Gelsolin Amyloidogenesis Is Effectively Modulated by Curcumin and Emetine Conjugated PLGA Nanoparticles

<div><p>Small molecule based therapeutic intervention of amyloids has been limited by their low solubility and poor pharmacokinetic characteristics. We report here, the use of water soluble poly lactic-co-glycolic acid (PLGA)-encapsulated curcumin and emetine nanoparticles (Cm-NPs and Em-NPs, respectively), as potential modulators of gelsolin amyloidogenesis. Using the amyloid-specific dye Thioflavin T (ThT) as an indicator along with electron microscopic imaging we show that the presence of Cm-NPs augmented amyloid formation in gelsolin by skipping the pre-fibrillar assemblies, while Em-NPs induced non-fibrillar aggregates. These two types of aggregates differed in their morphologies, surface hydrophobicity and secondary structural signatures, confirming that they followed distinct pathways. In spite of differences, both these aggregates displayed reduced toxicity against SH-SY5Y human neuroblastoma cells as compared to control gelsolin amyloids. We conclude that the cytotoxicity of gelsolin amyloids can be reduced by either stalling or accelerating its fibrillation process. In addition, Cm-NPs increased the fibrillar bulk while Em-NPs defibrillated the pre-formed gelsolin amyloids. Moreover, amyloid modulation happened at a much lower concentration and at a faster rate by the PLGA encapsulated compounds as compared to their free forms. Thus, besides improving pharmacokinetic and biocompatible properties of curcumin and emetine, PLGA conjugation elevates the therapeutic potential of both small molecules against amyloid fibrillation and toxicity.</p></div

Kinetics and morphology of <i>f</i>AGel aggregation.

<p>(A) Amyloid formation kinetics of <i>f</i>AGel alone monitored by ThT fluorescence showing sigmoidal trend with 18 h lag phase. (B) TEM image of amyloids formed after 24 h incubation. Scale bar 500 nm. (C) AFM image taken after 60 h showing fibrillar aggregates of <i>f</i>AGel. Scale bar 200 nm. The <i>f</i>AGel concentration was 2 mg/ml.</p

Fourier transformed infrared spectra of nanoparticles.

<p>FTIR spectra of Cm-NPs (red), Em-NPs (blue) and free PLGA-NPs (black) showing overlapping peaks of C = O stretching, corresponding to PLGA. Distinct—OH and—NH stretching peaks characteristic of curcumin and emetine, respectively are also shown.</p

Kinetics and morphologies of NP- incubated <i>f</i>AGel aggregates.

<p><i>f</i>AGel aggregation kinetics monitored by ThT fluorescence in the absence (black trace) or in the presence (coloured traces) of different concentrations of NPs. (A) Acceleration of aggregation in the presence of increasing concentrations of Cm-NPs. The corresponding TEM (B) and AFM (C) images are shown. (D) Suppression of aggregation in the presence of increasing concentrations of Em-NPs with corresponding TEM (E) and AFM (F) images. In each case the TEM and the AFM images were taken after 24 h and 60 h of <i>f</i>AGel incubation with 0.1 mg/ml of respective NPs. Scale bars represent 500 nm for TEM and 200 nm for AFM images.</p

Physicochemical characteristics of nanoparticles.

<p>Physicochemical characteristics of nanoparticles.</p

Cytotoxicity profile of aggregates.

<p>Toxicity assessment of <i>f</i>AGel amyloids and aggregates formed at different concentrations of Cm-NPs or Em-NPs on SH-SY5Y cells, represented as percentage of untreated control. In each case the 24 h incubated aggregates were used.</p

Chemical and morphological characteristics of nanoparticles.

<p>The chemical structure of curcumin (A) and emetine (B), SEM (C) and AFM (D) images of Cm-NPs, SEM (E) and AFM (F) images of Em-NPs. The scale bar in SEM images represents 1 μm.</p

Schematic representation of differential effect of NPs on <i>f</i>AGel amyloidogenesis.

<p>Schematic representation of differential effect of NPs on <i>f</i>AGel amyloidogenesis.</p

Comparison of parameters between nano-encapsulated versus free compounds.

<p>* Concentration of free curcumin and emetine used in Arya P <i>et al</i>. 2014.</p><p>^# Reduction of lag phase (curcumin) and increase in lag phase (emetine). Free compounds data taken from Arya P <i>et al</i>. 2014.</p><p>Comparison of parameters between nano-encapsulated versus free compounds.</p