Photo-induced antileishmanial activity of indocyanine green: In vitro and in vivo studies

doi:10.21203/rs.3.rs-3723016/v1

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Photo-induced antileishmanial activity of indocyanine green: In vitro and in vivo studies

https://doi.org/10.21203/rs.3.rs-3723016/v1

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Background

Indocyanine green is a promising dye for photodynamic and photothermal therapy. However, ICG tends to aggregate in aqueous media, which limits its use in light therapy. Loading ICG onto a biocompatible structure can improve its aqueous stability. The aim of this study was to investigate the synergistic effect of PDT/PTT on leishmanial activity in the presence of micelles loaded with ICG.

Methods

After synthesizing micelles containing ICG, the dark toxicity of the pharmaceutical agents and in vitro phototoxicity by a cw 808 nm laser on promastigotes were determined via MTS assay. Finally, the efficacy of the treatments was assessed by measuring the diameter of the lesion every three days in a study conducted on 33 female BALB/c mice aged 4-6 weeks.

Results

At 808 nm, the absorbance of ICG inside the micelles was approximately2.5 times that of free ICG. The optimal concentration of ICG was determined to be 100 μM based on the toxicity of the medicinal agents topromastigotes. In the in vitro experiment, the groups containing ICG showed a significant decrease in survival compared to the control group with increasing light dose. In the animal model study, the simultaneous presence of medicinal agents and the application of a laser created a significant difference in the relative area of the lesion compared to the control group.

Conclusion

The findings of this study show that PTT/PDT mediated by ICG can be considered an inexpensive, safe, easy to administer and efficient treatment against Leishmania L. major both in vitro and in vivo. Moreover, this treatment does not cause any adverse effects when compared to other treatments.

Leishmania major

Nanomicelle

Indocyanine green (ICG)

Photodynamic therapy (PDT)

Photothermal therapy (PTT)

Leishmaniasis is a neglected infectious disease that is caused by different species of flagellated protozoan Leishmania and is transmitted by the bite of a female phlebotomus mosquito (1). The disease affects 98 tropical and subtropical countries and three regions in more than one billion at-risk populations worldwide (2). Although kala-azar or visceral leishmaniasis is the most lethal type and causes death if left untreated, cutaneous leishmaniasis is the most common type of leishmaniasis (3, 4). This disease imposes a significant burden on human health and is a major global problem.

Currently, common antiparasitic drugs such as pentavalent antimony, miltefosine, isothionic pentamidine, and amphotericin B are used for the treatment of leishmaniasis. Some of these drugs are relatively expensive and lead to severe side effects and parasite resistance. Therefore, they are not always recommended for treatment. Therefore, the development of new drugs or alternative treatments is necessary for leishmaniasis (5–7).

In recent years, physical methods such as cryotherapy, hyperthermia, electrolysis, and photodynamic therapy (PDT) have been suggested for dealing with leishmaniasis (8–11). Photothermal therapy (PTT) is a type of heat therapy that uses near-infrared light. In this method, the light absorbed by photothermal factors induces local heat. After the absorption of light by these agents, the electrons move from the ground level to the excited level. The excitation energy is transferred to the surrounding environment through a nonradiative transition in the form of kinetic energy, which causes heat generation (12). PDT is a relatively new method in which a certain wavelength of light is used along with a photosensitizing drug to damage biological structures. The combination of an appropriate concentration of photosensitizer and a light dose in the lesion can cause the immediate death of microorganisms with minimal damage to normal cells (13). In addition, PDT can be used without developing resistance in microorganisms because reactive oxygen species will interact with several cellular structures (proteins, lipid membranes and nucleic acids) through multiple mechanisms. Therefore, a therapeutic window should be found so that the microorganisms are destroyed without damaging the healthy tissue. The interaction of photons with photosensitizers in the presence of oxygen molecules leads to the formation of oxygen radicals that destroy tissues under radiation (14).

Indocyanine green (ICG) has been approved by the US Food and Drug Administration for use in medical diagnostics. ICG is a hydrophilic compound with low toxicity, and its high absorption in the near-infrared region makes it a reliable dye for tumor imaging and a photothermal/photodynamic agent. This allows for the penetration of deeper tissues to produce ROS without inducing substantial heating. However, its poor stability and lack of targeting limit its application (15–17).

