Engineering Poly(lactic Acid)-Based Scaffolds for Abundant, Sustained, and Prolonged Lactate Release

Abstract

Recent studies have revealed that cardiac tissue regeneration is promoted by administering an initial dose of exogenous lactate and locally maintaining an abundant concentration of this compound for a prolonged period (i.e., around 10-14 days) through sustained release. The aim of this study is to develop a scaffold based on poly-(lactic acid) (PLA) for achieving a sustained daily release of lactate from the first day to the end of the recommended period. First, a five-layered electroresponsive scaffold has been engineered using three PLA layers (first, third, and fifth), each composed of electrospun microfibers (MFs), separated by spin coated lactate (second) and poly-(3,4-ethylenedioxythiophene):poly-(styrenesulfonate) (PEDOT:PSS) (fourth) intermediate layers. The hydrophobicity of the outer PLA layers (first and fifth) has been used to maintain the release of lactate from the intermediate second layer over 3 days, while the conducting fourth PEDOT:PSS layer has ensured a complete lactate release by electrostimulation. After that, in a second step, the same scaffold has been re-engineered to maintain the sustained release not only for a short period (3 days) but also for a prolonged period (>10 days). For this purpose, the PLA MFs of the intermediate third layer have been substituted by plasma-treated proteinase K-containing PLA MFs, obtained by electrospinning a PLA:enzyme mixture. The activity of the enzyme, which decomposes the ester bonds of PLA, combined with the effect of the plasma on the PLA structure, results in a prolonged sustained release that, in addition, can be modulated.

Keywords: cardiac tissue regeneration; conducting polymer; electroresponsive scaffolds; electrospinning; enzymatic degradation; poly(lactic acid).

1

(a) Sketch of the PLA/Lact/PLA/PEDOT/PLA five-layered scaffold. (b) Electrospinning of PLA MF layers. (c) Spin-coating of the lactate layer. (d) Plasma treatment applied to the PLA/Lact/PLA system. (e) Spin-coating of the PEDOT:PSS layer.

2

(a) Digital photographs of PLA MFs (a1), PLA/Lact (a2), plasma-treated PLA/Lact/PLA (a3), and PLA/Lact/PLA/PEDOT/PLA (a4) scaffolds. (b–e) SEM micrographs of the (b) PLA MFs mat (the diameter distribution graphic is included), (c) PLA/Lact, (d) plasma-treated PLA/Lact/PLA, and (e) PLA/Lact/PLA/PEDOT:PSS four-layered system. The red boxes in (c) and the blue ellipses in (e) show protuberances associated with lactate aggregates and interfiber PEDOT:PSS aggregates, respectively.

3

(a) Minimum energy conformations found for 4-PLA and 8-PEDOT model molecules. (b) Two lowest energy assemblies, which are isoenergetic, found for the 4-PLA···8-PEDOT complexes.

4

(a) FTIR and (b) Raman spectra recorded for PLA MFs, a lactate solution, a PEDOT:PSS film, the PLA/Lact two-layered system, and the complete PLA/Lact/PLA/PEDOT/PLA scaffold. Transmittance and Raman intensities are given in arbitrary units.

5

Optical micrographs (a1 and b1) and micro-Raman mapping images (a2–a4, b2–b4) of PLA/Lact and PLA/PEDOT, respectively. A typical background map (corresponding to the pink area) is displayed in a2 for PLA/Lact, while b4 combines the Raman mapping images collected for PLA and PEDOT.

6

(a) Average values of the water contact angle (WCA) for PLA, PLA/Lact, and PLA/PEDOT (standard deviations are indicated). (b) Control cyclic voltammograms recorded for PLA/Lact/PLA/PLA and PLA/Lact/PLA/PEDOT/PLA. (c) Cyclic voltammograms recorded for PLA/Lact/PLA/PEDOT/PLA after 3, 10, 20, 30, 40, and 50 redox cycles. The scan rate used in (b) and (c) was 100 mV/s. (d, e) Stress–strain curves recorded for (d) PLA/Lact and (e) PLA/Lact/PLA/PEDOT/PLA (eight independent samples for each one).

7

(a) Calibration curve used to quantify the amount of lactate released. (b) Cumulative lactate release profiles from PLA/Lact/PLA/PEDOT/PLA scaffolds without and with electrical stimulation (ST). Electrical stimuli consisted of the application of a voltage of −0.5 V for a time interval of 30 min. (c) Strategically chosen times (5 min, 1h, 2 h, 6 h, 24 h, and48 h) at which the electrostimulation protocol was applied. Standard deviations are displayed in parts (a) and (b).

8

Representative SEM micrographs of PLA­(K) (a) before and (b) after plasma treatment.

9

(a) Accumulated and (b) daily release profiles obtained at 25 (room temperature) and 37 °C (physiological temperature), with and without electrical stimulation. (c) Daily average amount of released lactate considering 10.5 day assays.

10

Cell viability results after (a) 1 and (b) 3 days in a leachable medium from PLA MFs, PLA/Lact/PLA­(K) after plasma treatment, and the complete PLA/Lact/PLA­(K)/PEDOT/PLA platform. (c) Confocal microscopy images obtained after 1 and 3 days in the leachable medium using a dilution factor of 1.