Exploring the potential of curcumin-loaded PLGA nanoparticles for angiogenesis and antioxidant proficiency in zebrafish embryo (Danio rerio)

Background

Curcumin is an age old traditional medicine. Although curcumin has several advantages, its water solubility and bioavailability limit its use as a natural therapeutic agent. Polymeric nano curcumin could be an excellent option to overcome these challenges to augment its therapeutic efficacy. This work aimed to synthesize curcumin-loaded PLGA nanoparticles and assess their angiogenic and antioxidant potential in the zebrafish model.

Results

The double emulsion solvent evaporation process was employed to make curcumin-loaded PLGA nanoparticles. Curcumin showed ~ 28.23 (± 2.49) encapsulation efficacy with an average diameter of PLGA nanoparticles 168.5 (± 2.5) nm and curcumin nanoparticles about 281.6 (± 17.2) nm, respectively. The curcumin nanoparticles showed no developmental toxicity to the zebrafish embryos while reduced toxicity compared to the native curcumin. Further, the curcumin nanoparticles reduced the generation of reactive oxygen species and improved angiogenesis in the model system. All these results confirmed that the nanoparticle has had higher bio-efficacy than that of native curcumin.

Conclusion

This study shows that PLGA curcumin nanoparticles hold an excellent therapeutic promise for wound healing, tissue regeneration and other biomedical applications where angiogenesis and ROS play critical role.

Since ancient times, people have been using plants and their bioactive metabolites for various medical purposes. Numerous phytochemicals like resveratrol, linamarin, cyanidin, curcumin, apigenin, epigallocatechin gallate, indole-3-carbinol, and flavopiridol displayed potent bioactive properties that can help to manage the onset and progression of illnesses [1]. Curcumin, a natural polyphenolic bioactive secondary metabolite from the rhizome of Curcumin longa with a wide variety of pharmacological actions such as anti-inflammatory, antioxidant, anticancer, antidiabetic, antimicrobial, tissue regeneration, and angiogenesis potentials [2,3,4]. Despite numerous advantages, hydrophobicity, low pharmacokinetics, low bioavailability, and poor absorption prevent its therapeutic applications [5]. Several researchers reported that lowering the size of bioactive substances improved their solubility and bioavailability and could lead to an improvement in their therapeutic potential like garlic acid nanoparticles [6] and date seed nanoparticles [7]. The synthesis of polymeric curcumin nanoparticles might improve the solubility and bio-efficacy of curcumin.

The polymeric nanoparticles have emerged as a cutting-edge technology for improving the solubility and bio-efficacy of drugs [8]. Furthermore, the polymeric nanoparticles showed limited toxicity, immunogenicity, increased solubility, biocompatibility, biodegradability, and extended drug circulation time [9]. The use of biodegradable polymers in tissue engineering, gene transfer, and biomedical devices has received more interest in the past few years. Among these polymers, polylactic-co-glycolic acid (PLGA) is a widely used biodegradable polymer approved by the US Food and Drug Administration (FDA). The PLGA polymer is an effective drug carrier because of its advantageous characteristics, such as lack of toxicity, biodegradability, compatibility with biological systems, and increased drug bioavailability [10].

Zebrafish (Danio rerio) have become an essential animal model due to their genetic similarities to humans, making them an effective in-vivo model for studying human biology and illnesses. Approximately, 70% of genes between humans and zebrafish are found similar, and because of their transparent embryos, simplicity of handling, affordability, and rapid development, they are a useful in-vivo model organism for various studies [11]. A study on zebrafish embryos examined the impact of Curcuma longa extract on the embryos, and the results revealed deformity, irregular heartbeats, reduced hatching rate, and increased mortality [12]. An in-vitro study on human hepatoma G2 cells revealed that curcumin did not have any mutagenic effects at low concentrations (2.5 μg/mL); however, at higher concentrations (10–40 μg/mL), it showed nuclear and mitochondrial DNA damage [13]. Thus, limiting toxicity and improving bio-efficacy could represent the most effective strategy for developing curcumin as a better therapeutic candidate.

The ultimate goal of this study was to synthesize and characterize curcumin nanoparticles, simultaneously investigating their toxicological, antioxidant, and angiogenic properties. The particular aims were to (a) assess the developmental toxicity of curcumin nanoparticles, (b) assess their antioxidant activity using H2DCFDA staining, and (c) assess the effect on inter-segmental blood vessel development in the zebrafish model.