Colloidal drug delivery systems, such as nanomicelles, can improve the therapeutic index of drugs. They achieve this by increasing their effectiveness and reducing their unwanted side effects (18, 19). These nanocarriers can also be used as delivery agents for photosensitizers, potentially increasing the efficacy and safety of PDT (20). Nanomicelles offer several advantages over conventional formulations, including improved solubility, reduced toxicity, improved pharmaceutical activity and stability, and protection against physical and chemical degradation (21).

To the best of our knowledge, no studies have been conducted on the use of ICG for treating cutaneous leishmaniasis. Therefore, the aim of this study was to introduce a nanostructure that carries ICG and investigate the synergistic effect of photodynamic therapy (PDT) and photothermal therapy (PTT) on leishmaniasis in the presence of ICG-loaded nanomicelles.

Chemicals

ICG purchased from ACROS (USA, 412541000), MTS/PMS (3-(4,5 dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium/phenazine methosulfate), penicillin, trypsin–EDTA, and streptomycin were purchased from Sigma‒Aldrich (St. Louis, MO, USA). Roswell Park Memorial Institute-1640 (RPMI-1640) medium and fetal bovine serum (FBS) were obtained from Gibco (Invitrogen Corporation, Carlsbad, CA). Surfactants (Tween 80 and Span 80) were procured from Sigma‒Aldrich (St. Louis, MO, USA). All other chemicals were of analytical grade.

Preparation and characterization of ICG-loaded micelles

ICG is prone to clumping and forming dimeric structures in water, which reduces its ability to produce singlet oxygen. To improve its photophysical properties, ICG was encapsulated in nanomicelles as a biocompatible carrier. This micellar structure is prepared from the combination of two nonionic surfactants with two completely different HLB. In this way, the solution of 0.002 mM ICG in micelles was prepared with two surfactants, SPAN-80 (HLB of 4.3) with 60% and TWEEN-80 (HLB of 15) with 40% of the total portion, so that from the combination of these two micellar structures, HLB 8.58 can be obtained, the final concentration of SPAN- 80 is equal to 2.5 mM and the concentration of TWEEN-80 is equal to 1.5 mM.

To investigate the role of the prepared nanostructure in improving the optical properties of ICG, UV–vis absorption spectra (UNICO 2100-UV, China) with 5 nm accuracy were recorded (the concentration of ICG in both samples was 0.002 mM).

The size distribution and zeta potential of ICG-loaded micelles were measured by a SZ-100 instrument (HORIBA, Japan) equipped with a helium-neon (He-Ne) laser (633 nm).

To determine the emission spectrum of the 808 nm laser, a spectrometer was used (AvaSpec, Netherlands).

Parasite cultivation

promastigotes of Leishmania major (MRHO/IR/75/ER) provided as a gift from the Research Centre of Skin Disease and Leprosy, Tehran University of Medical Sciences, kept by passage in BALB/c mice were used in this study. To induce parasite proliferation, amastigotes from mouse spleen were cultured on Novy-MacNeal-Nicolle (NNN) medium containing agar (4 mg/100 mL) and defibrinated rabbit blood (10%). After transformation of amastigotes to promastigotes, they were cultured in RPMI-1640 containing 100 IU/mL penicillin and 100 µg/mL streptomycin as well as 20% heat-inactivated FBS in an incubator at 28°C without CO₂ for two weeks. During several passages, the parasite suspension was precipitated in a refrigerated centrifuge for 10 min at 4500 rpm, and the supernatant was removed. Thus, the parasites achieved a fixed growth phase (stationary-phase) after 4 days of being kept in an incubator and were ready to be subjected to the designed experiments (22, 23). It should be noted that promastigotes have shown more intense infectivity during the stationary phase compared to the logarithmic phase. During this stage, the number of metacyclic promastigotes is higher than that of other forms. However, repeated cultivations of promastigotes reduce their infectivity property. To confirm the stationary phase, parasites at the stationary phase with 3 or 4 consecutive counts were quantified, and an unchanged or decreased count of parasites was the criterion. At each passage, parasite sampling was performed, and the growth of parasites was examined. In this study, the third passage of promastigotes was used to inoculate the lesions (9).

Cytotoxicity of the agents

In this phase of the research, the dark toxicity of free and micelle-loaded ICG on L. major parasites was investigated at five concentrations. The following steps were taken to perform the drug toxicity test and in vitro treatment:

Initially, a suspension with $2\times$10⁶ parasites per milliliter was prepared.

A total of 100,000 parasites were counted and cultured in each well of a 96-well plate.

