A powerful combination of copper-cysteamine nanoparticles with potassium iodide for bacterial destruction

Abstract

Herein, for the first time, we demonstrate that the combination of copper-cysteamine (Cu-Cy) nanoparticles (NPs) and potassium iodide (KI) can significantly inactivate both Gram-positive MRSA and Gram-negative E. coli. To uncover the mystery of the killing, the interaction of KI with Cu-Cy NPs was investigated systematically and the products from their interaction were identified. No copper ions were released after adding KI to Cu-Cy NPs in cell-free medium and, therefore, it is reasonable to conclude that the Fenton reaction induced by copper ions is not responsible for the bacterial killing. Based on the observations, we propose that the major killing mechanism involves the generation of toxic species, such as hydrogen peroxide, triiodide ions, iodide ions, singlet oxygen, and iodine molecules. Overall, the powerful combination of Cu-Cy NPs and KI has good potential as an independent treatment or a complementary antibiotic treatment to infectious diseases.

1. Introduction

Diseases caused by Bacteria and viruses have always been a big threat to human beings. Excessive consumption and reliance upon antibiotics increase the chance of antibiotic resistance in different bacterial species, motivating several researchers to talk about ‘the end of the antibiotic epidemic’ [1,2]. Bacterial resistance to antibiotics is a public health problem [2]. Finding more effective antibacterial therapies to combat resistant strains is an ongoing subject of pertinence that can help stave epidemic [3]. For example, a pathogenic Gram-negative bacterial species, such as E. coli, has a unique capability to live on surfaces for long periods, is a potential threat to patients [4]. Therefore, researchers are focusing on the development of new solutions that can be effective against multidrug-resistant strains responsible for fatal infections [5–7].

Photodynamic therapy involves photosensitizers (PS) that can be activated by a suitable light source to produce reactive oxidative species (ROS) to kill bacteria or viruses. These ROS can neutralize many biomolecules in microorganisms regardless of structure or resistance [5–7]. The photodynamic efficiency of a PS depends upon various factors, such as the concentration of PS, oxygen level, wavelength, and intensity of light [8–12]. It is known that a PS can be highly selective for bacteria by proper chemical design to ensure that molecules selectively attach to bacterial cells than normal human cells during short incubation times. Recently, we have reported copper-cysteamine (Cu-Cy), Cu₃Cl(SR)₂ (R = CH₂CH₂NH₂) [13], as a new type of sensitizer that has strong luminescence [13,14] and can produce ROS under UV light [13,15], X-rays [16–18], microwave radiation [19,20], and ultrasound [21]. All these results indicate that the Cu-Cy is a novel sensitizer with potential applications for anti-infection and antitumor therapy. Our previous study revealed that Cu-Cy NPs alone can effectively kill gram-positive bacteria MRSA, but was not effective against gram-negative bacteria E. coli under UV light [6]. In the present work, we report that when KI is added to Cu-Cy NPs, they can significantly inactivate both Gram-positive MRSA and Gram-negative E. coli. Additionally, the interaction of KI with Cu-Cy NPs and the killing mechanisms were studied in detail to illustrate the phenomena. The mechanism studies revealed that Cu-Cy NPs + KI under UV light produces singlet oxygen, H₂O₂, and triiodide ions, leading to the destruction of both gram-positive and gram-negative bacteria.

2. Materials and methods

2.1. Materials preparation and characterization

Synthesis and complete characterization of Cu-Cy NPs have been described in our recent publication [13]. Starch was obtained from RICCA Chemical Co. (Arlington, TX, USA). The Amplex Red hydrogen peroxide/peroxidase assay kit was bought from Invitrogen (Carlsbad, CA, USA). p-nitrosodimethylaniline (RNO) and imidazole (ID) were obtained from Sigma, USA. Brain heart infusion (BHI) broth was purchased from Becton, Dickinson, and Company, Franklin Lakes, NJ. Potassium iodide (KI) was purchased from Sigma. For the illumination of bacterial cells, UV light (365 nm, 10 J/cm²) from an Omnilux Clear-U light-emitting diode (LED) array (Photo Therapeutics, Inc., Carlsbad, CA) was used.