Chemicals and reagents

Poly-(lactic-co-glycolic acid) (PLGA), polyvinyl alcohol (PVA) with an average molecular weight ranging from 89,000 to 98,000, Pluronic F-127, acridine orange, tricaine, phenylthiourea (PTU), and 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) procured from Sigma-Aldrich, Germany. The author has procured the standard curcumin from Abcam Biochemicals, UK. The chemicals used in this study, including dimethylsulfoxide (DMSO), dichloromethane (DCM), sodium dodecyl sulphate (SDS), and sodium hydroxide (NaOH), were obtained from HiMedia, India.

Synthesis of PLGA curcumin nanoparticles

The water-in-oil–water (W1/O/W2) double emulsion solvent evaporation method was employed to prepare curcumin-loaded PLGA nanoparticles (PLGA, 7000–17000 MW, Sigma-Aldrich) with slight alterations [14]. Briefly, 100 mg PLGA solution was ready in the organic phase (dichloromethane), followed by the addition of 10 mg of curcumin and 5 mg of Pluronic F-127 into PLGA solution and the initial emulsion was performed by sonicating for 10 min (Qsonica, USA) on ice with 5 mL polyvinyl alcohol (25 mg/ml). The secondary emulsion was prepared by adding the primary emulsion to 40 ml of polyvinyl alcohol and replicating it on ice for the next 15 min. The suspension was placed at room temperature and stirred overnight for complete evaporation of the organic solvent. The curcumin nanoparticles were collected by ultracentrifugation (Beckman Coulter, Optima XPN Ultracentrifuge, USA) at 27,000 rpm, 4 °C for 30 min and washed twice with distilled water. Finally, the pellets were dispersed in distilled water and lyophilized for 48 h (Zirbus Technology, Germany). The empty PLGA nanoparticles were prepared following the same protocol without adding curcumin. In the freezer, the lyophilized product was stored until further use.

Characterization of nanoparticles

Dynamic light scattering

The hydrodynamic diameter, poly dispersity index (PDI), and zeta value of the nanoparticles were determined using the zeta sizer (SZ-100V2, Horiba Instruments Ltd, Japan). In short, the lyophilized samples were prepared by dissolving them in distilled water and dispensed into dynamic light scattering (DLS) cuvettes. The experiment was performed in a controlled environment at 25 °C temperature with a scattering angle 90° [15].

Surface morphology by scanning electron microscopy

The vacuum ion sputtering equipment applied a thin layer of gold to the dried samples, and the sample was kept in the chamber for 15 min. After the gold coating, the images were taken at various magnifications (20.00–30.00 KX magnification, 200 nm scale) [16].

Molecular characterization by FT-IR spectroscopy

The Fourier transform infrared spectroscopy (FT-IR) technique was used to examine the molecular composition of the samples. The experiment was conducted in a controlled environment using a scanned wave range of 4000–400 cm⁻¹ (Shimadzu Kyoto, Japan) [15].

Evaluation of encapsulation efficiency (EE)

The curcumin encapsulation efficiency (EE) and loading capacity (LC) were confirmed by comparing the peak area of standard curcumin (R2 = 0.998) at a wavelength of 425 nm using UV–vis spectrophotometer (BioSpectrometer, Eppendorf, Germany). The drug encapsulation efficiency and drug loading capacity of nanoparticles were assessed using the following formula [17]:

where A is the total amount of curcumin used, B is the amount of free curcumin present in the supernatant.

In vitro release studies

In short, the nanoparticle (~ 5.0 mg) was dispersed in 3 ml of phosphate-buffered saline (PBS) and divided equally into three micro-centrifuge tubes, each containing 1 ml of the nanoparticle suspension. The tubes were placed in a shaker incubator (RivoTEK, Orbital Shaker, India) at 37 °C temperature with a rotational speed of 100 rpm. The nanoparticle suspension was collected by centrifugation at 12,000 rpm, four °C temperature for 10 min (Eppendorf 5424R, Germany), with an interval of every 3 h, followed by replacement with an equal volume of fresh phosphate-buffered saline (PBS). The concentration of curcumin released from the nanoparticle was measured using a UV–vis spectrophotometer at 425 nm wavelength (BioSpectrometer, Eppendorf, Germany) [18].

