HSA-curcumin nanoparticles: a promising substitution for Curcumin as a Cancer chemoprevention and therapy

Abstract

Background

Many solutions have been evaluated to deal with “chemotherapy and radiation-resistant cancer cells’ as well as “severe complications of chemotherapy drugs”. One of these solutions is the use of herbal compounds with antioxidant properties. Among these antioxidant compounds, curcumin is identified as the strongest one to inhibit cancerous cells proliferation. However, its clinical trials have encountered many constraints, because curcumin is insoluble in water and unstable in physiological conditions. To overcome these limitations, in this study, curcumin was conjugated with human serum albumin (HSA) and its effects on breast cancer cell lines were also measured.

Methods

After making of HSA-curcumin nanoparticles (NPs) by the desolvation technique, they were characterized by the FTIR, DLS, TEM, and SEM method. At the end, its anticancer effects have been examined using MTT test and apoptosis assay.

Results

The FTIR graph confirmed that curcumin and HSA have been conjugated along with each other. Particles size was reported to be 220 nm and 180 nm by DLS and SEM, respectively. The zeta potential of HSA-curcumin NPs was −7 mV, while it was −37 mV for curcumin. The MTT and apoptosis assay results indicated that the toxicity of HSA-curcumin NPs on the normal cell are less than curcumin; however, its anti-cancer effects on the cancer cells are much greater, compared to curcumin.

Conclusion

HSA-curcumin NPs increase curcumin solubility in water as well as its stability in physiological and acidic conditions. These factors have the ability of overwhelming the limitations on using curcumin alone, and they could result in a significant increase in the toxicity of curcumin on the cancer cells without increasing its toxicity on the normal cells.

Grapical abstract

Introduction

The advent of chemotherapy-resistant and radiation-resistant cancer cells along with severe complications of chemotherapy drugs, have led researchers to explore alternative therapies [1]. The use of herbal compounds, which have antioxidant properties as chemoprevention and can reduce the dose of these drugs as well as their side effects, has been considered very much along with chemotherapy drugs [1, 2]. Among the herbal antioxidants, curcumin is one of the most potent antioxidants to inhibit the proliferation of cancer cells, and also induce apoptosis in them [3–5].

Curcumin (Fig. 1) is a natural yellow compound obtained from plant Curcuma longa. Curcumin can be found in two forms of ketone and enol, which in pH > 8, enol form is predominant. Regarding, in enol form, curcumin acts as a potent antioxidant by losing its proton and reduction of its free radicals. However, curcumin has a very low dissolution in water, and its ketone form is predominant in acidic conditions [6] (the pH of the environment surrounding the tumor is acidic [7–10]). In addition, the enzymatic degradation rate of curcumin and its elimination from the blood stream is very high; therefore, the antioxidant effects of curcumin have significantly reduced in the body [11].

Fig. 1.

Curcumin (a) Chemical structure (b) Properties

In recent years, the nanotechnology advancement has led to the production of various nano-drugs for the treatment of certain diseases such as cancer [12–15]. This has also resulted in the production and testing of a wide range of curcumin nanoparticles to overcome its problems [16–21]. Various nano-carriers have been used to make curcumin-nanoparticles such as solid lipid nanoparticles [22–24], micelles and liposomes [25, 26], dendrimers [27–29], and various polymers [30–34]. Each of these curcumin-nanoparticles has some advantages and disadvantages. The important issue is that the nano-substances must be harmless, maintain the anti-cancer properties of curcumin, and also increase its solubility and stability in water and in blood, respectively [35–38].

Human serum albumin (HAS) (Fig. 2) is a natural compound that has recently been used as a nanoparticle in the production of nano-drugs [39–45]. Firstly, one of the albumin duties is the transfer of almost insoluble fatty acids in the blood [46, 47] and secondly, curcumin structure is roughly similar to fatty acids, so albumin can be easily attached to curcumin [48, 49]. In cancer, blood albumin level is lower than usual [50–53], so the successful production of albumin-curcumin NPs cannot only significantly increase the anti-cancer effect of curcumin, but also the released-albumin after curcumin delivery may also balance the level of albumin in the patients. In addition, albumin is naturally antioxidant and this property can enhance the effectiveness of the nano-drug [54, 55].

Fig. 2.

Human serum albumin (HSA) (a) 3D structure (b) Properties

In the present study, curcumin was conjugated with HSA, which was also characterized. Then, its anti-cancer effects were evaluated on two breast cancer cell lines (MCF7 and SKBR3), and were compared with curcumin.