2.2. Bacterial strains and cultures

Gram-positive bacterium (methicillin-resistant Staphylococcus aureus (MRSA) US300) and Gram-negative bacterium (E. coli K-12 (ATCC 33780)) were used as microbial strains. Bacteria were colonized by suspending in 25 mL of BHI broth and then incubated overnight in a shaker incubator at 37 °C at 120 rpm. 1 mL of this suspension was transferred into freshly made BHI for 2 h at 37 °C to mid-log growth phase. In order to estimate the cell concentration, optical density (OD) of the suspension was measured at 600 nm (OD of 0.8 = 10⁸ colony-forming unit (CFU) cells/mL). The suspension was subsequently centrifuged, washed, and suspended in PBS to stop microbial growth and used (10⁸ CFU) again.

2.3. Photodynamic therapy (PDT) studies

Initially, we carried out PDT studies on MRSA and E. coli cells (10⁸ CFU; 500 μL) that were incubated with different concentrations of Cu-Cy NPs (1, 10, 50, and 100 μM) for 30 min with or without UV light. Afterward, 10 μL of the suspension was used as the control from each sample, while 200 μL of the suspension was added into a 96 well plate and then irradiated with the UV light (365 nm,10 J/cm²). To calculate the survival fraction, the cell samples were serially diluted up to 10⁻⁵ times of the initial concentration in PBS and 10 μL of each dilution was streaked horizontally on square BHI agar plates for bacteria. Plates were then streaked in triplicate and incubated for 12–18 h at 37 °C in the dark. The survival fractions were expressed as ratios of CFU of microbial cells of the experimental group and control group. To investigate the effect of KI on bacterial inactivation, we performed a similar experiment using the mixture of Cu-Cy NPs (10 and 50 μM) and KI (0, 50, and 100 mM). Finally, we compared the bactericidal effect of Cu-Cy NPs with or without KI mediated PDT treatment on MRSA and E. coli under UV light.

2.4. Detection of Cu²⁺

In order to understand the possible mechanism of bacteria-killing, Cu²⁺ was determined by electron spin resonance (ESR) measured at X band by a Bruker ER-200DSRC Analytic ESR spectrometer at room temperature. The experiment was performed at 9.64 GHz with a microwave power of 63 μW. 1 mL of Cu-Cy (1 mg/mL) and 1 mL of Cu-Cy (1 mg/mL) + KI (600 mg/mL) were prepared and kept in liquid nitrogen overnight.

2.5. Singlet oxygen detection

We used the RNO-ID method for singlet oxygen measurement [22]. Briefly, RNO (0.225 mg) and ID (16.34 mg) were dispersed into DI water (30 mL). The solution was oxygen saturated by air bubbling for about 20 min just before the experiment. The sample was made by adding 0.5 mL of Cu-Cy (200 μM) and 0.5 mL of KI (50 mM) into 2 mL of the above RNO-ID solution. The control experiment was performed by using 0.5 mL of DI water instead of 0.5 mL KI while keeping other experimental conditions the same. Meanwhile, the two groups were exposed to UV light (10 J/cm²) for various time intervals (0–8 min). The RNO absorbance at 440 nm was recorded using a Shimadzu UV-2450 spectrophotometer.

2.6. Hydrogen peroxide determination using Amplex red assay

Amplex Red hydrogen peroxide assay was used to measure the generation of H₂O₂ from Cu-Cy NPs plus KI-mediated PDT. In the presence of peroxidase, the non-fluorescent Amplex Red can react with H₂O₂ to produce fluorescent resorufin. The reaction systems containing Cu-Cy NPs with or without KI were illuminated with various doses of the UV light (365 nm). The illuminated samples were then transferred to Amplex Red reagent (50 μM) and horseradish peroxidase (0.1 U/mL) in Krebs-Ringer phosphate. Following a half-hour incubation period, a fluorescence microplate reader (Exi/Emi at 530 nm/590 nm) was employed to quantify the fluorescence intensity changes upon UV stimulation (365 nm) on Cu-Cy NPs and Cu-Cy NPs + KI. Sodium azide (NaN₃), a physical scavenger of singlet oxygen [23], was used to determine if H₂O₂ played a role in singlet oxygen production in the mixture.

2.7. Iodine starch test

Cu-Cy NPs (100 μM) and Cu-Cy NPs (100 μM) + KI (100 mM) were irradiated with the UV light at different intensities for 5 min, and aliquots (50 μL) were taken after each illumination to measure iodine by adding 50 μL of the starch indicator. Afterward, absorbance was monitored at 610 nm using a microplate reader.