In-vivo zebrafish study

Zebrafish maintenance and rearing

The adult zebrafish were raised in the aquaculture system with an ambient environmental condition like 28 °C temperature and a fixed 14/10 h a day/night cycle. The fishes were kept at a 1:2 (female: male) ratio in the breeding chambers overnight before matting. The fertilized embryos were collected and cleaned with distilled water the following day. The cleaned embryos were then transferred in the fresh Petri dishes containing E3 media and were grown at 28 °C temperature [12]. Healthy embryos were chosen for the experiments, and all the experiments were performed with the permission of Nitte University Centre for Science Education & Research, Mangalore, following the institutional standard of animal ethical guidelines.

In-vivo toxicity study

In this experiment, the wild-type AB line embryos (10/well) were treated with drugs at various concentrations (2–16 µg/ml) for 24 h. After twenty-four hours of incubation, the mortality was assessed by recording images with the help of a stereomicroscope. The experiment was carried out in triplicates for each concentration.

Evaluation of developmental and organ toxicity

The developmental toxicity of curcumin and curcumin nanoparticles in zebrafish was evaluated by examining the embryos for five days. Cardiotoxicity, hepatotoxicity, hatchability, mortality, somite formations, and body abnormalities were all recorded. In short, ten healthy embryos were divided into four groups: control (no exposure), curcumin, PLGA nanoparticles, and curcumin nanoparticles. The embryos were exposed to a concentration of 1 µg/ml, and every 24 h of incubation, the morphological changes were documented using a stereomicroscope (Leica S9D, Germany). After taking the images, the embryos were then incubated again with fresh E3 media containing the drug (1 µg/ml), and the cycle was repeated for five days. The experiment was carried out in triplicates [19].

Evaluation of antioxidant activity

Briefly, at 6 h post-fertilization (hpf), the embryos were exposed (10 embryos in each group) to H₂O₂ (100 mg/L) for ROS induction in the body, and 0.015% PTU was used for preventing the pigmentation of embryos. All the groups were then incubated with the compounds (1 µg/ml) for 24 h. To assess ROS generation, a ten µM H2DCFDA fluorescent probe (Sigma, USA) was used, and the samples were incubated for 1 h at room temperature in dark conditions. The embryos were anaesthesia with tricane (0.04%) before image acquisition. The images were taken at 10× magnification) using a fluorescence microscope (Leica DM2500, Germany). The ROS generation was estimated at two different time intervals (24 and 48 h). The fluorescence intensity of each fish was quantified using ImageJ software, and the data was compared with the control group. The experiment was conducted in triplicates [20].

Evaluation of angiogenic activity

For the angiogenesis study, a transgenic zebrafish (kdrl::GFP) was used. Briefly, at six hpf, the embryos (10 embryos in each group) were exposed to 0.015% of PTU for depigmentation, and the embryos were exposed to the tested compounds and subsequently evaluated for their effects on inter-segmental blood vessels (ISVs) after 72 h of post-fertilization. Before imaging, tricane (0.04%) was used to anaesthetize the embryos. All images were captured with a fluorescent microscope (Leica DM2500, Germany) at a 10× magnification. The experiment was performed in triplicates [21].

Statistical analysis

Statistical analysis was performed using one-way ANOVA and paired t-test; a p-value less than 0.05 was considered statistically significant. Data are represented as mean ± SD of three independent replicates for each experiment. P < 0.05, P < 0.01, and P < 0.001 are represented as *, **, and ***, respectively.

Nanoparticles characterizations

In this study, we synthesized curcumin-loaded PLGA nanoparticles using a double emulsion solvent evaporation method and investigated their antioxidant and angiogenic properties in an in-vivo zebrafish model system. The curcumin nanoparticles had an average size of approximately 281.6 (± 17.2) nm. In contrast, the PLGA blank nanoparticles had an average size of 168.5 (± 2.5) nm and the zeta value of curcumin nanoparticles + 4.62 (± 1.87) mV and PLGA nanoparticle − 6.05 (± 3.27) mV (Table 1).