Methods and materials

Preparation of HSA-curcumin NPs

HSA-curcumin NPs were prepared using the desolvation technique because it was a simple method, and required no advanced devices [56]. Briefly, 4 ml of 33 mM solutions of curcumin (minimum 94% curcuminoid content, gifted from basic sciences school of Tarbiat Modares University, Tehran, Iran) in ethanol (purity >96%, Merck) was mixed with 2 ml of 20% solution of HSA (at least 96% albumin, Iranian Blood Transfusion Organisation, Tehran, Iran) in purified distilled water, and 40 μL glutaraldehyde (25% Aqueous Solution, Merck) dropwise was added to the mixture. The mixture was well stirred for about 24 h at room temperature.

After 24 h, the resultant mixture was centrifuged (Model: Z38HK, Hermel Labortechnik GmbH, Germany) at 14000 rpm for 10 min. Then, the supernatant was discarded. The deposition was re-dissolved in distilled water, and after that was sonicated (Model: WUC-DIOH Wise clean, DAIHAN Scientific, Korea) for 5 min in bath sonicator. In the next step, it was again centrifuged for 10 min at 14000 rpm. The wash steps were repeated five times. HSA-curcumin NPs was lyophilized and kept in dark [57].

Characterization of HSA-curcumin NPs

1-Fourier-transform infrared spectroscopy (FTIR)

FTIR spectra of curcumin, albumin, and HSA-curcumin NPs were used to characterize the HSA-curcumin conjugate (PerkinElmer – Frontier, USA). The spectra were recorded in the range of 400 to 4000 cm − 1, and the scans have been done 3 times.

2-Particle size and zeta potential analysis

The particle diameter and zeta potential of curcumin NPs were measured three times using dynamic light scattering (DLS) (Malvern Instruments, UK). The size measurement was carried out at a concentration of 1.0 mg/mL of HSA-curcumin NPs in DMSO 10% at 25°^C.

3-Scanning electron microscopy (SEM) and transmission electron microscopy (TEM)

The HSA-curcumin NPs were gold coated by sputter coater (SBS 12, KYKY, China) for 30 s, and their morphology features and microstructures were determined by SEM (EM3200, KYKY, China) at an accelerating voltage of 20–26 kV with 40.0KX magnitude. Concurrently, these NPs were added to a carbon grid. After drying at room temperature, TEM was performed using TEM (ZIESS, EM900, PHILPS, Germany) at an accelerating voltage of 80 kV.

4-Water solubility analysis

To determine the water solubility of HSA-curcumin NPs, 1 mg of curcumin and an equivalent amount of HSA-curcumin NPs were added to 1 ml of deionized water. Then, it was vortexed for 10 min, and centrifuged at 14,000 rpm for another 10 min. Supernatants were mixed with ethyl acetate. Finally, the spectrophotometric detection of curcumin levels was carried out at 427 nm in triplicate.

5-Determination of loading and yield

The percent yield was calculated using the following equation:

$$\text{Yield}\%=\frac{\text{Weight of}\mathrm{HSA}\_\text{curcumin}\mathrm{NPs}}{\text{Total weight of Curcumin}+\mathrm{HSA}}\times 100$$

1 mg of HSA-curcumin NPs was dissolved in 10 ml of ethyl acetate/propanol (9:1, v/v) to determine the curcumin loading efficiencies in NPs. Immediately, the absorbance was measured at 427 nm free curcumin concentration. Next, the solvent was sonicated for 30 min to completely extract curcumin. Afterward, curcumin concentration in solution was determined by spectrophotometer at 427 nm, and also by the use of the standard curve. At the end, to determine the loading, the concentration of free curcumin was deducted from the final amount of curcumin that existed in the solution.

6-Determination of curcumin release

To simulate the physiological conditions and the tumor environment, the release mediums were phosphate-buffered saline (PBS, pH 7.4) and sodium citrate buffer (pH 5.6), respectively.

To determine the curcumin release, an innovation method was used as following:

The tubes containing 1 mg HSA-curcumin NPs in 1 ml release medium, were placed in a shaker incubator at 37°^C. The amount of curcumin was read by spectrophotometer at 427 nm after 1 min, 1 h, 2 h, 4 h, 8 h, 16 h, 1 day, 2 days, 4 days, and 8 days.

For sodium citrate buffer release medium, the progress was similar to that was mentioned above, except the temperature, which was 40 °^C due to the temperature difference of tumor environment.

