{"id":608594,"date":"2024-06-07T20:00:00","date_gmt":"2024-06-08T00:00:00","guid":{"rendered":"https:\/\/platohealth.ai\/polymers-of-functionalized-diaminopropionic-acid-are-efficient-mediators-of-active-exogenous-enzyme-delivery-into-cells-scientific-reports\/"},"modified":"2024-06-08T04:18:03","modified_gmt":"2024-06-08T08:18:03","slug":"polymers-of-functionalized-diaminopropionic-acid-are-efficient-mediators-of-active-exogenous-enzyme-delivery-into-cells-scientific-reports","status":"publish","type":"post","link":"https:\/\/platohealth.ai\/polymers-of-functionalized-diaminopropionic-acid-are-efficient-mediators-of-active-exogenous-enzyme-delivery-into-cells-scientific-reports\/","title":{"rendered":"Polymers of functionalized diaminopropionic acid are efficient mediators of active exogenous enzyme delivery into cells – Scientific Reports","gt_translate_keys":[{"key":"rendered","format":"text"}]},"content":{"rendered":"

Synthesis<\/h3>\n

The peptidomimetics were synthesized according to a procedure described previously by our team14<\/a><\/sup>. The biotin moiety was attached to the N-terminal amino group using a molar excess of Bt-NHS:DIPEA (3:1.5) in NMP. The completeness of the reaction was monitored by the Kaiser test.<\/p>\n

Cell lines<\/h3>\n

The healthy cell line HB2 (human breast epithelial cells) was obtained from Merck (Germany), the cancer cell line MDA-MB-231 (human breast cancer epithelial cells) was obtained from ATCC (United Kingdom), the cancer cell line SKBR3 (human mammary gland cancer) was obtained from CLS (Germany), and the cancer cell line T47D (human breast cancer epithelial cells) was obtained from CLS (Germany). All cell lines were cultured at 37 \u00b0C in 5% CO2<\/sub> in Dulbecco\u2019s modified Eagle medium (DMEM) supplemented with 4.5 g\/L glucose, 10% fetal bovine serum (FBS) and 1% penicillin\u2012streptomycin solution (100 units of penicillin and 100 \u00b5g\/mL streptomycin). The HB2 cell line requires the addition of 5 \u00b5g\/mL insulin and 5 \u00b5g\/mL alcoholic hydrocortisone solution.<\/p>\n

Complex formation and electrophoresis assay<\/h3>\n

FITC-Strp: Bt-O2Oc-[Dap(GO2)]6<\/sub>-O2Oc-NH2<\/sub>\/Bt-O2Oc-[Dap(GO2)]8<\/sub>-O2Oc-NH2<\/sub>
\n <\/h4>\n

FITC-streptavidin (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was incubated with increasing concentrations of polymers with N-terminal biotin (compound 1 or 2) or free amino groups (compound 1a or 2a) at molar ratios of 1:1, 1:2, and 1:4 for one hour at RT. Each complex was separated on a native polyacrylamide gel at a concentration of 0.1 or 0.2 \u00b5g per lane with polymer and FITC-Strp as controls. A 15-well Native PAGE 4\u201316%, Bis\u2013Tris, 1.0 mm, Mini Protein Gel (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was used. The separation conditions were as follows: 50 mM Bis\u2013Tris, 50 mM tricine, pH 6.8 (running buffer), and constant voltage of 150 V for 2 h at 24 \u00b0C. Loading buffer composition: 200 mM Bis\u2013Tris, 6 N HCl, 200 mM NaCl, 40% w\/v glycerol, 0.004% bromophenol blue, pH 7.2. Cathode buffer composition: 0.4% Coomassie G-250 diluted 10 times in double distilled water in running buffer. At the final step, the gel was placed in 100 mL of fix solution (40% methanol and 10% acetic acid in water) and microwaved at 1100 watts for 45 s. Next, the gel was shaken for 30 min at room temperature. The fixing solution was removed, and the whole procedure was repeated. After this, the gel was ready for visualization.<\/p>\n