The surface morphology of curcumin PLGA nanoparticles and PLGA blank nanoparticles showed that they were more or less uniform in size, which might help as an ideal drug delivery vehicle (Fig. 1A and B). Figure 1C illustrates the FT-IR spectra of PLGA and curcumin nanoparticles and the structural link between the two molecules. The curcumin spectra revealed peaks at 1509 cm⁻¹ (C=O and C=C vibrations), 1428 cm⁻¹ (olefinic C–H bending vibrations), and 1280 cm⁻¹ (aromatic C–O stretching vibrations) [19], and this is also evident in our investigation. PLGA’s distinctive peak between 3500 and 3400 cm⁻¹ represents a weak peak due to O–H stretching (broad), and the peak around 2850–3000 cm⁻¹ represents the C–H stretching in the aliphatic chain. The peak at 1081 cm-1 suggests the presence of C–O and the 1380 and 880 cm⁻¹ peaks may be connected with O–H vibrations [22]. Drug encapsulation plays a vital role in the encapsulation process. Encapsulated curcumin has shown more effective therapeutic efficacy than that of natural curcumin [23]. We found that the encapsulation efficacy of curcumin was about 28.23 (± 2.49), and the curcumin PLGA nanoparticles were dispersed in an aqueous solution without any aggregation. The study showed that ~ 58.62% of the curcumin was released within the first three hours, which is found almost similar to previous findings [24]. After 3 h, the release rate considerably slowed, and ~ 88.56% of the curcumin was released from the nanoparticles within 24 h. The initial increase in curcumin release within the first three hours might be attributed to the adsorbed curcumin to the surface of the nanoparticles, and curcumin began to release from the inner matrix of the nanoparticles as the surface-associated curcumin depleted (Fig. 1D).

Physicochemical characterization of curcumin-loaded PLGA nanoparticles. The surface morphology of the A PLGA nanoparticle and B curcumin nanoparticle was investigated using scanning electron microscopy at 100 nm scale with 20.00–30.00 k X magnifications. C The FT-IR spectra of standard curcumin, PLGA nanoparticles, and curcumin nanoparticles. The FT-IR spectra of curcumin nanoparticles show 1428 cm⁻¹ and 1280 cm⁻¹ peaks in the standard curcumin spectrum, representing the C–H bending and aromatic C–O stretching vibrations. D The cumulative curcumin release from the nanoparticles was investigated in PBS (pH 7.4), and the absorbance was taken at 425 nm using UV–vis spectrophotometry. All the experiments were performed in triplicate

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The cytotoxicity of curcumin on the zebrafish larvae was investigated over 24 h of incubation. The impact of curcumin on the survival rate of zebrafish embryos was examined by observing the coagulation of eggs and the heartbeat of the embryos. The zebrafish embryos showed no toxicity at ~ 2.71 µM of concentration and showed 100% survivability. Further, we also found that increasing the concentration of curcumin (~ 10.85 µM/ml) reduces the rate of survivability, which was also found similar to the result reported by another research group (~ 10.00 µM/ml) [25]. The survival rate of curcumin-treated embryos decreased significantly at values above ten µM. In contrast, no mortality was recorded with the curcumin nanoparticle group carried out on zebrafish embryos at the same concentration, and a similar report was published by Abdullah et al. [25]. We also found that the mortality rate increased with the increasing concentration of native curcumin. However, the PLGA blank and curcumin nanoparticles were nontoxic at the same concentration (24 h of exposure). 1 mg/L concentration of drugs showed no embryotoxicity, and to further confirm whether the dose was safe for the organism, the embryos were exposed, and five days of developmental study was performed. We found that the concentration (1 mg/L) showed no teratogenic toxicity like bend tail, bend trunk, body curvature, or yolk sac oedema, and the organs like the heart and liver were also found to be expected (no oedema) after five days of exposure (Fig. 2). The heart rates of all groups were in the normal range (Control: 170 ± (4), curcumin: 164 ± (2), PLGA nanoparticle: 162 ± (2) and curcumin nanoparticle: 172 ± (2)). Further, no deformities like bend tail, oedema, yolk sac extension, and somite formation were reported. So, it was confirmed that the dose (1 mg/L) had no teratogenic effects on the model organism. Further, the embryonic hatching rate was assessed between 48 and 72 hpf, and it is interesting to note that by 72 hpf, all embryos had hatched, hence no delayed hatching.