Cell culture

Human breast adenocarcinoma cell lines namely MCF7 and SK-BR3 were obtained from Iranian Biological Research Centre (Tehran, Iran). The MCF7 cells were cultured in Dulbecco modified Eagle medium (DMEM) F12 (Bioidea, Iran) and SK-BR3 cells were cultured in DMEM- high glucose (Bioidea, Iran), supplemented with 10% FBS (Gibco, Invitrogen), 100 units/ml penicillin, and 100 μg/ml streptomycin. Peripheral blood mononuclear cells (PBMC) were obtained from fresh blood and cultured in Roswell Park Memorial Institute medium (RPMI) (Bioidea, Iran), supplemented with 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were maintained in a humidified incubator at 37°^C with 5% CO2 (BINDER, Germany). Also, Seeding and propagation of cancer cells were performed by trypsinization.

Cytotoxicity assay

Analysis of cytotoxicity was performed by standard 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Cells were seeded in 96 well plates (SPL life sciences, Korea) at a density of 8000 per well, and were then grown for 24 h. After that, Cells were exposed to various concentrations of curcumin, and HSA-curcumin NPs in 10 μl DMSO 10% for 24, 48, and 72 h. MTT assay solution (0.5 mg/ml) was then added to each well, and the plates were incubated at 37°^C for 3 h. After incubation, media was carefully removed and the purple Formosan precipitate was dissolved in DMSO on a rotary shaker for 20 min. The absorbance was read at the wavelength of 540 nm in a plate reader (ELISA reader, Labsystem Multiskan MS 352, Finland). Moreover, the assays were performed in triplicates. The data was plotted against the drug concentration and the relative cell viability (%) related to the control containing cell culture medium without the drug. This test was performed on PBMC after 24 h, to evaluate the toxicity of drugs on normal cells.

Apoptosis assay

Apoptosis was evaluated using Flowcytometry with Annexin V/ propidium iodide (PI) staining kit (Biovision). For this purpose, cells were seeded in a 12- well plate (SPL life sciences, Korea) at a density of 75,000 per well, and were then grown for 24 h. Then, the cells were treated with the appropriate dose of curcumin and HSA-curcumin NPs obtained from the MTT test (20 μg/ml for curcumin and 50 μg/ml for HSA-curcumin). Apoptosis assay was performed using flow cytometry (BD FACS, USA) for MCF7 and SK-BR3 cells after 24 and 36 h, respectively, in terms of the manufacturer protocol. To evaluate apoptosis in normal cells, apoptosis assay was performed on PBMC after 24 h. the obtained Findings were analysed by Flowjo software version 2.2.

Statistics analysis

IC50 (half maximal inhibitory concentration) for curcumin and HSA-curcumin NPs were calculated by Probit Regression analysis using SPSS version 14. The t-test was used for analysing apoptosis data, and P value less than 0.05 was considered as statistically significant. Using SPSS version 14, all results were analyzed and the graphs were drawn.

RESULTS and discussion

Preparation and characterization of HSA-curcumin NPs

FTIR

The FTIR spectra of curcumin, albumin, and HSA-curcumin NPs are shown in Fig. 3. As shown in Fig. 1, O-H bond at 3420 cm⁻¹ in HSA-curcumin NPs is slightly stronger than curcumin. It was shown that a new hydrogen bond between C=O of curcumin and amine group in albumin has been created; therefore, an associated peak of carbonyl groups (C=O) of curcumin at 1749 cm⁻¹ has almost disappeared in HSA-curcumin NPs. Also, a new bond at 2371 cm⁻¹ in HSA-curcumin NPs was created, which was the illustration of C ≡ N bond (weak) between curcumin and albumin. These data have confirmed that the conjugation of HSA-curcumin was made.

Fig. 3.

The FTIR graph of albumin, Curcumin and HSA-curcumin NPs

Particle size and zeta potential

The results of DLS and zeta potential are shown in Figs. 4 and 5, respectively.

Fig. 4.

The DLS results (a) Curcumin (b) HSA-curcumin NPs

Fig. 5.

The zeta potential results. a Curcumin b HSA-curcumin NPs

As shown in Fig. 2, the results of DLS have indicated that the binding of curcumin to albumin reduced the size of particles in the solution, due to the reduction in the amount of aggregation of curcumin particles (220 ± 2 nm vs. ~ 519 ± 2 nm).

