16]

Curcumin has been shown to possess significant amoebicidal activities against Acanthamoeba castellanii.[26] Curcumin can also affect the cysts of A. castellanii.[27] However, the amoebicidal activities of curcumin against N. fowleri and B. mandrillaris are yet to be evaluated. We hypothesized that curcumin possesses antiamoebic activities against N. fowleri and B. mandrillaris. Therefore, we evaluated the amoebicidal properties of curcumin against N. fowleri and B. mandrillaris. We also conjugated curcumin with gold nanoparticles (AuNPs) to further enhance its amoebicidal properties.

Results and Discussion

Pathogenic free-living amoebae, including N. fowleri and B. mandrillaris, are opportunistic protists that cause infection of the CNS.[1−3] The lack of effective treatments against those virulent pathogens is clearly indicated by the fact that infections with free-living amoebae almost always leads to death.[4−6]N. fowleri is known to infect the CNS, causing PAM, while B. mandrillaris also targets the CNS, causing GAE.[7,11]

Curcumin expressed antioxidant activities by inhibiting lipid peroxidation, superoxides, and hydroxyl radicals.[17,18] Curcumin is known to exhibit antimicrobial, anti-inflammatory, and anticarcinogenic activities as well as therapeutic efficacy against various cardiovascular diseases, neurological disease, diabetes, and arthritis.[16] Also clearly established are the myocardial infarction protective, hepatoprotective, nephroprotective, thrombosis suppressing, and antirheumatic effects of curcumin.[16] The ability of curcumin to express neuroprotective abilities has been reported.[28] This would indicate the ability of curcumin to penetrate the blood–brain barrier, and hence, it might be a treatment option for brain infections. Wound healing and tissue repair are intricate processes, involving inflammation, tissue remodeling, and granulation, that are enhanced by curcumin.[29] This might help the patient in recovery from infections. Curcumin has been shown to possess significant amoebicidal activities against A. castellanii trophozoites and cysts.[26,27] Therefore, here, we evaluated the amoebicidal properties of curcumin against N. fowleri and B. mandrillaris.

Curcumin was Successfully Conjugated with AuNPs

The size of curcumin after conjugation with AuNPs was determined using dynamic light scattering. The particle size analyzer revealed that the particles formed were in the nanometer range with an average size of 53 nm (Figure ). This confirms that the curcumin conjugated with AuNPs was successfully synthesized.

Figure 1

Size distribution of curcumin–AuNPs. The figure shows the sizes of curcumin after conjugation with AuNPs determined by dynamic light scattering.

Curcumin Exhibits No Cytotoxicity toward HaCaT Cells at Concentrations below 10 μM

At 10 μM and lower, curcumin did not exhibit cytotoxic activities against human cells. At 12.5 μM, 2% cytotoxicity was recorded, while 6 and 8% were observed for 25 and 50 μM of curcumin, respectively (Figure ). While exhibiting 11% cytotoxicity at 100 μM, curcumin exhibited 20% cytotoxicity at 200 μM. Solvent controls did not exhibit cytotoxic activities against human cells. Gold nanoparticles at both 5 and 10 μM did not exhibit cytotoxic activities against human cells. Curcumin conjugated with AuNPs at both 5 and 10 μM did not exhibit cytotoxic activities against human cells (Figure ). Curcumin is known to be safe for humans, and it has been shown that even at doses of 8000 mg/day, curcumin did not cause any noticeable toxicities, except diarrhea and mild nausea in some cases.[30]

Figure 2

(i) Curcumin did not express cytotoxic activities at concentrations lower than 10 μM. The cytotoxic activities of curcumin against HaCaT cells were determined at a range of concentrations. The cytotoxic activities of the solvent control (ethanol), AuNPs, and curcumin–AuNPs against HaCaT cells are also shown. Briefly, curcumin was incubated with HaCaT cells for 24 h at 37 °C in a 5% CO2 incubator. The results showed that curcumin has limited host cell damage. The data are presented as the mean ± standard error of two experiments conducted in duplicates. (ii) Representative microscopic images of HaCaT cells incubated with and without curcumin. (A) Untreated HaCaT cells. (B) HaCaT cells treated with Triton X-100 (positive control). (C) HaCaT cells treated with 200 μM curcumin. (D) HaCaT cells treated with 10 μM curcumin–AuNPs.

Curcumin Expressed Significant Amoebicidal Activity at 50 μM against B. mandrillaris

Amoebicidal activities of curcumin against B. mandrillaris were investigated at a range of concentrations (Figure ). The solvent control expressed 17% activity, while curcumin expressed 19, 28, 34, 37, and 55% amoebicidal activities at 12.5, 25, 50, 100, and 200 μM, respectively (Figure a). As compared to the solvent control, 4% ethanol (M = 17, SE = 4), curcumin exhibited significant amoebicidal activities against B. mandrillaris at 50 (M = 34, SE = 4; t(4) = 2.83, P = 0.05) and 200 μM (M = 55, SE = 6 t(4) = 5.33, P = 0.006). The activity of curcumin against B. mandrillaris is concentration dependent (Figure b). According to the Quest Graph IC50 calculator, the AC50 of curcumin against B. mandrillaris was 172 μM.

