Biosourced Amphiphilic Degradable Elastomers of Poly(glycerol sebacate): Synthesis and Network and Oligomer Characterization

Glycerol (G, a triol) and sebacic acid (S, an α,ω-dicarboxylic acid) were condensed in the bulk to obtain poly­(glycerol sebacate) (PGS) cross-linked elastomers which were characterized in terms of their swelling, thermal, and mechanical properties. The soluble precursors to the elastomers were characterized in terms of their size, size distribution, and composition. In particular, G–S mixtures of five different compositions (molar G:S ratio = 2:1, 2:2, 2:3, 2:4, and 2:5) were copolymerized in the bulk at 120 °C in a three-step strategy (first step under inert gas atmosphere, followed by two steps <i>in vacuo</i>). When the G:S molar ratio was equal to (2:3) or close to (2:4), the stoichiometrically matched, network formation took place from the second condensation step, whereas three reaction steps were necessary for network formation far from stoichiometry, at G:S molar ratios equal to 2:2 and 2:5; at a G:S molar ratio of 2:1, no network formation was observed at all. Network composition also proved to be an important structural property, directly influencing the swelling and thermomechanical behavior of the elastomers. In particular, at the stoichiometrically matched G:S ratio of 2:3, corresponding to the cross-linking density maximum, the sol fraction extracted from the elastomers and the elastomer degree of swelling in aqueous media and in organic solvents presented a minimum, whereas the storage moduli of PGS elastomeric membranes in the dry state, measured within the temperature range between 35 and 140 °C, exhibited a maximum. The molecular weights of all soluble network precursors were found to be below 5000 g mol^–1 (gel permeation chromatography), containing but traces of ring oligomers (electron-spray ionization mass spectrometry). ¹H NMR spectroscopy indicated that the precursor composition was close to that expected on the basis of the G:S feed ratio and that monomer-to-polymer conversion increased from the first to the second condensation step

¹⁹F MRI-based quantification in solutions and CPCs and determination of cellular detectability limit.

<p>¹⁹F MR spectroscopy, image-based quantification, and sensitivity detection limits: <b>(A, B)</b> Axial ¹H and ¹⁹F images from TFA phantoms of different concentrations (25–100 mM), and image<u>s</u> of a multivial sensitivity phantom containing 0.25, 0.5, 0.75, and 1 million labeled/transfected CT cells suspended in media for sensitivity limit detection (cell pellets resided at the bottom of the Eppendorf tubes) using the butterfly coil. <b>(C)</b> ¹H imaging indicates spatial B₁ fall off-effects (laterally and with depth, non-adiabatic excitation). ¹⁹F imaging indicates a minimum detectable cellular load of approximately 500k cells in a total acquisition of 4.4 min (white arrows). The ¹⁹F MRI in <b>(D)</b> shows cells over a slice thickness of 20 mm. As shown by the inserted schematic, the ¹H MRI in <b>(C)</b> shows cross-sections (from the middle of the Eppendorf tubes), while the ¹⁹F MRI in <b>(D)</b> shows the hyperintense cell pellets that were sometimes slightly displaced spatially given the tilting of some of the tubes and the dispersion of the cells on the walls of the tubes in instances where the acquisitions were prolonged. <b>(E)</b> Quantification of labelled CPCs using ¹⁹F MRS (solenoid). The linearity of the evoked fully relaxed spectral area versus cell number was independently confirmed using fast, direct, image-based SPGR using CPCs (butterfly coil) (results not shown).</p

CPC label confirmation using flow cytometry and in vitro ¹⁹F MRI/MRS validation.

<p><b>(A, B, D, E)</b> Ungated scatter plots of forward (FSC) and side scatter (SSC, singlets vs. doublets) and <b>(C, F)</b> gated, overlapped flow cytometry histograms of control <b>(C)</b> and labeled CT cells <b>(F)</b> confirming cellular uptake. Applied gates are indicated in the scatter plots as highlighted regions-of-interest. <b>(G, H)</b> Confocal microscopy images of PFCE labelled <b>(G)</b> CDC GFP+ (calcein [gray]), <b>(H)</b> Atto647 (red), and (<b>I)</b> merged calcein/Atto647 with a zoomed inlet indicating the heterogenous distribution of cellular label uptake. <b>(J, K)</b> Corresponding ¹⁹F and ¹H-¹⁹F merged MRI of labeled CT cells (~4.5 million) obtained using the solenoid coil showing excellent ¹⁹F signal localization. <b>(L)</b> ¹⁹F magnitude spectrum in labeled CTs using the solenoid coil (line broadening = 30 Hz, zero reference frequency set to the NP-labeled CT cell resonance).</p

Post-mortem and in vivo murine cardiac ¹⁹F MRI following intramyocardial CPC injections.