Strp-\u03b2-gal complex with Bt-peptidomimetic<\/h4>\n

The hybrid protein construct streptavidin-\u03b2-gal (Thermo Fisher Scientific, Waltham, Massachusetts, USA) at a concentration of 2 mg\/mL in H2<\/sub>O was incubated for 1 h at RT in molar ratios of 1:1, 1:2, or 1:4 with compound 1 or 2; nonbiotinylated compounds (1a or 2a) were used as controls. All samples were subjected to electrophoretic separation at a protein concentration equal to 7.5 \u00b5g per well.<\/p>\n

Acidic native gel<\/h4>\n

The polyacrylamide gel was cast and run as follows: stacking gel, 4% acrylamide, pH 5.0 (0.22 M acetic acid\/KOH buffer); resolving gel, 8% acrylamide, pH 4.0 (0.075 M acetic acid\/KOH buffer); running buffer, 3.55% \u03b2-alanine\/acetic acid in water, pH 4.0; loading buffer, 50% glycerol in 0.22 M acetic acid\/KOH buffer, pH 5.0 with 0.0005% crystal violet.<\/p>\n

The gel was run with reverse polarization at 5 mA initially (45 min); then, after the samples migrated out from the stacking gel, it was run at 20 mA (2 h). Images of the gel were taken using a Fusion Fx (Vilber Lourmat, France) system.<\/p>\n

Cell studies<\/h3>\n

FITC-streptavidin: Bt-peptidomimetic complex cellular uptake<\/h4>\n

Labeled FITC-streptavidin (Thermo Fisher Scientific, Waltham, Massachusetts, USA) at a concentration of 0.2 mg\/mL in H2<\/sub>O was incubated with peptidomimetic (biotinylated (1 or 2) or lacking biotin (1a or 2a)) at molar ratios of 1:2 and 1:4 for one hour at RT.<\/p>\n

All cells were seeded in 24-well plates (initial number of cells: 104<\/sup>) and cultured in 0.5 mL of appropriate medium for 24 h. Next, the cells were washed three times with PBS, and 0.5 mL of peptidomimetic (1, 2 or 1a, 2a) in medium was added. The concentration of the all peptidomimetic (1, 2 or 1a, 2a) was 10 \u00b5M (molar ratio 1:2) or 20 \u00b5M (molar ratio 1:4) respectively. After the indicated time period (2 h, 4 h or 24 h), the cells were washed with PBS and cultured in FluoroBrite DMEM (Gibco, New York, USA).<\/p>\n

An ECLIPSE Ti-E inverted fluorescence microscope (Nikon, Tokyo, Japan) was used to evaluate the complex distribution in the cells. The appropriate filters were used for the detection of DAPI and FITC.<\/p>\n

Streptavidin-\u03b2-gal: Bt-peptidomimetic complex cellular uptake<\/h4>\n

Streptavidin-\u03b2-gal (Invitrogen by Thermo Fisher Scientific, Waltham, Massachusetts, USA) at a concentration of 0.2 mg\/mL in H2<\/sub>O was incubated with peptidomimetic (biotinylated (1,2) or lacking biotin (1a, 2a)) at molar ratios of 1:2 and 1:4 for 1 h at RT.<\/p>\n

All cells were seeded in 96-well plates (initial number of cells: 103<\/sup>) and cultured in 0.1 mL of appropriate medium for 48 h. Next, the cells were washed with PBS (three times), and 50 \u00b5L of appropriate peptidomimetic (1 or 2) in medium was added. The concentration of each peptidomimetic was 10 \u00b5M (molar ratio 1:2) or 20 \u00b5M (molar ratio 1:4). Cells were cultured for 24 h and then subjected to PBS washes (2 times). After the selected time period, 0 h (t24h<\/sub>) or 2 h (t24h+2h<\/sub>) (see scheme on Fig. 2<\/a>) the cells were washed with PBS and lysed with Reporter Lysis Buffer RLB (Promega, Madison, Wisconsin, USA) according to the manufacturer\u2019s manual. All lysates were immediately snap frozen at \u2212 80 \u00b0C.<\/p>\n