2.8. Superoxide detection by nitroblue tetrazolium (NBT) assay

For the purpose of superoxide measurement, the superoxide assay NBT (20 mM), Cu-Cy NPs (10 μM), and KI (50 mM) were mixed in PBS immediately before the experiment. Then, the absorbance of the resulting blue product was recorded using an optical microplate reader at 560 nm after each UV illumination on Cu-Cy and Cu-Cy + KI, respectively.

2.9. Statistical analyses

A one-way analysis of variance (ANOVA) was used to evaluate statistical significance. P < 0.05 were considered as statistically significant. The data represented as the mean ± standard deviation was conducted at least three times independently.

3. Results

3.1. Characterization of Cu-Cy

Fig. 1a presents the images of Cu-Cy NPs suspended in DI water under ambient light (left) and UV exposure (right). The Cu-Cy NPs have strong red luminescence as displayed in Fig. 1a and b. The emission spectrum has doublet peaks at 607 and 633 nm as shown in Fig. 1b. Two emission peaks are from the two different copper ions-Cu(1) and Cu(2), differing from each other by different coordination. The emission peak at 633 nm corresponds to Cu(1) as it has shorter distances to neighboring copper ions (2.81 Å and 2.89 Å) when compared with the distances of Cu(2) with neighboring copper ions (3.31 Å and 3.74 Å), respectively; thus, the emission at 607 nm corresponds to Cu(2) ions [13,24]. Fig. 1c and d depict TEM images of the Cu-Cy NPs used in the present study. Fig. 1e exhibits the size distribution of Cu-Cy NPs with an average diameter of 93 ± 41 nm. The electron diffraction pattern (Fig. 1f) reveals the single-crystal nature of the Cu-Cy NPs.

Fig. 1.

(a) Photos of Cu-Cy NPs dispersed in DI water under room light (left) and UV light (right); (b) Photoluminescence emission spectrum (right) of Cu-Cy following 360 nm excitation. Excitation spectrum is shown by monitoring emission at 607 nm (left); (c and d) Representative TEM image of Cu-Cy NPs used in this study; (e) Particle size distribution of Cu-Cy NPs used in the present study; (f) SAED of Cu-Cy crystal.

3.2. ROS measurement

Fig. 2a shows the absorption spectrum of Cu-Cy NPs suspended in DI water. Cu-Cy has strong absorption in the UV region (peak at 365 nm), whereas very little absorption in the visible range. This phenomenon makes Cu-Cy NPs different from conventional photosensitizers having strong absorptions in both UV and visible regions [25,26]. Owing to strong absorption in the UV range, Cu-Cy can be used for treating bacterial diseases upon UV light exposure. As displayed in Fig. 2b, Cu-Cy NPs produced ROS under UV light as reported previously [13,15]. Meanwhile, the decrease in RNO absorbance was not seen in the control group (DI water) (Fig. 2b), indicating that UV light alone could not generate singlet oxygen.

Fig. 2.

(a) Optical absorption spectrum of Cu-Cy NPs in aqueous solution. (b) RNO absorption curves of DI water (control) and Cu-Cy NPs upon UV light irradiation.

3.3. PDT effect of Cu-Cy NPs on MRSA and E. coli under UV light

We evaluated the PDT effect of Cu-Cy under UV-light illumination and the results are presented in Fig. 3. The results showed that Cu-Cy NPs did not pose noticeable toxicity to any kind of bacteria in the absence of light. However, under UV light illumination, the Cu-Cy NPs showed obvious antibacterial ability on MRSA in a dose-dependent manner (Fig. 3a). As depicted in Fig. 3a, the cytotoxic effect increased significantly, even at 50 μM of Cu-Cy. When 100 μM of Cu-Cy was used, the cytotoxicity to MRSA increased by > 5 log. On the other hand, Cu-Cy NPs could not induce noticeable killing to E. coli; even at 100 μM Cu-Cy concentration, the survival fraction declined only by less than < 1 log (Fig. 3b). From these results, we can conclude that Cu-Cy under UV light has a significant effect on MRSA, but has almost no effect on E. coli as we reported previously [6].

Fig. 3.

(a) Survival fraction of Cu-Cy on MRSA with or without UV light. (b) Survival fraction of Cu-Cy on E. coli with or without UV light.