HSA-curcumin NPs have been 30 mV more positive than curcumin (−37.3 ± 1 vs. 7.22 ± 1). The larger size and high negative charge in the particles can cause opsonisation and their rapid removal by macrophages [58]. Therefore, size reduction and the negative charge of curcumin in HSA-curcumin NP can reduce their phagocytosis, as well as increasing the duration of the curcumin presence in the bloodstream [59]. Also, the high negative charge of free curcumin increases its absorption by normal cells, and thus enhances its cytotoxicity [58]. In cancer cells, unlike normal cells, the main pathway of energy metabolism is glycolysis rather than oxidative phosphorylation. Due to this reason, lactate levels increase in extracellular space of cancer cells that leads to reduction in pH level [7, 60, 61].

Also in cancer cells, due to changes in the levels of sialic acid and lipid peroxidation products, the charge of cell membrane is more negative than normal cells [62]. These factors increase the charge difference between the cancer cells membrane and nanoparticles, which results in their proper absorption of HSA-curcumin NP. However, they reduce the absorption of curcumin due to reduction of difference in charge between cancer cells membrane and curcumin. The curcumin conjugation with albumin leads to a reduction in the contact of curcumin molecules with the acidic environment around the tumor, which reduces their presence in the vicinity of cancer cells in the form of ketones. While in free curcumin, molecules can be rapidly converted to their ketone form, due to contact with acidic environment around the tumor [11], which have not only no antioxidant properties, but also are rapidly aggregated and removed by phagocytosis [36, 58].

TEM and SEM

The results of the TEM image (Fig. 6a) in a 15-nm scale showed that each albumin molecule has bonded to about eight molecules of curcumin, which some of these curcumins have also attached to adjacent albumin molecules.

Fig. 6.

The TEM of HSA-curcumin NPs. The white arrow represents albumin particle and the yellow one represents Curcumin particles

As shown in the Fig. 4b, the particle coating is in which the albumin molecules are more in outside, and the curcumin molecules are inside.

This particle subset (Including about 10 albumin molecules with related curcumin molecules) has probably formed a spherical blend with a diameter of about 180 ± 2 nm, which was observed in the SEM (Fig. 7a). While curcumin particles had the lack of the spherical state (Fig. 7b).

Fig. 7.

The SEM result for (a) HSA-curcumin NPs (b) curcumin particles

Thus, the contact surface of the hydrophobic area with the solvent was reduced. This molecular arrangement model increases the solubility and stability of HSA-curcumin NPs in the blood, because it protects curcumin from enzymatic degradation until it reachesto the tumor site. In the environment of tumor, some of curcumin molecules are released, due to not only increasing of the temperature, but also due to pH reduction. Because they are small hydrophobic molecules, they directly pass through the membrane of cancer cells. Albumin molecules after binding to their receptors (GP60), were entered into endosomes of cancer cells by endocytosis [42, 43, 63]. In endosome, curcumin molecules that are still attached to the albumin, have released and escaped into the cytosol; and therefore they acted as antioxidant. Since amount of albumin receptors on cancer cells are more than normal cells [63–65], the endocytosis of the albumin and consequently its bonded curcumin in the normal cells is less than that of the cancer cells.

Loading, yield, release and water solubility

The amount of curcumin loaded in the nanoparticles was equal to 12%, and the efficiency was 70%. The solubility test results showed that the solubility of curcumin in water was 4.03 μg / ml, while the solubility of HSA-curcumin NPs in water was 68.29 μg / ml. The rates of the release of curcumin from albumin in PBS and citrate buffer were 94 h and 47 h, respectively.

The loading rate showed that there were 160 μg curcumin (40 μg free curcumin+120 μg curcumin bonded to albumin) and 840 μg albumin in 1 mg of the drug. According to the molecular weight of approximately 200 times the albumin (molecular mass of 66.5 kDa) to curcumin (molar mass of 368.385 g·mol⁻¹), this obtained result is justifiable.

The result of release test showed that release of curcumin has increased in acidic condition relative to physiological condition. Due to the fact that the environment around the cancer cells is acidic, so this result is desirable [8–10].

Based on the low rate of curcumin release from HSA-curcumin NPs in PBS, we anticipated that its release will be low in the blood as well; and consequently, most of the curcumin will remain in combination with albumin before reaching the tumor side, and its toxic effects on the normal cells in the body will also decrease. Since we checked the rate of curcumin release in citrate buffer whit pH 5.6 in vitro and we observed that its release rate increases in acidic pH, therefore we can anticipate that in the vicinity of cancer cells, the rate of curcumin release from HSA-curcumin NPs will also increase, due to the reduction of pH in tumor microenvironment [8–10]. The relatively long release of curcumin from albumin, compared to free curcumin, increases the exposure time of curcumin with cancer cells, and as a result, its anti-cancer effects are increased (It should be noted that free curcumin is rapidly removed from the blood by phagocytosis). However, all of these can also decrease the toxic effects on the normal cells of the body compared with free curcumin.