Different concentrations of ICG (10, 20, 40, 80, and 100 µM) were prepared and added to each well. The mixture was then kept at 28°C for 24 hours.

After incubation, the samples were centrifuged at 4000 rpm for 10 min, and the supernatant solution was removed.

RPMI 1640 culture medium (without phenol red) supplemented with 10% FBS was added to each well and incubated for 48 hours.

To determine the survival percentage, a mixture of the resulting MTS/PMS solution (at a ratio of 5:1) with a volume of 20 µl was added to each well. For this assay, 3 replicates were performed (24).

To estimate background absorbance from light scattering, we analyzed wells without parasites but with the same sample concentration.

Finally, after 3 hours, light absorption at a wavelength of 492 nm was obtained by using a Stat Fax 2100 microplate reader (Awareness Technology, Palm City, FL) equipped with a printer (LQ-300 EPSON, Nagano, Japan).

The survival rate is derived from this relationship:$\frac{\text{O}\text{p}\text{t}\text{i}\text{c}\text{a}\text{l} \text{d}\text{e}\text{n}\text{s}\text{i}\text{t}\text{y} \text{o}\text{f} \text{d}\text{r}\text{u}\text{g} \text{t}\text{r}\text{e}\text{a}\text{t}\text{e}\text{d} \text{g}\text{r}\text{o}\text{u}\text{p}\text{s}}{\text{O}\text{p}\text{t}\text{i}\text{c}\text{a}\text{l} \text{d}\text{e}\text{n}\text{s}\text{i}\text{t}\text{y} \text{o}\text{f} \text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l} \text{g}\text{r}\text{o}\text{u}\text{p}}\times 100$

Animal models

Female BALB/c mice were purchased from the Pasteur Institute, Karaj Branch, Iran and used at 6–8 weeks of age (n = 24, mean weight 20 g, standard deviation 1 g). Animals were maintained under specific pathogen-free conditions at 22–25°C and in lighting conditions of 12 h darkness and 12 h light. The third or fourth passage of promastigotes in the stationary phase of growth was used to infect mice. After proliferation and harvesting, parasites were washed with PBS, and the concentration was adjusted to a PBS so that 1 ml contained 40 × 10⁶ L. major. One hundred microliters of solution containing live promastigotes (containing 4 × 10⁶ parasites) was subcutaneously injected into the right hind paw of each animal. After approximately 20 days, the wound was established. After a random sampling of some animals and confirmation of cutaneous leishmaniasis by a pathologist, the mice were randomly stored into six groups, each consisting of four animals as follows:

Control: Untreated group, including treated mice with injection of 100 µL sterile PBS into the right hind paw of mice.
ICG: The mice were treated only with an injection of 100 µL ICG (100 µg mL^− 1) solution, which was injected into the right hind paw of the mice. The dosage was 0.5 mg kg^{− 1 mouse− 1}.
ICG micelles: The mice were treated only with an injection of 100 µL of ICG-loaded micelle (100 µg mL^− 1) solution, which was injected into the right hind paw of the mice. The dosage was 0.5 mg kg^{− 1 mouse−1}.
Laser: Treated mice with laser irradiation at 2.5 W cm^-2 for 10 min and 100 µL of sterile PBS was injected.
ICG + Laser: The mice were treated with an injection of 100 µL ICG (100 µg mL^− 1) solution into the right hind paw. The dosage was 10 mg kg^− 1 mouse^− 1. Then, the mice were treated with laser irradiation at 2.5 W cm^-2 for 10 min.
ICG micelles + Laser: Mice treated with an injection of 100 µL of ICG-loaded micelle (100 µg mL^− 1) solution into the right hind paw. The dosage was 10 mg kg^− 1 mouse^− 1. Then, the mice were treated with laser irradiation at 2.5 W cm^-2 for 10 min.

ICG-NIR irradiation

For in vitro ICG-NIR irradiation, we used ICG at a concentration of 100 µM with and without micelles and a cw diode laser at 808 nm (MDL-III-808, CNI Laser, China) at a specified nominal power of 1 W. According to the beam spot size of 40 mm², a nominal intensity of 2.5 W/cm² was obtained. The exposure time for all groups was selected as 0, 5 and 10 minutes, which corresponded to light doses of 750 and 1500 J/cm². Samples were placed in wells of a 96-well plate, at least one-well spaced, and individually irradiated from bottom upwards.