Evaluation of developmental toxicity study of curcumin, PLGA nanoparticles, and curcumin nanoparticles on zebrafish (Danio rerio). A 5-day morphological study for detecting oedema, pigmentation, tail deformities and mortality. The organ toxicity, like cardiotoxicity, hepatotoxicity, heart rate, and hatching ability of zebrafish embryos, was evaluated after exposure to the compounds. B Represent the morphology of the heart, liver and swim bladder, and the result showed that the curcumin nanoparticles had no adverse effects during the organogenesis of zebrafish embryos. C and D, the bar graph represents embryos' heart rate and hatching percentage after exposure to the compounds. It was found that all the groups showed average heart rate and hatching ability. The experiment was performed in triplicates, and the P < 0.05, P < 0.01, and P < 0.001 are denoted as *, **, and ***, respectively

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Several toxicological studies have already used the zebrafish to investigate the in-vivo effect of drugs or nanoparticles on redox equilibrium, indicating that this species is suitable as an animal model for the field of drug development and oxidative stress. The antioxidant effect of curcumin nanoparticles was assessed by measuring the reduction in ROS signal due to H₂O₂. ROS production has been observed using H2DCFDA, a cell-permeable dye that converts from a nonfluorescent to a fluorescent state in the presence of an oxidative stress response. The protective effect of curcumin nanoparticles against H₂O₂-induced oxidative stress was evaluated by using H2DCFDA staining. This study employed H₂O₂ as a positive control, and the ROS level was measured at two different periods (24 and 48 h). The results showed that curcumin nanoparticles did not cause substantial amounts of ROS generation in the body, whereas curcumin did (Fig. 3). These findings indicated that curcumin nanoparticles protect against H₂O₂-induced oxidative stress in the zebrafish model.

Assessment of the antioxidant potential of standard curcumin, PLGA nanoparticles, and curcumin nanoparticles. A After 24 and 48 h of exposure, the curcumin nanoparticle displayed minimal generation of ROS in the embryos compared to the regular curcumin and positive control (H₂O₂), and B The bar graph represents the fluorescence intensity of all treatment groups after 24 and 48 h of exposure. P < 0.05, P < 0.01, and P < 0.001 are denoted as *, **, and ***, respectively (n = 3)

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Angiogenesis has been recognized as a critical step in the regeneration and restoration of many tissues. Curcumin has already been reported as an efficient natural agent in wound healing, and the angiogenic property of the curcumin nanoparticles was assessed in a transgenic zebrafish (kdrl::GFP) that expresses GFP in the blood vessels, allowing real-time monitoring of the progression of angiogenesis. As shown in Fig. 4, the curcumin nanoparticles improved the structure and generation of new blood vessels compared to native curcumin (Fig. 4). The elevated sprouting of angiogenesis refers to an increase in the formation of new blood vessels that confirm curcumin nanoparticle has better angiogenesis potential compare to the native curcumin.

Evaluation of angiogenic sprouting of inter-segmental blood vessels (ISVs) on the transgenic zebrafish (kdrl::EGFP) embryos. The result showed that after exposure to curcumin nanoparticles, the sprouting of inter-segmental blood vessels increases compared to the standard curcumin, respectively (n = 3)

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The nanomedicine and nano drug delivery systems are relatively new research fields that aim to increase the solubility and bio-efficacy of pharmaceutical molecules [26]. Many studies have been conducted to develop more effective drug delivery methods, and the preparation and characterization of drug-encapsulated nanoparticles are necessary in this field. Particle size is crucial since it immediately impacts the nanoparticles' physical stability, cellular absorption, biodistribution, and drug release [27]. It was found that small nanoparticles (NPs) have higher penetration than larger nanoparticles [28]. Several researchers reported the size of the PLGA curcumin nanoparticles to range from 150 to 495 nm [29], and we found that our developed curcumin-loaded PLGA nanoparticles (281.6 ± 17.2 nm) are within the specified range. This smaller particle size could penetrate the mucus layer and improve its bio efficiency. Generally, the PLGA nanoparticles exhibited negative zeta values [30], also observed in our investigation. We found that the zeta potential of the PLGA blank nanoparticle was about -6.05 mV (± 3.2), and the curcumin nanoparticle was + 4.62 mV (± 1.87). The low and reverse zeta potential of the nanoparticles might be because the curcumin molecules may adsorb onto the surface of the nanoparticles, altering the surface charge; likewise, PVA can also adsorb onto the surface of the nanoparticles, potentially masking the carboxyl groups of PLGA, resulting in a lower or even reversed zeta potential.