3.4. PDT effect of Cu-Cy NPs plus KI against MRSA

In order to evaluate the PDT effect of Cu-Cy plus KI, 10 μM of Cu-Cy NPs with different concentrations of KI was applied to MRSA (Fig. 4a). The result showed that the bactericidal effect becomes progressively stronger with the increase of the concentration of KI. Furthermore, when the 50 μM of Cu-Cy NPs was used, bactericidal ability increased significantly after adding 50 mM of KI (P < 0.01). The results indicate that KI can enhance the PDT mediated killing effect of Cu-Cy NPs. Notably, when 100 mM of KI was used instead of 50 mM, no significant difference (P > 0.05) in the survival fraction of bacteria was observed, indicating that KI is non-toxic. These results encouraged us to investigate the PDT effect of Cu-Cy + KI against gram-negative bacteria, such as E-Coli.

Fig. 4.

PDT effect study of Cu-Cy + KI. (a) Killing effect of Cu-Cy NPs (10 μM) and (50 μM) with or without KI on PDT against MRSA. *versus control, P < 0.01, ^#versus in group, P < 0.05. (b) Killing effect of Cu-Cy NPs (50 μM) with or without KI (50 mM) against MRSA and E. coli. *versus MRSA and E. coli in group, P < 0.01, ^#versus in different group, P < 0.01.

3.5. PDT effect of Cu-Cy NPs plus KI on MRSA and E. coli

The MRSA and E. coli bacteria were divided into the following treatment groups: UV, Cu-Cy + UV, and Cu-Cy + UV + KI. As displayed in Fig. 4b, the survival fraction of bacteria was not affected by UV light (10 J/cm²) alone. However, in the presence of Cu-Cy NPs (50 μM), UV irradiation induced significant killing effect on MRSA bacteria than E. coli (MRSA vs. E. coli: 0.08 ± 0.025 vs. 0.52 ± 0.127; P < 0.005). It should further be noted that Cu-Cy NPs + UV induced significant killing on MRSA than UV only group, but not on E. coli, which is consistent with our previous result [6]. Most interestingly, after adding KI (50 mM) to Cu-Cy NPs, there was a dramatic increase in the killing effect on both MRSA and E. coli, with the latter being more pronounced (> 6 log). On MRSA (Cu-Cy only vs. Cu-Cy + KI: 0.143 ± 0.065 vs. e⁻⁹ ± e⁻¹⁰; P = 0.014 < 0.05); On E. coli (Cu-Cy only vs. Cu-Cy + KI: 0.523 ± 0.127 vs. 0.029 ± 0.016; P < 0.001). Furthermore, Cu-Cy + KI group caused statistically similar killing effect on gram-negative bacteria and gram-positive bacteria (P > 0.05) (MRSA vs. E. coli: e⁻⁹ ± e⁻¹⁰ vs. 0.029 ± 0.016; P = 0.575 > 0.005), inferring that Cu-Cy NPs + KI was efficient in killing both types of bacteria. This result confirms that the bactericidal killing ability of Cu-Cy NPs obviously improved after adding KI, especially on E. coli.

4. Discussion

To reveal the mechanisms behind the powerful combination of Cu-Cy NPs and KI, we carried out a series of experiments. The Gram-negative bacteria have strongly negative charge on their outer covering due to the existence of phospholipids and lipopolysaccharides [27]. Therefore, cationic NPs have a superiority for killing Gram-negative cells, though cationic PS are often much better at killing Gram-positive bacteria compared to Gram-negatives [28–31]. It has been proposed that the cationic PS penetrate the outer membrane of Gram-negatives by the “self-promoted uptake pathway” in which the divalent metal cations Ca²⁺ and Mg²⁺ are gradually displaced by the PS, and the lipopolysaccharide in the outer membrane permeability barrier is destabilized [32]. Cu-Cy NPs were coated with negatively charged poly (ethylene glycol) methyl ether thiol [13], making them difficult to attach to the bacterial membranes or cell walls. This is one of the reasons that the killing of gram-negative bacteria is not effective. Secondly, the Cu-Cy NPs were not able to penetrate the cell walls due to their large size. Gram-negative bacteria have a family of proteins called porins at the outer membranes. The porins function as molecular filters for hydrophilic molecules [33]. They work as ‘molecular sieves’ at the outer membrane. Usually, particles < 600 Da or about 2 nm in size can diffuse through porins [30,34]. The Cu-Cy NPs in this study are 93 ± 41 nm in size, which are much larger than the channels of porins, therefore, they cannot go through the porin channels. As a result, the majority of killing is perhaps from the ROS and other toxins produced by the interaction of Cu-Cy NPs with KI upon light activation.