For in vivo ICG-NIR irradiation, twenty-four hours after injection, irradiation was performed with 808 nm laser light at a power density of 2.5 W/cm² for 10 min (light dose of 1500 J/cm²). After NIR irradiation, the mice were returned to the colony. The dimensions of the lesions were recorded twice a week by a digital caliper, and their changes were normalized compared to the day before the treatment for a total of 30 days following the first treatment.

Statistical analysis

Data were derived from at least three independent experiments and are presented as the mean ± standard error of the mean. Data from different stages of the study were entered into SPSS version 26 software (IBM Corp., Armonk, NY, USA) for statistical analysis. The normality of the data distribution was checked using the Kolmogorov‒Smirnov test. Changes in the relative diameter of the lesion between the groups were compared at the 95% confidence level. The Kruskal‒Wallis and Mann‒Whitney tests were used to compare the relative area of the lesion in the comparison of two groups. P values less than 0.05 were considered statistically significant.

To compare the efficiency of the treatment, the combination index (CI) was defined to compare the groups. For this purpose, possible synergistic effects in treatment were calculated in the presence of pharmaceutical agents along with laser radiation. In this way, if two factors A and B affect parasite survival separately or in combination with each other, the Combination Index is defined as follows:

$$\text{CI}\text{ =}\frac{\text{V}\text{A}\text{×V}\text{B}}{\text{V}\text{AB}}$$

where V_A, V_B and V_AB are the microorganism viabilities under the effects of A, B (alone) and their combination. Here, CI > 1, CI = 1 or CI < 1 indicate the dominance of synergistic, additive or antagonistic effects of A and B agents (25).

On the other hand, to compare two treatment groups, one with nanoparticles and the other without nanoparticles (both groups were irradiated), the relative lethal synergism (RLS) index was used, which is equal to the ratio of cell death following treatment in the presence of nanoparticles to cell death after using the same treatment method but in the absence of nanoparticles. In this case, if the RLS is more than 1, there is a synergic effect.

Additionally, to compare two treatment groups, one with micelles and the other without micelles (both groups were irradiated), the relative lethal synergism (RLS) index was used. RLS is equal to the ratio of cell death following treatment with micelles to cell death after the same treatment without nanoparticles. An RLS greater than 1 indicates a synergistic effect (26).

Characterization of ICG-loaded micelles

Figure 1 shows the emission spectrum of the 808 nm laser, whose peak is at the wavelength of 807 nm, which is very close to the claim of the laser manufacturer.

The absorption spectra of ICG in water and nanocarrier were recorded using a UV‒visible spectrophotometer (the concentration of ICG in both samples was 0.002 mM). From Fig. 2, it can be seen that the absorption of ICG at 808 nm increased significantly when it entered the nanocarrier.

In Fig. 3 (a) and (b), the size distribution and zeta potential of nanoparticles were studied using a (HORIBA, Japan, model SZ100), with a mean hydrodynamic diameter of 25 ± 10 nm, PDI of 0.65 ± 0.1 and zeta potential of -2.3 ± 1 mV (the values depict the mean ± SD based on three individual measurements taken in triplicate).

In vitro antileishmanial activity

The difference in the average percentage of survival in the groups containing free ICG was not significant compared with the control group. However, the difference in the average percentage of survival in micelle groups was significant compared to the control group. On the other hand, the difference in the average percentage of survival in groups containing micelles (except for 10 µM concentration) was significant compared to free ICG. Additionally, using the linear equation to fit the toxicity graph of ICG with and without micelles, the IC₂₀ was calculated, which was equal to the concentrations (in µM) of 102 and 390, respectively. Therefore, the optimal concentration of ICG was considered to be 100 µM.

The laser irradiation results showed that groups containing ICG had a significant decrease in survival compared to the control group (according to the toxicity test) with increasing light dose. In control groups with only laser irradiation, there was no significant decrease in cell survival compared to the control group without laser irradiation after exposure, indicating the lack of phototoxicity of the 808 nm wavelength. It should also be added that the difference in the average percentage of survival in the ICG-loaded micelles group compared to free ICG was not significant.

Additionally, the combination index of 808 nm laser with ICG were calculated for different light doses and showed in Table 1.