\n
Figure 2<\/b><\/figcaption>
\n
\"figure<\/a><\/div>\n
\n

Incubation scheme for Strp-\u03b2-gal:peptidomimetics. Additional two hours of culturing allow the membrane bound complex to dissociate.<\/p>\n<\/div>\n<\/div>\n

Full size image<\/span><\/a><\/div>\n<\/figure>\n<\/div>\n

Activity studies<\/h3>\n

The enzymatic activity of \u03b2-gal was monitored using resorufin \u03b2-d<\/span>-galactopyranoside (Thermo Fisher Scientific, Waltham, Massachusetts, USA) (final concentration\u2009=\u20092.5 mg\/mL dimethyl sulfoxide (DMSO)). The increase in fluorescence was monitored in triplicate using a FluoroStar OMEGA microplate reader (BMG LABTECH, Ortenberg, Germany) with extinction and emission wavelengths of 541 nm and 585 nm. Assay conditions were as follows: 2.5 \u00b5L of lysate, 100 \u00b5L assay buffer (0.1 M phosphate buffer, pH 7.0) and 5 \u00b5L of substrate. The assay was run for 120 min at 37 \u00b0C.<\/p>\n

In-cell \u03b2-gal activity detection<\/h3>\n

Cells were incubated with the formed complexes for 24 h in culture medium washed by PBS and cultured for additional 2 h or directly subjected to the next step. After this, two washes with PBS were applied to each system; then, the cells were fixed in 3.7% paraformaldehyde in PBS for 15 min. Next, the cells were washed twice in PBS, and 125 \u00b5L of reaction mixture was added to each system. The reaction buffer consisted of 40 mM citric acid, 40 mM sodium phosphate, 300 mM NaCl, 10 mM mercaptoethanol, and 4 mM MgCl2<\/sub> (pH 6.0) with 2 mM X-Gal (5-bromo-4-chloro-3-indolyl \u03b2-d<\/span>-galactopyranoside) added immediately prior to use from a 34 mM stock in DMSO17<\/a><\/sup>. The plate was incubated for two hours at 37 \u00b0C and observed under an Olympus IX51 microscope (Olympus, Tokyo, Japan). Application of the X-Gal reagent in the presence of \u03b2-gal activity results in a blue, insoluble product that precipitates and can be monitored under an optical microscope.<\/p>\n

Cytotoxicity studies<\/h3>\n

All cell lines were seeded at a concentration of 5\u2009\u00d7\u2009103<\/sup> per well in 96-well plates and cultured for 48 h in 100 \u00b5L of medium. Next, the medium was replaced with new medium containing the target amount of tested compounds or their complexes.<\/p>\n

The following compounds were tested: Bt-O2Oc-[Dap(GO2)]6<\/sub>-O2Oc-NH2<\/sub> (Compound 1<\/b>), where Bt\u2014biotin, O2Oc\u20148-amino-3,6-dioxaoctanoic acid, Dap\u2014L-2,3-diaminopropionic acid, GO2\u20148-amidino-3,6-dioxaoctanoic acid, Bt-O2Oc-[Dap(GO2)]8<\/sub>-O2Oc-NH2<\/sub> (Compound 2<\/b>), O2Oc-[Dap(GO2)]6<\/sub>-O2Oc-NH2<\/sub> (Compound 1a<\/b>) and O2Oc-[Dap(GO2)]8<\/sub>-O2Oc-NH2<\/sub> (Compound 2a<\/b>) at concentrations of 1, 5, 10, 20, 50, and 100 \u00b5M in the well;<\/p>\n