4.1. Cu²⁺ detection using ESR

One possibility is when Cu-Cy NPs interact with KI, copper ions could be released from Cu-Cy NPs to induce Fenton reaction that produces ROS for bacterial killing. Copper is a redox-active transition metal and can accelerate reactive oxygen species (ROS) formation. In reducing conditions, Cu²⁺ may be reduced to the powerful and reactive Cu⁺ ion that can produce ROS, such as hydroxyl radical, for tissue damage [35]. Copper ions released from Cu-Cy NPs may enhance ROS generation through the Fenton reaction and the Haber-Weiss reaction [35,36] as shown below:

$${\text{Cu}}^{+}{+\text{H}}_2\text{O}\to {\text{Cu}}^{2+}+{}^{•}\text{O}\text{H}+{}^{-}\text{O}\text{H}\left(\text{Fenton}\text{Reaction}\right)$$ (1)

$${\text{O}}_2{}^{{•}_{-}}+{\text{H}}_2{\text{O}}_2\to {\text{O}}_2+{}^{•}\text{O}\text{H}+{}^{-}\text{O}\text{H}\left(\text{Haber–Weiss}\text{Reaction}\right)$$ (2)

If the above Fenton reactions occurred, Cu²⁺ ions would be detected by ESR as reported in our previous work [19]. As shown in Fig. 5a, the ESR spectra for both Cu-Cy and Cu-Cy + KI show a very weak Cu²⁺ signal around g = 2.06. Compared to the signal intensity of a Cu²⁺ 1 mM standard, this signal would account for 8 μM and 2 μM total Cu²⁺ concentration for Cu-Cy and Cu-Cy + KI samples, respectively. The result suggests that KI might not trigger the release of Cu from Cu-Cy NPs in aqueous solution and, therefore, copper release and hence Fenton reaction may not be one of the mechanisms by which Cu-Cy NPs + KI cause cytotoxicity for bacterial inactivation. There must be some other toxic species produced from the interaction of Cu-Cy NPs with KI that are lethal to bacteria, as discussed below.

Fig. 5.

(a) ESR spectra of Cu-Cy NPs (black) and Cu-Cy NPs + KI (red). (b) RNO absorption curves of DI water (control), Cu-Cy NPs (100 μM) + KI (100 mM), and Cu-Cy NPs (100 μM) under UV irradiation. (c) Cu-Cy NPs, Cu-Cy NPs + KI, and Cu-Cy + NaN₃ irradiated with UV light and aliquots were withdrawn and added to Amplex Red reagent. (d) Cu-Cy NPs (100 μM) and Cu-Cy NPs (100 μM) + KI (100 mM) were illuminated with increasing fluence of UV light and aliquots were withdrawn and added to starch solution. (e) Absorption spectra of Cu-Cy NPs + KI after different incubation time. (f) The photoluminecence spectra of Cu-Cy NPs after adding different concentration of KI to Cu-Cy NPs after 2 h. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

4.2. The influence of KI on singlet oxygen production from Cu-Cy NPs

RNO-ID method [22] was used to measure singlet oxygen as described in our previous publication [13,20]. To determine whether singlet oxygen is the major factor of the potentiated microbial killing, we measured the singlet oxygen production from Cu-Cy NPs with and without KI. As can be seen in Fig. 5b, KI quenched the singlet oxygen from Cu-Cy NPs. So, it is reasonable to believe that singlet oxygen does not play a dominant role in the PDT mediated killing.

4.3. Other possible products from Cu-Cy NPs and KI interaction

We further investigated the production of H₂O₂ using the Amplex red assay [5]. As can be seen in Fig. 5c, the fluorescence intensity of the Amplex red exhibited a linear enhancement with increasing fluence of UV light to the solution of Cu-Cy NPs (50 μM) and KI (400 mM), while without KI there was only a little increment in the fluorescence intensity. Therefore, we concluded that a significant level of H₂O₂ formed after adding KI to Cu-Cy NPs. Interestingly, there were almost no H₂O₂ produced when singlet oxygen was quenched by NaN₃, indicating that singlet oxygen plays a key role in H₂O₂ formation.

ROS generated in the photodynamic reaction may oxidize the iodide anion to molecular iodine. We further carried out a starch-iodine assay to detect if molecular iodine was formed after mixing Cu-Cy NPs and KI (Fig. 5d). However, no molecular iodine was detected by the assay at various light doses (0–32 J/cm²).