Table 1

Combination index of the 808 nm laser with ICG for different light doses and the same dye concentration
Light dose (J/cm²)	combination index
Light dose (J/cm²)	Free ICG	ICG- loaded micelles
750	1.32 ± 0.13	1.33 ± 0.13
1500	2.62 ± 0.26	3.86 ± 0.38
Mean combination index ± SD	1.97 ± 0.2	2.59 ± 0.25

On the other hand, according to the results of the MTS assay, the RLS index was calculated for the groups receiving laser along with ICG with and without micelles at two light doses of 750 and 1500 J/cm², which were 1.23 and 1.41, respectively. Was obtained. Since the RLS was greater than 1 for both light doses, it indicates a synergistic effect.

In vivo antileishmanial activity

The effects of 808 nm laser irradiation alone and in the presence of dye on the right foot lesions in BALB/c mice were studied. At the end of the 30-day period during and after the start of treatment, compared to the day before the first treatment, the size of lesions increased almost more than 2 times in the control groups and almost 2 times in the groups receiving pharmaceutical agents alone and laser alone. Fifteen days after the treatment, the lesion size in the groups containing laser and medicinal agents had a noticeable decrease, but in the control group, the increase in the size reached more than 50% compared to the day before the treatment. Nine days after treatment, the lesion size was not significantly increased in the laser, ICG and ICG micelle groups, but in the control group, the lesion size increased nearly twice as much the day before the treatment. No increase in lesion size was observed in the ICG + laser and ICG micelles + laser groups at the end of the 15-day treatment. It should be noted that the lesion size in the ICG micelles + laser group showed a declining trend until day 18. In the group that received laser irradiation alone, no reduction in lesion size was observed until the third day after irradiation. Furthermore, during the follow-up period, there was no reduction in lesion size until the end of the study. However, on the 15th day after treatment, the lesion size increased by approximately 40% compared to the day before the first treatment. It is worth noting that in most of the treatment and control groups, the lesion surface increased during the last 6 days after the end of the treatment.

Additionally, the average relative size of the lesion was calculated within 30 days for the studied animal groups and is shown in Fig. 7. Based on this analysis, the application of medicinal agents and laser simultaneously resulted in a significant difference in the relative size of the lesion compared to the control group. Additionally, the ICG and micelles + ICG groups did not show any significant difference in the relative size of the lesion compared to the control group. On the other hand, there was no significant difference in the relative size of the lesion between the two groups, namely, ICG + laser and micelles + ICG + laser. Additionally, a significant difference was observed between the group receiving laser and the group receiving combined treatment (drugs plus laser) in favor of combined treatment. A significant difference was observed between the groups receiving combined treatment and the groups receiving medicinal agents without laser irradiation.

Over the past several decades, the incidence of cutaneous leishmaniasis has almost reached epidemic proportions. The current drugs used to treat leishmaniasis are expensive and cause severe side effects and parasite resistance (27). The PDT/PTT method is proving to be highly effective in fighting bacterial, fungal, and leishmanial infections, as well as aiding in wound healing. This approach has demonstrated that it can directly eliminate microorganisms and promote wound healing through the use of both ROS and thermal action. As a result, the PDT/PTT approach shows great potential in the medical field (28, 29). ICG, an FDA-approved photosensitizer, is commonly used due to its hydrophilic nature, low toxicity, and high absorption in the NIR. It can produce ROS, making it a valuable PTT and PDT agent (12). The use of ICG in PDT has two primary drawbacks, namely, its quick elimination from the body due to its binding to lipoproteins and its instability in aqueous solutions. To overcome these challenges, a possible solution is to employ a nanoparticulate system that is both biocompatible and biodegradable, which can trap ICG. In recent years, micellar nanostructures have emerged as potential colloidal drug carriers that can address these issues effectively. Therefore, micelles-ICG-NPs were synthesized using SPAN-80 and TWEEN-80 as biocompatible adjustment agents. The particles were characterized and employed for the study.

According to Fig. 2, the reduction of dye aggregation after entering the nanomiclle can justify the redshift of the ICG in the aqueous medium (30). This finding was also observed in the study of Yan and Qiu, who investigated the photothermal effect of free and loaded ICG in micelles on nasopharyngeal cancer (31). However, they did not state the reason for this finding. Based on this, the role of the prepared micellar nanostructure in improving the optical properties of ICG was confirmed. It was observed that the absorption of ICG at 808 nm increased significantly when it was loaded in micelles. ICG has a high tendency to form aggregates and dimeric structures in water, which requires a suitable encapsulation system to increase the quantum efficiency of the excited level and thus produce singlet oxygen.