Spherical nanoparticles have many advantages because of their uniform shape, huge surface area, and ease of production that make them ideal for a wide range of applications, like drug delivery. The surface of the curcumin nanoparticles was found to be spherical as analysed by scanning electron microscopy, and this shape of the nanoparticles is also an essential parameter for sustained drug release and the nanoparticle's toxicity [31]. It was reported that rod-shaped Fe₂O₃ nanoparticles produced significantly higher cytotoxic levels than sphere-shaped Fe₂O₃ nanoparticles [32], and this spherical-shaped nanoparticle also plays a crucial role in drug circulation [33]. Encapsulation efficiency (EE) of the drug is another critical parameter in the formulation study, and the EE is affected by several factors, including polymer content, surfactant type, aqueous-organic phase volume ratio, and others [34]. Encapsulating curcumin in PLGA (poly(lactic-co-glycolic acid)) nanoparticles is a promising strategy to improve the solubility, stability, and bioavailability of curcumin. The emulsion solvent evaporation method was commonly used for this purpose, providing nanoparticles with sustained release properties. The drug's release study was examined in a PBS solution at pH 7.4. We found that the initial release of curcumin from the formulation was a burst release, possibly due to the drug molecule's weak bonding on the nanoparticles’ surface. However, a prolonged drug delivery system retains plasma drug concentration, improves bio-efficacy [35], and reduces toxicity [36]. We found that after 3 h, the release rate considerably slowed, and ~ 88.56% of the curcumin was released from the nanoparticles within 24 h (30 h: ~ 95.64%). The initial increase in curcumin release within the first three hours might be attributed to the adsorbed curcumin to the surface of the nanoparticles, and curcumin began to release from the inner matrix of the nanoparticles as the surface-associated curcumin depleted. The rate of drug release from nanoparticles correlates with the formulation composition, drug molecular structure and the relative affinities of the drug and polymer, as well as the aqueous phase. It has been reported that nanoparticles protect their payload from early enzymatic and chemical degradation, increasing bioavailability and cellular absorption [37].

Curcumin, a bioactive substance, has several limitations, including poor absorption, limited water solubility, instability at physiological pH, and harmful effects on normal cells. Because of these difficulties, curcumin's therapeutic value has significantly decreased [38]. It was reported that at 10 µM dose curcumin induced deformities in embryos with apparent curvature body, bent tail, yolk sac oedema, etc. [39], and we also observed almost similar results in our study (~ 10.85 µM). Zebrafish embryos are structurally and functionally identical to humans [38]. It is generally assumed that the cytotoxic impacts of curcumin nanoparticles in zebrafish embryos are strongly associated with human embryonic development [25]. The toxicity profile was investigated by examining the developmental alterations that occurred after the drug exposure. The curcumin nanoparticles showed delayed cytotoxicity, and a similar report was published by Shamsi et al., claiming that curcumin nanoparticles had a delayed cytotoxic effect on HepG2 cells compared to curcumin. The delayed cytotoxicity observed with curcumin-loaded PLGA nanoparticles is primarily due to the controlled and sustained release of curcumin from the nanoparticles, protection of curcumin from degradation, gradual cellular uptake and internalization of nanoparticles [40, 41]. These findings also represent that prolonged exposure to curcumin nanoparticles may be needed to achieve its full therapeutic effect at the cellular level. Using zebrafish as a model to investigate developmental study of curcumin-loaded PLGA nanoparticles offers a comprehensive approach to understanding their biological effects, safety, and therapeutic potential. The embryos exposed to curcumin solution developed a yellowish tint, indicating that the substance had passed past the chorion and affected the embryonic cells. High concentration of curcumin is harmful for zebrafish survivability and embryonic development, and we found that, at the same concentration (1 µg/ml), both the curcumin and curcumin nanoparticles showed no teratogenic effects to the model system. The organs like heart and liver showed no oedema and no abnormalities like curvature body, yolk sac oedema, low hatching rate, and abnormal heart rate were absent.