Figure 3

Curcumin showed amoebicidal activities against B. mandrillaris. Briefly, B. mandrillaris was incubated with various concentrations of curcumin for 24 h, and viability was determined using trypan blue exclusion assay. The percentage amoebicidal activity was calculated by [((RPMI cell count – cell count for the sample)/RPMI cell count) × 100)]. The results revealed that curcumin caused a reduction in the number of viable B. mandrillaris cells. The results are representative of three independent experiments performed in duplicates. The data are presented as the mean ± standard error (*: P < 0.05, **: P < 0.01, using Student’s t test; two-tailed distribution). (a) Bar chart showing the activity of individual concentrations and controls. (b) Curve showing the concentration-dependent activity of curcumin against B. mandrillaris.

Curcumin Expressed a Significant Amoebicidal Activity at 50 μM against N. fowleri

Amoebicidal activities of curcumin against N. fowleri were investigated at a range of concentrations (Figure ). At 200 μM, curcumin expressed 66% amoebicidal activity against N. fowleri (Figure a). As compared to the solvent control, 4% ethanol (M = 23, SE = 5), 50 (M = 41, SE = 4), 100 (M = 51, SE = 5), and 200 μM (M = 66, SE = 4) of curcumin resulted in significant (t(4) = 2.81, P = 0.05; t(4) = 4.06, P = 0.02; t(4) = 6.56, P = 0.003) amoebicidal activities against N. fowleri. At 6.25, 12.5, and 25 μM, curcumin expressed 22, 30, and 35% amoebicidal activities, respectively, against N. fowleri. The activity of curcumin against N. fowleri is concentration dependent (Figure b). The AC50 of curcumin against N. fowleri was 74 μM, according to the Quest Graph IC50 calculator. At 50 μM, curcumin exhibited potent amoebicidal effects against N. fowleri (Figure c).

Figure 4

Curcumin showed amoebicidal activities against N. fowleri. Briefly, N. fowleri was incubated with various concentrations of curcumin for 24 h, and viability was determined using trypan blue exclusion assay. The percentage amoebicidal activity was calculated by [((RPMI cell count – cell count for the sample)/RPMI cell count) × 100)]. A significant reduction in the number of viable N. fowleri cells was observed for cells treated with curcumin at 50 μM and above. The results are representative of three independent experiments performed in duplicates. The data are presented as the mean ± standard error (*: P < 0.05, **: P < 0.01, ***: P < 0.001 using Student’s t test; two-tailed distribution). (a) Bar chart showing the activity of individual concentrations and controls. (b) Curve showing the concentration-dependent activity of curcumin against N. fowleri. (c) Representative effects of curcumin on N. fowleri.

When tested against another amoeba, Dictyostelium discoideum, it was shown that curcumin acts by inhibiting cell signaling, proliferation, and adhesion while increasing reactive oxygen species (ROS) by inducing the expression of glutathione S-transferase.[31] The increase in ROS induced by curcumin results in both mitochondrial and nuclear DNA damage.[32] Curcumin can also induce deformation due to swelling, morphological changes in cytoplasmic membrane, and cell agglutination, and provoke apoptosis-like changes.[33] Moreover, the ability of curcumin to cause size changes and cell membrane damage, leading to loss of cellular integrity in amoebae, has been reported.[34] Curcumin has also been shown to affect gene transcription and induce apoptosis in cancer cells.[35] Other molecular targets of curcumin include growth factors, transcription factors, protein kinases, and inflammatory cytokines.[36] Hence, curcumin may express amoebicidal activities against B. mandrillaris and N. fowleri using one or several of these mechanisms.

Despite its safety at high concentrations, curcumin has been shown to exhibit poor bioavailability in humans due to rapid metabolism and poor absorption.[16] Nanoparticles can enhance the bioavailability of curcumin, a hydrophobic compound, by overcoming its low aqueous solubility.[16] Hence, we conjugated curcumin with gold nanoparticles and tested its amoebicidal activities against B. mandrillaris and N. fowleri at 5 and 10 μM.