The reason for choosing the SPAN-80 surfactant as the dominant surfactant in this study is its hydrophobic property, which increases the probability of passing through the phospholipid membrane of protozoa. SPAN-80 has low toxicity and a very low CMC, which prevents premature micelle separation even in blood and tissue. The micelles formed in this study are vesicles because SPAN-80 is a vesicle-forming surfactant. To optimize the loading, release, size distribution, and PDI characteristics of the vesicle nanostructure, edge-activating surfactants such as TWEEN-80 are used in a suitable ratio with vesicle-forming surfactants. This leads to the modification of fluidity, elasticity, and integrity of the vesicles (32, 33). The encapsulation efficiency (EE) of micelles is affected by the HLB value. Micelles exhibit a high EE at an HLB of 8.6 (34, 35). It is important to note that in the present study, a micellar structure with an HLB of 8.58 was obtained by combining these two structures. This approach is expected to increase cellular uptake and circulation in the blood under in vivo conditions, ultimately enhancing the kinetic stability of the drug carrier.

According to Fig. 3 (a), an average size of 25 nm was obtained for the studied micellar nanocarrier. This small size is beneficial for achieving a higher circulation time (t _1/2) and for parenteral delivery, as the chance of capillary blockage by nanosized droplets is minimal. The dimensions of the nanocarrier were much smaller than the diameter of the smallest blood capillary (400 nm), making it unlikely to cause capillary occlusion (36). Additionally, as shown in Fig. 3 (b), the zeta potential of the nanomicelles indicates a neutral electrostatic potential, with a zeta potential between + 10 and − 10 mV considered neutral. The nonionic nature of the surfactants used is one of the main reasons for the neutralization of this potential (37, 38).

In a study conducted by Kirchherr et al., with the aim of stabilizing ICG by encapsulation in micellar systems, the average diameter was 12 nm, the zeta potential was − 1.2 mV and the PDI was approximately 0.05 for ICG loaded in micelles prepared with surfactant. Additionally, the absorption range of ICG in these micelles increased by 40% compared to the free aqueous solution and showed a redshift of 25 nm (39). Among the reasons for the differences in the absorption range, diameter, and PDI of their study structure with the current study, we can mention the difference in the surfactant used (CMC, molecular structure, and HLB), synthesis method, and ICG concentration. In their study, the concentration of ICG was 0.0128 mM, but in the present study, the concentration of ICG was 0.002 mM. On the other hand, the HLB of the surfactant used in the study of Kirchherr et al. was reported to be between 14 and 16, while the HLB of the nanostructures in the present study was approximately 8.6.

According to the results presented in Fig. 4, the difference in average survival in all concentrations of ICG loaded micelles groups was significant compared to the control group. However, the difference in average survival in all groups containing free ICG compared to the control group was not significant. On the other hand, the average percentage of survival in the 100 µM ICG-loaded micelle groups was not significantly different from that in the 10 and 20 µM ICG-loaded micelle groups, and it was also not significantly different from that in the 40 and 80 µM ICG-loaded micelle groups. These results indicate that the micellar structure carrying ICG had higher toxicity than the free dye, suggesting that the micelles are more effective in targeting Leishmania parasite promastigotes. This is likely due to the hydrophobic property of the prepared nanomicelles, which increases the rate of passage through the phospholipid membrane. Accordingly, the concentration of 100 µM, which induced less than 20% inhibition of parasite growth, was used to investigate the photodynamic/photothermal effect, as shown in the survival curve of the Leishmania parasite in the presence of ICG with and without micelles. It should be noted that this low toxicity of ICG was also reported by other researchers (17, 40). On the other hand, one of the main advantages of ICG over porphyrins is its minimal absorption in the 400–600 nm range, which results in lower phototoxicity on the skin (41).

According to the results shown in Fig. 5 during the in vitro phase, the average combination index for the micellar groups was significantly higher than that of the free ICG group. This indicates that the micellar nanostructure is more efficient in treatment. Additionally, both the combined index and RLS calculations showed a synergistic effect in the groups containing ICG. It appears that the reason for the better performance of the nanostructure compared to the free dye is due to the enhancement of the absorption peak of ICG at the wavelength of 808 nm, which is 2.5 times higher in the nanomicelles compared to the free dye. The micellar structure was used in this study for several reasons. First, it increased the stability of the dye and its dispersion in water. Additionally, it improved the photophysical and photochemical properties of the dye, all of which are related to reducing the clumping of the dye. Finally, it may lead to better long-term blood circulation in in vivo conditions. Considering the superficiality of leishmaniasis lesions, it was expected that PDT could be effective in treating or at least preventing the progression of lesions. This is because PDT leads to more ROS production, which in turn causes more oxidative stress and a local increase in temperature.