The reactive oxygen species (ROS) are produced through various cellular oxidation processes, impair cellular homeostasis and structures, and lead to oxidative stress [42]; further, the overproduction of ROS causes cellular death by damaging protein, DNA, and lipid structures [25]. ROS, such as superoxide and hydrogen peroxide, are commonly produced during H₂O₂ exposure, and a decrease in ROS signal in embryos treated with test compounds compared with just H₂O₂-treated embryos suggests that the test compound might possess antioxidant effects. Curcumin has ten times antioxidant properties of vitamin E, depending on the dose or concentration of curcumin [43]. H2DCFDA is oxidized by reactive oxygen species (ROS), such as hydrogen peroxide (H₂O₂), hydroxyl radicals (•OH), and peroxynitrite (ONOO–), to form the highly fluorescent compound DCF [44]. Higher the fluorescence intensity signifies higher generation of ROS. We observed that after exposure with curcumin nanoparticles, the fluorescence intensity significantly reduced in the zebrafish embryos compared to native curcumin, resulting in increased curcumin antioxidant properties. It could be because the curcumin exhibited sustained release from the encapsulated nanoparticles. Curcumin has long been recognized as a useful phytochemical used in Indian households to treat various skin injuries, diseases, and other inflammatory diseases [45]. Curcumin can promote wound healing by accelerating the angiogenesis by activating growth factors, modulating inflammatory responses, stimulating endothelial cell activities, and enhancing cell survival [46, 47]. Curcumin also promotes neovascularization and small capillary growth in the rat model [48], as well as in a streptozotocin-induced diabetes model [49]. Curcumin concentrations of 1–5 µM/ml were displayed to promote the development of new blood vessels and the expression of angiogenesis-related genes [4], and in our study, we also found the similar result between the reported curcumin concentrations (~ 2.7 µM/ml). The result confirmed that the curcumin nanoparticle has been established as a potent therapeutic agent in wound healing and tissue regeneration.

In a nutshell, the study demonstrated that curcumin nanoparticles had no significant impact on the survivability of fish embryos while could reduce curcumin's toxicity with higher antioxidant and angiogenic activity indicating their safe pharmacological applications. Only curcumin generated higher ROS than curcumin nanoparticles, and it was correlated with the cytotoxic response in zebrafish. The data given in this study may serve as the foundation for developing curcumin nanoparticles as a more biocompatible alternative to curcumin in medicinal applications.

The data that support the findings of this study are available from the corresponding authors, upon reasonable request.

ROS:

Reactive oxygen species

PLGA:

Polylactic-co-glycolic acid

FDA:

Food and Drug Administration

H2DCFDA:

2′,7′-Dichlorodihydrofluorescein diacetate

PVA:

Polyvinyl alcohol

PTU:

Phenylthiourea

DCM:

Dichloromethane

SDS:

Sodium dodecyl sulphate

NaOH:

Sodium hydroxide

PDI:

Particle dispersion index

DLS:

Dynamic light scattering

FT-IR:

Fourier transform infrared spectroscopy

EE:

Encapsulation efficiency

PBS:

Phosphate-buffered saline

hpf:

Hours post-fertilization

kdrl:

Kinase insert domain receptor like

GFP:

Green fluorescent protein

The authors thank to NITTE University (Deemed to be University) for providing the resources and facilities to conduct the experiments. The authors also thank the Central Instrument Centre of Tripura University for all the instrumentation facility.

This research was not funded by any specific grants from funding agencies in the public, commercial, or not-for-profit sectors.

Authors and Affiliations

1. Cancer Biology, Cell Signalling and Molecular Genetics Lab, Department of Human Physiology, Tripura University, Suryamaninagar, Madhupur, 799022, India

   Achinta Singha & Samir Kumar Sil

2. Department of Bio and Nano Technology, NITTE University Centre for Science Education and Research, Nitte (Deemed to be University), Paneer Campus, Deralakatte, Mangalore, 575018, India

   Mave Harshitha, Biswajit Maiti & Akshath Uchangi Satyaprasad

3. Department of Molecular Genetics and Cancer, Nitte University Centre for Science Education and Research, Nitte (Deemed to be University), Paneer Campus, Deralakatte, Mangalore, 575018, India

   Krithika Kalladka, Gunimala Chakraborty & Anirban Chakraborty

Contributions

AS (Achinta Singha) contributed to conceptualization, methodology, validation, software, formal analysis, investigation, data curation, writing—original draft. HM (Mave Harshitha) was involved in investigation, data curation, writing—review & editing, visualization. KK (Krithika Kalladka) done software, formal analysis, investigation, data curation. GC (Gunimala Chakraborty), BM (Biswajit Maiti), and AUS (Akshath Uchangi Satyaprasad) were involved in formal analysis and writing—review & editing. AC (Anirban Chakraborty) and SKS (Samir Kumar Sil) contributed to conceptualization, formal analysis, writing—review & editing, project administration.

Corresponding authors

Correspondence to Anirban Chakraborty or Samir Kumar Sil.

Ethics approval and consent to participate

All of the in-vivo zebrafish studies were carried out with the approval of the NITTE University Centre for Science Education & Research in Mangalore, Karnataka, India, in accordance with institutional animal ethics regulations.

Competing interests

The authors declare that they have no competing interests.

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