Interaction of curcumin nanoformulations with human plasma proteins and erythrocytes

Abstract

Background: Recent studies report curcumin nanoformulation(s) based on polylactic-co-glycolic acid (PLGA), β-cyclodextrin, cellulose, nanogel, and dendrimers to have anticancer potential. However, no comparative data are currently available for the interaction of curcumin nanoformulations with blood proteins and erythrocytes. The objective of this study was to examine the interaction of curcumin nanoformulations with cancer cells, serum proteins, and human red blood cells, and to assess their potential application for in vivo preclinical and clinical studies.

Methods: The cellular uptake of curcumin nanoformulations was assessed by measuring curcumin levels in cancer cells using ultraviolet-visible spectrophotometry. Protein interaction studies were conducted using particle size analysis, zeta potential, and Western blot techniques. Curcumin nanoformulations were incubated with human red blood cells to evaluate their acute toxicity and hemocompatibility.

Results: Cellular uptake of curcumin nanoformulations by cancer cells demonstrated preferential uptake versus free curcumin. Particle sizes and zeta potentials of curucumin nanoformulations were varied after human serum albumin adsorption. A remarkable capacity of the dendrimer curcumin nanoformulation to bind to plasma protein was observed, while the other formulations showed minimal binding capacity. Dendrimer curcumin nanoformulations also showed higher toxicity to red blood cells compared with the other curcumin nanoformulations.

Conclusion: PLGA and nanogel curcumin nanoformulations appear to be very compatible with erythrocytes and have low serum protein binding characteristics, which suggests that they may be suitable for application in the treatment of malignancy. These findings advance our understanding of the characteristics of curcumin nanoformulations, a necessary component in harnessing and implementing improved in vivo effects of curcumin.

Keywords: cellular uptake; chemotherapy; curcumin; hemocompatibility; nanoparticle; protein binding.

Figure 1

Structural variations of the curcumin nanoformulations and their uptake in cancer cells. (A) Chemical structure of curcumin. (B) Different types of curcumin nanoformulations and their structures based on chemical composition. Structures do not represent exact size or orientation, and the solid background is intended to distinguish the different formulations. Entire polymer chains, drugs (curcumin), and other structures are defined in the figure itself. (C) Cellular uptake of curcumin nanoformulations in SKBR-3, MDA-MB-231, and HPAF-II cancer cells. Cancer cells were treated with curcumin 20 μg or curcumin nanoformulations (20 μg equivalent to curcumin) for 6 hours. Uptake was determined by recording absorption of acetone cell lysates at λmax 450 nm using UV-vis spectrophotometer. Notes: Data are reported as the mean of three repeats for each uptake (mean ± standard error of the mean deviation; *P < 0.05, curcumin nanoformulations versus free curcumin). Abbreviations: CD, β-cyclodextrin; CUR, curcumin; PLGA, polylactic-co-glycolic acid; PVA, polyvinyl alcohol; PLL, poly-L-lysine.

Figure 2

Human serum albumin binding alters the physicochemical properties of the curcumin nanoformulations, ie, PLGA, β-cyclodextrin, cellulose, nanogel, and dendrimecurcumin nanoparticles. (A) Transmission electron microscopic images of the curcumin nanoformulations (1 mg/mL) before and after incubation with human serum albumin 100 μg. Incubation with human serum albumin was performed for 2 hours, after which the nanoparticles were deposited on transmission electron microscopy grids, stained with uranyl acetate, and imaged under transmission electron microscopy. Red arrows represent uranyl acetate over stain. Blue arrows indicate aliened HSA networks. (B–C) Change in particle size and zeta potential of curcumin nanoformulations before and after incubation with human serum albumin. Particle size was measured for 3 minutes and the zeta potential was measured for 90 runs (9 minutes). Note: Data are reported as the mean ± standard error of the mean for three repeats for each incubation. Abbreviations: CD, β-cyclodextrin; CUR, curcumin; PLGA, polylactic-co-glycolic acid; HSA, human serum albumin.

Figure 3

Human serum proteins binding to curcumin nanoformulations (PLGA, β-cyclodextrin, cellulose, nanogel, and dendrimer). (A) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of curcumin nanoformulations (20 μM) incubated in 100 μg human plasma proteins (fibrinogen, immunoglobulin G, transferrin, and serum albumin). After 2 hours of incubation, bound or adsorbed proteins were separated, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis was run at 150 V for 60 minutes and stained with Coomassie^® G-250 stain. (B) Adsorbed protein bands were quantified by densitometry using AlphaEase Fc software. Data are reported as the mean of three repeats for each incubation (mean ± standard error of the mean, *P < 0.05, compared with PLGA curcumin nanoformulation). (C) Adsorption of curcumin nanoparticles on red blood cells using different concentrations of curcumin nanoformulations (10–40 μM). Notes: Data are reported as the mean of three repeats for each adsorption (mean ± standard error of the mean deviation, *P < 0.05, compared with free curcumin or PLGA curcumin nanoformulation). Abbreviations: CD, β-cyclodextrin; CUR, curcumin; PLGA, polylactic-co-glycolic acid; HSA, human serum albumin.

Figure 4

Hemolysis of curcumin nanoformulations incubated in red blood cells. (A) Optical image of supernatants from hemolysis study. Hemolysis was performed by incubating curcumin nanoformulations in red blood cells for two hours, centrifuging and collecting the supernatant for analysis. Phosphate-buffered solution and sodium dodecyl sulfate were considered as negative and positive controls in the experiment, respectively. (B) The extent of hemolysis was recorded by measuring optical density at λmax 570 nm using an ultraviolet-visible spectrophotometer. The graph was normalized with respect to the optical density of sodium dodecyl sulfate (positive control, 100% hemolysis). Note: Data are presented as the mean of three repeats for each incubation (mean ± standard error of the mean, *P < 0.05, compared with the PLGA curcumin nanoformulation). Abbreviations: CD, β-cyclodextrin; CUR, curcumin; PLGA, polylactic-co-glycolic acid; HSA, human serum albumin; PBS, phosphate-buffered solution; SDS, sodium dodecyl sulfate.

Figure 5

Morphological variation of red blood cells incubated with curcumin nanoformulations for 2 hours. Phase contrast images of red blood cells incubated with (A) controls (phosphate-buffered solution and sodium dodecyl sulfate), (B) curcumin nanoformulations (50 μM), and (C) curcumin nanoformulations (100 μM). Bar equals 20 microns.

Figure 6

Attachment of curcumin nanoparticles to red blood cells. Transmission electron microscopic images of cross-sections of red blood cells after incubation with phosphate-buffered solution and curcumin nanoformulations (100 μM). Bar equals 5 microns.