Cytotoxicity of curcumin and HSA-curcumin NPs

The results of MTT tests and the IC50 values obtained for MCF7, SK-BR3, and PBMC cells are presented in Figs. 8 and 9, and The results of MCF7 cells showed that in the first 24 h, curcumin toxicity was higher than HSA-curcumin NPs. After 48 h, the result was similar and in treatment for 72 h, the toxicity of HSA-curcumin NPs was much higher than that of curcumin.

Fig. 8.

In vitro cytotoxicity of Curcumin and HSA-curcumin NPs on MCF7, SK-BR3 at 24, 48,72 h and PBMC at 24 h (n = 10) Means±2SDs

Fig. 9.

The IC50 of Curcumin and HSA-curcumin NPs on MCF7, SK-BR3 at 24, 48,72 h and PBMC at 24 h

Table 1 respectively.

Table 1.

The IC50 of curcumin and HSA-curcumin NPs on MCF7, SK-BR3 at 24, 48,72 h and PBMC at 24 h

Cell	Time (h)	Curcumin	HSA-curcumin NPs
MCF7	24	71.195	92.778
	48	101.036	62.464
	72	109.785	53.421
SK-BR3	24	138.265	140.795
	48	149.653	104.531
	72	140.103	94.609
PBMC	24	127.250	149.882

The results of MCF7 cells showed that in the first 24 h, curcumin toxicity was higher than HSA-curcumin NPs. After 48 h, the result was similar and in treatment for 72 h, the toxicity of HSA-curcumin NPs was much higher than that of curcumin.

In the results of SK-BR3 cells, it was observed that in first 24 h neither curcumin nor HSA-curcumin NPs had a significant effect on these cells. This result can be justified by the fact that the main mechanism of anti-cancer activity of curcumin and its compounds, is the inhibition of proliferation and induction of apoptosis. Because the doubling time of these cells is 59 h, in the first 24 h, none of the curcumin or HSA-curcumin NPs can have significant anticancer effects on them. After 72 h, due to acidification of the culture medium, the findings showed that HSA-curcumin NPs had more toxic effects on SK-BR3 cells compared to curcumin, since curcumin lost its stability due to the acidification of the culture medium. [11]

The results on normal cells showed that this dose had no significant effect on them. Especially the immune cells in the blood stream, which have the highest exposure to drugs until being transmitted into the tumor site.

The results of the apoptotic assay are shown in Figs. 10, 11, and Table 2, respectively. Based on these results, one of the main causes of death of cancer cells by curcumin or HSA-curcumin NPs is the increase in apoptosis in these cells, which can be explained by inhibition of the NF-κB signaling pathway by the curcumin [3, 66–69], and by the relationship between curcumin and the reduction of Bcl2 expression [70–72].These results confirm the results of the MTT test.

Fig. 10.

Apoptosis assay (a) PBMC 24 h (control = 6363, Curcumin = 5110 and HAS-curcumin NPs = 6571 cells), b) MCF7 24 h (control = 8195, Curcumin = 3604 and HAS-curcumin NPs = 4643 cells) and c SK-BR3 36 h((control = 3080, Curcumin = 2433 and HAS-curcumin NPs = 2255 cell

Fig. 11.

Apoptosis assay results of Curcumin and HAS-curcumin NPs on MCF7, SK-BR3 and PBMC

Table 2.

The P value of apoptosis assay results

		MCF7	SK-BR3	PBMC
One Sample T test	Curcumin	0.011	0.115	0.306
	HSA-curcumin NPs	0.004	0.03	0.677
Independent Sample T test	Curcumin/HSA-curcumin NPs	< 0.05	< 0.05	0.437

Conclusion

Curcumin-loaded HSA nanoparticles could increase curcumin solubility in water, as well as its stability in physiological and acidic conditions. These factors have the ability to overcome the limitations on the use of curcumin alone, and have also led to a significant increase in the toxicity of curcumin on the cancer cells without increasing its toxicity on the normal cells. In addition, HSA is the natural protein of the blood, so the nanoparticles themselves have no unwanted side effects. Therefore, this new nano-formulation can be a potential substitution for curcumin in breast cancer therapy.

Conflict of interest

The authors declare that there are no conflicts of interest regarding the publication of this paper.