4.4. Iodine oxidation and H₂O₂ formation

One of the possible mechanisms of H₂O₂ production is presented in (Eqs. (3)–(5)). In this mechanism, the singlet oxygen can oxidize the iodide ion to generate iodide radical and superoxide radical (Eq. (3)). The iodide radical dimerize to make diiodine (Eq. (4)), while superoxide anions can yield H₂O₂ through dismutation (Eq. (5)) [5,37].

$${}^1{\text{O}}_2+{\text{I}}^{-}\to {\text{O}}_2{}^{{•}_{-}}+{\text{I}}^{•}$$ (3)

$${\text{2I}}^{•}\to {\text{I}}_2$$ (4)

$${\text{2O}}_2{}^{{•}_{-}}+{\text{2H}}^{+}\to {\text{O}}_2{+\text{H}}_2{\text{O}}_2$$ (5)

To determine if the above mechanism was followed, we used the nitroblue tetrazolium (NBT) assay to detect if superoxide is produced in the Cu-Cy + KI. However, no superoxide was detected by NBT assay, indicating that H₂O₂ formation did not follow the proposed mechanism (Eqs. (3)–(5)).

The second possible mechanism for H₂O₂ production is through the interaction of singlet oxygen and iodide.

$${}^1\text{O}{}_2{+\text{I}}^{-}{+\text{H}}_2\text{O}\to \text{IOOH}{+\text{HO}}^{-}$$ (6)

$$\text{IOOH}+{\text{I}}^{-}\to {\text{HOOI}}_2{}^{-}$$ (7)

$${\text{HOOI}}_2{}^{-}\to {\text{I}}_2+{\text{HO}}_2{}^{-}$$ (8)

$${\text{I}}_2{+\text{I}}^{-}\to \text{I}{}_3{}^{-}$$ (9)

$${\text{HO}}_2{}^{-}+{\text{H}}_2\text{O}\to {\text{H}}_2{\text{O}}_2{+\text{HO}}^{-}$$ (10)

The reaction schemes (Eqs. (6)–(10)) present the detailed reaction steps that lead to H₂O₂ formation in the Cu-Cy + KI mixture. These reaction schemes can be combined to one equation (Eq. (11)). As presented in Eq. (11), singlet oxygen produced by Cu-Cy upon UV excitation can react with iodide ions to yield triiodide ions and H₂O₂.

$${}^1\text{O}{}_2+{\text{3I}}^{-}+{\text{2H}}_2\text{O}\to {\text{I}}_3{}^{-}+{\text{2H}}_2{\text{O}}_2$$ (11)

It was reported that absorptions at the wavelengths of 288 and 352 nm are from a contingent of the I₃⁻ anion in solution and all molecular iodine was converted to I₃⁻ [38]. Therefore, to confirm the existence of triiodide species in the Cu-Cy NPs + KI mixture, we monitored the absorbance of Cu-Cy NPs + KI mixture in the UV wavelength region. As expected, after adding KI to Cu-Cy NPs, a new absorption peak at 280–290 nm appeared, which became more and more pronounced over time (Fig. 5e). The iodine molecule exists only in a transient state and immediately converts into triiodide species in the presence of singlet oxygen. Due to this reason, the iodine molecule could not be detected by a starch assay. Consequently, the possible products of Cu-Cy NPs + KI solution that are responsible for the enhanced antimicrobial activity are singlet oxygen, biocidal triiodide (I₃⁻), and H₂O₂. Our conclusion is consistent with the previous report that triiodide can potentiate microbial killing in combination with H₂O₂ [39].

4.5. KI influence on Cu-Cy NPs’s luminescence

To explore the effect of KI on Cu-Cy NP’s luminescence, different concentrations of KI were mixed with the same concentration of Cu-Cy NPs. After incubating for 2 h, the emission spectra were measured by using a spectrofluorophotometer and the result is shown in Fig. 5f. We found that luminescence intensity decreased with the increasing amount of KI (Fig. 5f). This is possibly related to the interaction between iodide ions and Cu-Cy NPs, as iodide ion has the ability to quench the luminescence of different luminescent dyes [4,5,7].