It should be noted that in the group without medicinal agents, the survival rate decreased slightly after laser exposure. It has been suggested that cytochrome c oxidase (Cox) is the primary light receptor in mammalian cells. Cox has the most absorption in the visible range and some in the NIR range. This result can also be applied to the mitochondria of parasites (42). Therefore, the findings obtained from irradiation of promastigotes at an 808 nm wavelength in the absence of ICG can be justified.

Onda et al.'s study on cancer cells (HT-29) showed that ICG accumulates mainly in lysosomes within 24 hours of incubation (43). Therefore, due to the short lifetime of singlet oxygen and the localization of ICG within the lysosome, their absence from the parasite nucleus means that DNA damage (genotoxicity) through ROS is unlikely to occur (13).

In vivo studies showed that the growth of cutaneous lesions was completely inhibited only after ICG-NIR irradiation (with and without micelles) and not after NIR irradiation alone. These findings indicate that ICG is necessary as both a photothermal agent and photosensitizer for effective ICG-NIR therapy. On the other hand, due to an increase in lesion size observed in the treatment groups from the 15th day, it is necessary to repeat the treatments. According to our measurements, the average depth of the lesion was almost 5 mm. It is widely believed that ICG-NIR therapy is a useful treatment option for cutaneous Leishmania, as the reported penetration depth of NIR light is approximately 10 mm (44, 45). The synergistic effect of ICG and NIR radiation is expected to primarily cause parasite destruction through apoptosis (44).

The use of a combination therapy involving PDT/ PTT, mediated by ICG may be a favorable choice for creating novel, affordable, and secure substances that can combat Leishmania L. major. This method shows great potential for future research. However, ICG has a tendency to clump together in aqueous solutions, which restricts its application in phototherapy. To encapsulate indocyanine green, a biocompatible surfactant micelle-based method was used, reducing dimerization of the cyanine dye compared to free ICG. This resulted in decreased self-quenching and a significant increase in absorbance, with a 26 nm redshift in the absorbance spectra. Micelle incorporation of ICG can prevent plasma protein binding, increasing its in vivo half-life. The surfactants used are approved for parenteral administration and have a lower temperature sensitivity than PEG surfactants due to their nonionic nature.

PDT: Photodynamic therapy

PTT: Photothermal therapy

ICG: Indocyanine green

L. major: Leishmania major

HLB: Hydrophilic–lipophilic balance

NIR: Near-Infrared

ROS: Reactive oxygen species

CMC: Critical micelle concentration

Cox: Cytochrome c oxidase

PDI: Polydispersity Index

IC₂₀: Inhibitory concentrations 20

RLS: Relative lethal synergism

CI: Combination index

Acknowledgments

We are grateful to Dr Ali Khamesipour (Center for Research and Training in Skin Diseases and Leprosy, Tehran University of Medical Sciences, Tehran, Iran) for kindly providing the L. major strain MRHO/IR/75/ER.

Author contributions

MH: Performing the experiments; analyzing and interpreting the data; writing the paper. SJ: participating in the design of the experiment; Revising and giving final approval. AS: Conceiving and designing the experiments; analyzing and interpreting the data; contributing reagents, materials, analysis tools, or data.

Funding

This research was funded by Department of Health, Rescue and Treatment of I.R. Iran Police Force, Applied Research Center (A-10-1929-1).

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Ethics approval and consent to participate

All animal experiments were approved by the Institutional Animal Care and Use Committee and were in compliance with National Institutes of Health guidelines. In addition, disease burden did not exceed the recommended dimensions and animals were anesthetized and sacrificed using acceptable method techniques.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Photo-induced antileishmanial activity of indocyanine green: In vitro and in vivo studies

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Abstract

Figures

Introduction

Materials and Methods

Chemicals

Preparation and characterization of ICG-loaded micelles

Parasite cultivation

Cytotoxicity of the agents

Animal models

ICG-NIR irradiation

Statistical analysis

Results

Characterization of ICG-loaded micelles

In vitro antileishmanial activity

In vivo antileishmanial activity

Discussion

Conclusion

Abbreviations

Declarations

References

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