Activity of Curcumin against B. mandrillaris was Significantly Enhanced after Conjugation with AuNPs

Amoebicidal activities of curcumin against B. mandrillaris after conjugation with AuNPs were investigated at 10 and 5 μM (Figure ) since curcumin did not exhibit cytotoxicity against human cells at concentrations below 10 μM. At 10 μM, curcumin–AuNPs expressed 78% amoebicidal activities against B. mandrillaris. The activity of 10 μM curcumin–AuNPs (M = 78, SE = 3) was statistically significant (t(4) = 17, P = 0.00008; t(4) = 19, P = 0.00005; t(4) = 21, P = 0.00003) as compared to 10 μM AuNPs (M = 18, SE = 2), 10 μM curcumin (M = 22, SE = 0.2), and the solvent control (M = 11, SE = 1). For curcumin–AuNPs at 5 μM, 64% amoebicidal activity was observed. The amoebicidal activity of 5 μM curcumin–AuNPs (M = 64, SE = 9) as compared to 5 μM AuNPs (M = 3, SE = 5), 5 μM curcumin (M = 15, SE = 0.2), and the solvent control (M = 0, SE = 10) was statistically significant (t(4) = 6, P = 0.004; t(4) = 5, P = 0.006; t(4) = 5, P = 0.01).

Figure 5

Activity of curcumin against B. mandrillaris was enhanced after conjugation with AuNPs. Briefly, B. mandrillaris was incubated with 5 and 10 μM curcumin and curcumin–AuNPs for 24 h, and viability was determined using trypan blue exclusion assay. The percentage amoebicidal activity was calculated by [((RPMI cell count – cell count for the sample)/RPMI cell count) × 100)]. The results revealed that the activity of curcumin against B. mandrillaris cells increased after conjugation with AuNPs. The results are representative of three independent experiments performed in duplicates. The data are presented as the mean ± standard error (**: P < 0.01, ***: P < 0.001 using Student’s t test; two-tailed distribution).

Activity of Curcumin against N. fowleri was Significantly Enhanced after Conjugation with AuNPs

Amoebicidal activities of curcumin conjugated with AuNPs were investigated at 5 and 10 μM. Ten-micromolar curcumin–AuNPs resulted in a 69% amoebicidal activity against N. fowleri (Figure ). As compared to 10 μM AuNPs (M = 0, SE = 0), ethanol (2%) (M = 20, SE = 2), and 10 μM curcumin (M = 32, SE = 3), 10 μM curcumin–AuNPs (M = 69, SE = 1) showed significant amoebicidal activity (t(4) = 84, P = 0.0000001; t(4) = 19, P = 0.00004; t(4) = 11, P = 0.0004). At 5 μM, 53% amoebicidal activity was observed for curcumin–AuNPs. The activity of 5 μM curcumin–AuNPs (M = 53, SE = 2) was statistically significant (t(4) = 28, P = 0.000009; t(4) = 13, P = 0.0002; t(4) = 15, P = 0.0001) as compared to 5 μM AuNPs (M = 0, SE = 0), 5 μM curcumin (M = 21, SE = 2), and solvent control (M = 3, SE = 3).

Figure 6

Amoebicidal properties of curcumin against N. fowleri were significantly enhanced after conjugation with AuNPs. Briefly, N. fowleri was incubated with 5 and 10 μM curcumin and curcumin–AuNPs for 24 h, and viability was determined using trypan blue exclusion assay. Percentage amoebicidal activity was calculated by [((RPMI cell count – cell count for sample)/RPMI cell count) × 100)]. A significant increase in the activity of curcumin against N. fowleri cells was observed after conjugation with AuNPs. The results are representative of three independent experiments performed in duplicates. The data are presented as the mean ± standard error (**: P < 0.01, ***: P < 0.001 using Student’s t test; two-tailed distribution).

Our cytotoxicity results show that curcumin did not exhibit any cytotoxic activities against human cells at 10 and 5 μM. AuNPs at both 5 and 10 μM did not exhibit any cytotoxic activities against human cells. Also, curcumin conjugated with AuNPs at both 5 and 10 μM did not exhibit cytotoxic activities against human cells. The amoebicidal activities of curcumin were enhanced from 15 and 22%, before conjugation, at 5 and 10 μM, to 64 and 78%, respectively, after conjugation, against B. mandrillaris. Against N. fowleri, the amoebicidal activities of curcumin was enhanced from 21 and 32%, before conjugation at 5 and 10 μM, to 53 and 69%, respectively, after conjugation.

The increase in activity of curcumin may be linked to the biological activity of gold nanoparticles. AuNPs have been shown to cause the formation of ROS that results in the disruption of the mitochondrial membrane potential and indicates apoptosis.[37] AuNPs also caused a downregulation in the expression of genes involved in cell cycle and DNA repair mechanisms.[37] Due to the drop in the mitochondrial function, the loss of mitochondrial membrane potential, the activation of caspase-3, the increase in the amount of intracellular calcium, and the increased levels of nuclear p53, AuNPs cause cell death by apoptosis.[37] Enhanced effects of Au-conjugated drugs may also be explained by the fact that metals such as Au affect the ability of the amoebae to replicate DNA and its expression of enzymes and ribosomal subunit proteins, which are involved in the ATP production and hence respiratory chain.[38]

In conclusion, curcumin expressed significant amoebicidal activities against both B. mandrillaris and N. fowleri at 50 μM while exhibiting limited cytotoxicity toward human cells. The activity of curcumin was significantly increased against both amoebae after conjugation with gold nanoparticles. Although the mechanism of action of curcumin against B. mandrillaris and N. fowleri is yet to be explored as well as its in vivo abilities, curcumin shows great promise as a future treatment option against those free-living amoebae.