4.6. Interaction of KI with Gram-negative and Gram-positive bacteria

KI is a harmless and nontoxic health-food supplement that can be used to treat bacterial infections [40]. We demonstrated that the addition of KI can dramatically potentiate the antibacterial effects obtained using in vitro PDT under UV light excitation of Cu-Cy, while Cu-Cy NPs alone have almost no effect on negative bacteria. The mechanism for the potentiation by adding KI is interesting but not completely understood yet. There could be several possibilities. First, iodide anions may oxidize photoexcited Cu-Cy NPs through electron transfer to form iodide radicals (I·) and some other reactive radical species that are bactericidal. Iodide anions could be used as a source of electrons to improve the type-1 electron-transfer photochemical mechanism, thereby producing more ROS. Furthermore, both type-1 and type-2 can readily oxidize iodide anion to molecular iodine I₂ or I₃⁻, which are toxic to bacteria and can effectively kill bacteria as shown in Fig. 6.

Fig. 6.

A schematic illustration for photodynamic killing on bacteria from the combination of Cu-Cy NPs with KI under UV light activation.

As reported previously, Photofrin (PF) is ineffective in PDT mediated killing of Gram-negative bacteria [5] and no uptake of PF by E. coli [41]. The inefficacy of PF towards Gram-negative bacteria can be attributed to its neutral or slightly anionic nature due to which it cannot attach to the outer membrane of the Gram-negative bacteria. Especially in solution, Gram-negative bacteria are protected from extracellular produced singlet oxygen [42]. This is also corroborated by our result: Cu-Cy NPs (50 μM) and UV light (10 J/cm²) did not produce killing effect on Gram-negative species of E. coli. However, after adding KI to Cu-Cy NPs under UV light, more than > 6 log of cells were destroyed. Cu-Cy NPs are inactive against Gram-negative bacteria because Cu-Cy NPs were neutral or negatively charged [13,24], which could not attach to the outer membrane of Gram-negative bacteria.

The case is different for Gram-positive bacteria: for Cu-Cy-mediated photodynamic anti Gram-positive MRSA treatment, there was inherent cytotoxicity with or without KI. Cu-Cy NPs and singlet oxygen are neutrally charged molecules, which can easily penetrate Gram-positive bacteria [43]. In addition, the porous nature of the Gram-positive cell wall allows various kinds of particles or radicals to penetrate the cells [44]. Undoubtedly, Gram-positive bacteria are more vulnerable to extracellular produced singlet oxygen [42,45]. The permeability of microbial cells to iodide anions have not been explored extensively. In the present study, we assume that Cu-Cy NPs generate singlet oxygen through PDT, and singlet oxygen or some other ROS react with iodide to form triiodide ions to enhance the bactericidal ability. These findings suggest that the combination of Cu-Cy NPs and KI under UV light irradiation could be used for developing a new antibiotic drug for the prevention and cure of many types of infectious diseases caused by bacteria and/or viruses, such as for wound healing, skin diseases like lepus, vitigilo, and water treatment as well as blood or medicine sterilization.

5. Conclusions

In summary, for the first time, we found that the combination of Cu-Cy NPs and KI is very effective for killing Gram-positive bacterium MRSA and Gram-negative bacterium E. coli. As the NPs are much larger than the channels of the bacterial outer members, Cu-Cy NPs cannot penetrate the cell walls to reach the inner cytosols to kill them. No copper ions were released after adding KI to Cu-Cy in DI water and the Fenton reaction induced by copper ions may not be responsible for bacterial killing. To uncover the possible mechanism, the interaction of KI with Cu-Cy NPs was investigated systematically, and the products from their interaction were identified. Based on our results, we conclude that the major killing mechanism involves the generation of toxic species, such as H₂O₂, triiodide ions, and singlet oxygen. The combination of Cu-Cy and KI has potential as an independent treatment or can potentially act as an adjunct modality for infection diseases.

Abbreviations

PDT

photodynamic therapy

BHI

brain heart infusion

CFU

colony-forming units

DC

dark control

KI

potassium iodiode

LED

lightemitting diode

mHCTPO

4-hydro-3-carbamoyl-2,2,5,5-tetraperdeu teromethylpyrrolin-1-oxyl

MRSA

methicillin-resistant Staphylococcus aureus

OD

optical density

Cu-Cy

copper-cysteamine nanoparticles with a formula of Cu₃Cl (SR)₂ (R = CH₂CH₂NH₂)

PS

photosensitizer

ROS

reactive oxygen species

YPD

yeast peptone dextrose

EDTA

ethylenediaminetetraacetic acid