<p>Post-mortem <b>(A-D)</b> and in vivo <b>(E-F)</b>, merged ¹H-¹⁹F images of approximately 1.5–2.5 million labeled CT cells administered in the <b>(A, B)</b> femoral skeletal (axial, sagittal views; both legs were injected), <b>(C, D)</b> post-mortem cardiac (pseudo-short and short-axis views, without <b>(C)</b> and with the anterior thorax <b>(D)</b>) from PHD3f/f, PHD2flox/flox, <b>(E)</b> ungated in vivo cardiac (coronal) views from a C57BL/6 mouse using the butterfly coil. <b>(F)</b> Corresponding ungated, unlocalized ¹⁹F MRS from the upper thorax showing the two isoflurane (ISO) and the labelled CT cell peaks. All ¹H images were acquired when the coil was tuned/matched at the ¹⁹F resonance. <b>(G)</b> Indicative optical bright field histological image from the mouse heart in <b>(D)</b> above. The dotted square box indicates the area where cells were localized within the left ventricular myocardium.</p

Comparison of acquisition parameters and cell detection limits in this and prior studies.

<p>Optimized sequence parameters for the current ¹⁹F MRI study led to fast imaging acquisitions (3–5 min) in comparison to prior published studies. The table summarizes the existing literature in reference to the total imaging time, cell detection limit, and the ultimate detection limit for ¹⁹F signal detection (an extended version of prior published studies can be retrieved from Srinivas et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190558#pone.0190558.ref036" target="_blank">36</a>]).</p

¹⁹F MRI validation in NP solution phantoms.

<p><b>(A)</b> Non-selective ¹⁹F magnitude spectrum of NP solutions in the presence of a 25 mM TFA phantom (as shown in B). (<b>B)</b> ¹H and ¹⁹F MRI of NP phantoms using <b>(C)</b> the SPGR, and <b>(D)</b> the echo-SSFP sequences. The TFA phantom does not appear in ¹⁹F MRI <b>(C, D)</b> since broadband excitation/narrowband receiver detection was used centered at the NP resonance. (<b>E</b>) Variation of the mean ¹⁹F SNR from phantom solutions in <b>(C, D)</b> above for the SPGR and SSFP sequences for different NP concentrations.</p

In vitro relaxation values of fluorinated compounds.

<p>Summary of ¹⁹F T₁ and T₂ relaxation values in phantoms and in labeled cells (immune, neural stem, and cardiac progenitor) from this and prior published studies. Significantly increased T₁ values (p<0.00047 (labeled), p<0.001 (FuGENE-labeled), α = 5%) were measured for the NP-labeled cells compared to NPs in solution.</p

Experimental pulse sequence comparison in phantoms.

<p>Pulse sequence SNR comparison using the birdcage coil based on two-dimensional (2D) acquisitions using a 100 mM TFA phantom in the same total imaging acquisition time. SNR values lied within the set scale bar shown on the right.</p

Pulse sequence simulations in parametric space.

<p><b>(A)</b> Theoretical normalized parametric signal-to-noise (SNR) plots for labeled CT cells (T₁ = 1.32 s and T₂ = 0.05 s) for a: <b>(A)</b> SPGR sequence (flip angle versus TR/T₁), <b>(B)</b> a rapid acquisition with relaxation enhancement (RARE) sequence [echo train length (ETL) versus TR/T₁ with flip-back], <b>(C, D)</b> balanced steady state free precession [free induction decay (fSSFP) and echo-SSFP (eSSFP)] (flip angle versus TR/T₁ without sign alteration). Optimal and selected acquisition zones are indicated. Optimized labeled cell imaging was based on the generation of the respective plots that used the estimated relaxation values as listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190558#pone.0190558.t001" target="_blank">Table 1</a>. All simulations assumed a total imaging acquisition of 4.5 min, NEX = 256, an acquisition matrix of 32×32, and an acquisition bandwidth of 4 kHz.</p

Axial ¹⁹F MRI images of phantoms using a surface coil.

<p><b>(A)</b> butterfly without, and <b>(B)</b> with the use of adiabatic hyperbolic secant adiabatic full passage (HS-AFP) using the spoiled gradient echo sequence (SPGR) and a 100 mM TFA phantom. Profiles depict signal intensities versus pixel values along the oblique orientation defined in <b>A</b>. <b>(C-E)</b> Corresponding axial images of a multivial TFA phantom containing 5 mM TFA solutions. <b>(C)</b> Axial ¹H image using the butterfly coil without, and <b>(D)</b> with HS-AFP adiabatic excitation, and <b>(E)</b> axial ¹⁹F with adiabatic excitation. The artifact observed in the right part of the phantom is attributed to fact that the adiabatic condition is not fully met owing to the butterfly’s B₁ assymetry (driving cables).</p