Short peptide based nanotubes capable of effective curcumin delivery for treating drug resistant malaria

Abstract

Background: Curcumin (Ccm) has shown immense potential as an antimalarial agent; however its low solubility and less bioavailability attenuate the in vivo efficacy of this potent compound. In order to increase Ccm's bioavailability, a number of organic/inorganic polymer based nanoparticles have been investigated. However, most of the present day nano based delivery systems pose a conundrum with respect to their complex synthesis procedures, poor in vivo stability and toxicity issues. Peptides due to their high biocompatibility could act as excellent materials for the synthesis of nanoparticulate drug delivery systems. Here, we have investigated dehydrophenylalanine (ΔPhe) di-peptide based self-assembled nanoparticles for the efficient delivery of Ccm as an antimalarial agent. The self-assembly and curcumin loading capacity of different ΔPhe dipeptides, phenylalanine-α,β-dehydrophenylalanine (FΔF), arginine-α,β-dehydrophenylalanine (RΔF), valine-α,β-dehydrophenylalanine (VΔF) and methonine-α,β-dehydrophenylalanine (MΔF) were investigated for achieving enhanced and effective delivery of the compound for potential anti-malarial therapy.

Results: FΔF, RΔF, VΔF and MΔF peptides formed different types of nanoparticles like nanotubes and nanovesicles under similar assembling conditions. Out of these, F∆F nanotubes showed maximum curcumin loading capacity of almost 68 % W/W. Ccm loaded F∆F nanotubes (Ccm-F∆F) showed comparatively higher (IC50, 3.0 µM) inhibition of Plasmodium falciparum (Indo strain) as compared to free Ccm (IC50, 13 µM). Ccm-F∆F nano formulation further demonstrated higher inhibition of parasite growth in malaria infected mice as compared to free Ccm. The dipeptide nanoparticles were highly biocompatible and didn't show any toxic effect on mammalian cell lines and normal blood cells.

Conclusion: This work provides a proof of principle of using highly biocompatible short peptide based nanoparticles for entrapment and in vivo delivery of Ccm leading to an enhancement in its efficacy as an antimalarial agent.

Keywords: Antimalarial; Curcumin; Nanotubes; Peptide; Self-assembly.

Fig. 1

Transmission electron micrographs of DNPs: TEM image of a F∆F, showing the formation of tubular structure with mean diameter of 25 nm and length in microns, b M∆F, demonstrating the formation of vesicular structures with mean diameter of 40 nm c V∆F, showing the formation of vesicular structures with mean diameter of 55 nm, d RΔF demonstrating the formation of vesicular structures with mean diameter of 62 nm and e Ccm-F∆F showing dense tubular structures

Fig. 2

In vitro cytotoxicity and haemolytic assay: cell toxicity was assessed using a MTT assay. L929 cells were treated with different concentrations i.e., from 0 to 4000 µM of the DNPs for 24 h. Viability was expressed as the percentage of media control. b LDH release assay: cells treated with 50 µM of DNPs showed almost similar release of LDH like PBS treated cells. Cells treated with DMSO as positive control showed the maximum LDH release. c Percentage haemolysis at three different concentrations. None of the DNPs showed haemolytic activity. Triton X-100 taken as a positive control showed 100 % haemolysis

Fig. 3

Release of curcumin from Ccm-FΔF: in vitro curcumin release from Ccm-FΔF nanoformulations, stored at room temperature for different time points (day 1, 14 and 90) in methanol: water (1:1 v/v). Curcumin content was estimated using (UV–Vis) spectrophotometer at a wavelength of 425 nm. (n = 3), error bar represent ± standard deviations

Fig. 4

Stability of Ccm-FΔF nanotubes: TEM photographs of curcumin loaded nanotubes at different time points. a–f represent images taken after 1,7,14, 28, 56 and 90 days of incubation at room temperature (25 ± 2 °C). Results demonstrated the stability of curcumin loaded nanotubes over the entire incubation period of 90 days

Fig. 5

Fluorescence emission spectra of curcumin: i fluorescence spectra of both curcumin (A) and Ccm-FΔF at two different points, (B) at 1 day and (C) after 90 days, in aqueous solution of methanol (1:1, v/v) at an excitation wavelength of 425 nm. ii Amount of curumin present in Ccm-FΔF nano-formulations during the incubation period. Curcumin content was determined at three different time points (1, 14 and 90 days). It was observed that the curcumin concentration inside the nanotubes remained almost constant even after 90 days of storage depicting the stability of the drug inside the nanotubes

Fig. 6

Malaria parasite (Pf indo) inhibition assays under in vitro conditions: Curcumin entrapped in nanotubes inhibited the growth of chloroquine-resistant P.falciparum (Pf indo) in culture, more efficiently (IC₅₀, 3 µM) than free curcumin (IC₅₀, 13 µM). Void nanotubes (F∆F) did not show any inhibitory effect

Fig. 7

Survival graph of P. bergi-infected mice treated with different groups. Most of the mice in the group treated with PBS and FΔF died with high parasitemia between 10 and 14 days of infection. Mice treated with free Ccm showed increased life span but died earlier than those treated with Ccm-FΔF

Fig. 8

Percentage parasitemia of different groups of mice: Mice treated with intra-peritoneal injection of the nanoformulations. a PBS and b FΔF treated group. These groups showed increase in parasitemia with time killing all the animals. c Ccm (50 mg/kg BW of curcumin) treated group, where parasitemia rose slowly and mice survived for the longer time as compared to the PBS control group. d Mice treated with Ccm-FΔF (equivalent to 50 mg/kg BW of curcumin) showed significant decrease in parasitemia and increase in life span

Fig. 9

In vivo antimalarial assay design. After infection with P. berghei (ANKA) mice were treated with different formulations in the corresponding group and parasitemia count was determined every alternative day