Materials and Methods

Nanoparticle Conjugation

Potassium gold (III) chloride and curcumin solutions were mixed in equal volume (1:1), and the solution was stirred magnetically at 200 × g for 2 h. Sodium borohydride was added to catalyze the formation of AuNPs from the potassium gold (III) chloride. As previously described, curcumin–AuNPs complex formation was indicted by color change of solution from colorless to pale pink.[39] Dynamic light scattering (Litesizer 500, Anton Paar) was performed at 25 °C and a constant angle of 90° to analyze the size distribution profile of curcumin–AuNPs in a suspension at 25 °C and a constant angle of 90°, as previously described.[40]

Henrietta Lacks Cervical Cancer Cells

Henrietta Lacks (HeLa) cervical cancer cells were used as a food source for amoeba cells.[41] HeLa cells were acquired from American Type Culture Collection (ATCC CCL-2, Singapore). Cells were cultured in a Roswell Park Memorial Institute (RPMI) 1640 medium (Serana, Germany), supplemented with 10% fetal bovine serum (FBS) (Sigma, United States), 1% antibiotics (penicillin–streptomycin) (Nacalai Tesque, Japan), 1% minimum essential medium amino acids (Nacalai Tesque, Japan), and 1% l-glutamine (Nacalai Tesque, Japan) at 5% CO2, 95% humidity, and 37 °C as previously described.[42]

Human Keratinized Skin Cells

Human keratinized skin cells (HaCaT) (CLS:300493) were obtained from CLS Cell Lines. HaCaT cells were grown in RPMI-1640 (Serana, Germany) complemented with 10% FBS (Sigma, United States), 1% penicillin–streptomycin (Nacalai Tesque, Japan), 1% minimum essential medium amino acids (Nacalai Tesque, Japan), and 1% l-glutamine (Nacalai Tesque, Japan) at 37 °C and 5% CO2.

N. fowleri Culture

N. fowleri was cultured as previously described.[41]N. fowleri cells (HB1 strain; ATCC 30174) were acquired from ATCC and cultured in an RPMI-1640 medium (Serana, Germany) supplemented with 1% antibiotics (penicillin–streptomycin) (Nacalai Tesque, Japan) with a monolayer of HeLa cells used as a food source at 5% CO2 and 37 °C.

B. mandrillaris Culture

B. mandrillaris cells (ATCC 50209) were acquired from ATCC and cultured in an RPMI-1640 (Serana, Germany) medium supplemented with 1% antibiotics (penicillin–streptomycin) (Nacalai Tesque, Japan) with a monolayer of HeLa cells used as a food source at 5% CO2 and 37 °C, as previously described.[43]

Cytotoxicity Assay

Cytotoxicity of curcumin against human cells was assessed as previously described.[42,44] Various concentrations of curcumin were incubated in RPMI-1640 (Serana, Germany) with HaCaT cells for 24 h at 5% CO2 and 37 °C. Cell-free supernatant media was collected, and the presence of LDH enzyme was assessed using a cytotoxicity detection kit (Roche Applied Science). Percentage cytotoxic effects were determined as follows: [(absorbance of media from cells treated with sample – absorbance of media from untreated cells)/(absorbance of media from cells with total LDH release – absorbance of media from untreated cells)] × 100% = percentage cytotoxic activity].

Amoebicidal Assay

The ability of the compounds to kill the amoebae was determined through amoebicidal assays, as previously described.[41,42] In 24-well plates in RPMI-1640 (Serana, Germany), 5 × 105 amoeba cells were incubated with curcumin, curcumin–AuNPs complexes, and solvents at 37 °C for 24 h. Amoebae incubated with RPMI-1640 served as a negative control. For the positive control, miltefosine was used. The percentage of viable amoebae was determined by counting unstained (live) amoeba cells using a hemocytometer, following the addition of 0.1% trypan blue. Statistical significance was calculated using Student’s t test with two-tailed distribution to compare the mean of experimental results.

Statistical Analysis

The data are illustrative of the mean ± standard error of several independent experiments accomplished in duplicates. Statistical significance for differences was evaluated using a two-sample t test with two-tailed distribution, contrasting the mean of two different experiments repeated using similar conditions. P values were determined for analysis.