PAMAM dendrimers of generation 4.5 loaded with curcumin interfere with α-synuclein aggregation

Curcumin (CUR) is a bioactive compound that has been proposed for the treatment of various neurodegenerative diseases. However, its use is limited due to its low solubility in aqueous media and chemical instability under physiological conditions. Herein, we propose a strategy to overcome these limitations by using PAMAM dendrimers of generation 4.5 (DG4.5). Using a combination of biophysical techniques together with in vitro models, we demonstrate that CUR-DG4.5 complexes: (i) increased the solubility and stability of CUR via internalization into dendrimer's pockets and interaction with terminal carboxylic groups; (ii) showed in vitro biocompatibility and increased CUR uptake; (iii) presented DPPH radical scavenging activity and in vitro inhibition of H2O2-induced stress; and (iv) interfere with α-synuclein aggregation. In conclusion, this work lays the foundation to use curcumin-loaded PAMAM dendrimers of generation 4.5 as nanodrugs capable of reducing oxidative stress and inhibiting α-synuclein aggregation to treat synucleinopathies

Study of cationic lipopolymer/DNA interaction by flow cytometry.

<p>Flow cytometry analysis results for (a) SSC-H versus FSC-H and (b) FL2 versus FL1 values for polymerized DMPC:DC_8,9PC:DOTAP (1:1:0.2)/SYBR^® Green I-labeled pDsRed plasmid DNA complexes and (c) SSC-H versus FSC-H and (d) FL2 versus FL1 values for polymerized DMPC:DC_8,9PC:MCL (1:1:0.2)/SYBR^® Green I-labeled pDsRed plasmid DNA complexes.</p

Stoichiometry of the cationic lipopolymer/DNA complex.

<p>Percentage of plasmid DNA association as a function of cationic lipopolymer/pCH110 (a and b) or pDsRed (c and d) plasmid DNA ratios (mol of lipids: mol of base pairs) for the DMPC:DC_8,9PC:DOTAP (1:1:0.2), DMPC:DC_8,9PC:SA (1:1:0.2), and DMPC:DC_8,9PC:MCL (1:1:0.2) formulations.</p

Polymerization confirmation.

<p>Absorbance as a function of wavelength (nm) for the DMPC:DC_8,9PC:DOTAP (1:1:0.2), DMPC:DC_8,9PC:MCL (1:1:0.2) and DMPC:DC_8,9PC:SA (1:1:0.2) mixtures, prepared with the copolymerization methodology, after 20 UV irradiation cycles. Peaks observed around 480 and 520 nm are indicative of polymer formation.</p

Z-potential measurements.

<p>Z-potential measurements.</p

Light-scattering measurements.

<p>Light-scattering measurements.</p

Serum nucleases digestion assay.

<p>The different cationic lipopolymers DMPC:DC_8,9PC:DOTAP (1:1:0.2) and DMPC:DC_8,9PC:MCL (1:1:0.2)/pDsRed plasmid DNA complexes (16:1 mol of lipids: mol of base pairs ratio) were formed in water, PBS or MEM (indicated on the right). Each lane was loaded with 1 μg of plasmid DNA. Lanes correspond to: (1) pDsRed alone with a 24-h incubation in the indicated medium, (2) pDsRed alone with a 24-h incubation in the presence of 50% v/v FBS, (3) cationic lipopolymer (stated above the gel picture)/pDsRed plasmid DNA complex formed in the indicated medium with a 24-h incubation in the indicated medium without FBS, and (4) cationic lipopolymer (stated above the gel picture)/pDsRed plasmid DNA complex formed in the indicated medium with a 24-h incubation in the presence of 50% v/v FBS. The two main topological plasmid conformations, relaxed and negatively supercoiled, are indicated with arrows on the left of the figure noted as relax and -supercoiled, respectively. Degraded DNA is also indicated with an arrow on the left of the figure.</p

Hemolysis percentage (% H).

<p>Hemolysis percentage (% H).</p

Effect of different incubation media on the cationic lipopolymer/DNA interaction.

<p>The different cationic lipopolymers were incubated with the pDsRed plasmid DNA in a 16:1 (mol of lipids: mol of base pairs) ratio in the medium (water, PBS or MEM) indicated on the right. Each lane was loaded with 1 μg of plasmid DNA. Lanes correspond to: (1) pDsRed alone with an additional 10-min incubation in the indicated medium, (2) pDsRed alone with an additional 10-min incubation in the presence of 10% v/v FBS, (3) cationic lipopolymer (stated above the gel picture)/pDsRed plasmid DNA complex formed in the indicated medium with an additional 10-min incubation without FBS, (4) cationic lipopolymer (stated above the gel picture)/pDsRed plasmid DNA complex formed in the indicated medium with an additional 10-min incubation in the presence of 10% v/v FBS, (5) cationic lipopolymer (stated above the gel picture)/pDsRed plasmid DNA complex formed in the indicated medium with an additional 10-min incubation in the presence of 50% v/v FBS, (6) cationic lipopolymer (stated above the gel picture)/pDsRed plasmid DNA complex formed in the indicated medium with 10% v/v FBS and with an additional 10-min incubation in the presence of 50% v/v FBS. The two main topological plasmid conformations, relaxed and negatively supercoiled, are indicated with arrows on the left of the figure noted as relax and -supercoiled, respectively.</p

Optimization of the study of cationic lipopolymer/DNA interaction by flow cytometry.

<p>Flow cytometry analysis results for non-polymerized DMPC:DC_8,9PC:MCL (1:1:0.2) liposomes, used to set control values of (a) SSC-H values (related to particle complexity) versus FSC-H values (related to particle size) and (b) FL1 values (where SYBR^® Green I-labeled plasmid DNA fluorescence is detected) and FL2 values (where cationic lipopolymer fluorescence is detected). (c) and (d) show the results for polymerized DMPC:DC_8,9PC:MCL (1:1:0.2) for SSC-H versus FSC-H and FL2 versus FL1 values respectively. (e) and (f) show the results for non-polymerized DMPC:DC_8,9PC:MCL (1:1:0.2)/SYBR^® Green I-labeled pDsRed plasmid DNA complexes for SSC-H and FSC-H values and FL2 versus FL1 values respectively.</p