Fluorescence imaging enabled poly(lactide-co-glycolide)

Abstract

Fluorescent biomaterials have attracted significant research efforts in the past decades. Herein, we report a new series of biodegradable, fluorescence imaging-enabled copolymers, biodegradable photoluminescent poly(lactide-co-glycolide) (BPLP-co-PLGA). Photoluminescence characterization shows that BPLP-co-PLGA solutions, films and nanoparticles all exhibit strong, tunable and stable photoluminescence. By adjusting the molar ratios of L-lactide (LA)/glycolide (GA) and (LA+GA)/BPLP, full degradation of BPLP-co-PLGA can be achieved in 8-16 weeks. The fluorescence decay behavior of BPLP-co-PLGA can be used for non-invasive monitoring of material degradation. In vitro cytotoxicity and in vivo foreign body response evaluations demonstrate that BPLP-co-PLGA exhibits similar biocompatibility to poly(lactide-co-glycolide) (PLGA). The imaging-enabled BPLP-co-PLGA was fabricated into porous scaffolds whose degradation can be monitored through non-invasive imaging and nanoparticles that show theranostic potential demonstrated by fluorescent cellular labeling, imaging and sustained 5-fluorouracil delivery. The development of inherently fluorescent PLGA copolymers is expected to impact the use of already widely accepted PLGA polymers for applications where fluorescent properties are highly desired but limited by the conventional use of cytotoxic quantum dots and photobleaching organic dyes.

Statement of significance: This manuscript describes a novel strategy of conferring intrinsic photoluminescence to the widely used biodegradable polymers, poly(lactide-co-glycolide) without introducing any cytotoxic quantum dots or photo-bleaching organic dyes, which may greatly expand the applications of these polymers in where fluorescent properties are highly desired. Given the already significant impact generated by the use of PLGA and alike, this work contributes to fluorescence chemistry and new functional biomaterial design and will potentially generate significant impact on many fields of applications such as tissue engineering, molecular imaging and labeling, and drug delivery.

Keywords: Biodegradable; Bioimaging; Drug delivery; PLGA; Photoluminescence; Tissue engineering.

Fig. 1

(a) Synthesis of BPLP-co-PLGA. Step 1: Synthesis of BPLP via polycondensation; Step 2: Synthesis of BPLP-co-PLGA via a ring-opening polymerization using BPLP as a macromolecular initiator. (b) Feeding ratios, molecular weights (Mw), polydispersity indexes (PDI), and intrinsic viscosities ([η]) of PLGA and BPLP-co-PLGA copolymers synthesized in this study.

Fig. 2

Chemical, mechanical and thermodynamic properties and wettability of BPLP-co-PLGA. 1H-NMR (a) and ATR-FTIR (b) spectra, Tensile strength and Young’s modulus (c), strain-stress curves (d), differential scanning calorimetry (DSC, e) and thermal gravity analysis (TGA, f) curves of representative PLGA and BPLP-co-PLGA. Water-in-air contact angle vs. time curves (g) of representative PLGA and BPLP-co-PLGA copolymers films.

Fig. 3

Photoluminescent (PL) properties of BPLP-co-PLGA solutions, films. (a) Photoluminescent excitation and emission spectra of BPLP-co-PLGA copolymers solutions (5 mg/mL in chloroform) (insert, optical imaging of BPLP-co-PLGA 75/25-100 solution under UV light), PLGA 75/25 was used as control. (b) Photoluminescence stability evaluation of representative BPLP-co-PLGA copolymers and Rhodamine B. (c) and (d) Quantum yields and extinction coefficients of BPLP-co-PLGA copolymers. (e) Imaging effect of PLGA 75/25 and BPLP-co-PLGA 75/25-100 films (1) under white or UV light, (2) under fluorescent microscope with mono filter, GFP filter, Cy3 filter and DAPI filter.

Fig. 4

In vitro degradation of BPLP-co-PLGA. (a) Weight loss in PBS (pH 7.4) of BPLP-co-PLGA and PLGA 75/25 copolymers at 37°C. (b) Photoluminescent emission spectra of BPLP-co-PLGA 75/25-100 copolymers (5 mg/mL in chloroform) at various times degradation in PBS. (c) Photoluminescence emission spectra of degradation solutions (DS) of BPLP-co-PLGA 75/25-100 (insert: emission intensity vs. degradation time). (d) ¹H-NMR spectra of original and degrading (6 weeks in PBS) BPLP-co-PLGA 75/25-100.

Fig. 5

In vitro cytotoxicity and in vivo foreign body responses of BPLP-co-PLGA. (a) MTT assay (570 nm) against human mesenchymal stem cells (hMSCs) cultured on copolymer films for 24 hours (insert, SEM image of hMSCs cultured for 24 hours on BPLP-co-PLGA 75/25-100 film). (b) Normalized cell viability of hMSCs cultured with the presence of different dilutions of BPLP-co-PLGA degradation products, PLGA 75/25 was used as control. (c) Live/Dead assay images of hMSCs cultured with 10× diluted degradation products of PLGA 75/25 and BPLP-co-PLGA 75/25-100 for 1, 3 and 7 days. (d) Foreign body response evaluations: (1) Representative H&E staining images of surrounding tissues of BPLP-co-PLGA and PLGA copolymer films after 1 month of implantation (P: polymer, C: fibrous capsule, M: muscle); (2) Cell numbers in a 200 × 200 µm² square region (from 400× images of H & E staining) of the skin-side tissue near the implants.

Fig. 6

Scaffold and nanoparticle fabrication of BPLP-co-PLGA. (a) SEM images of BPLP-co-PLGA 75/25-100 scaffolds with pore sizes of 50-100 µm (1), and 200-250 µm (2). (b) The particle sizes and distributions of representative PLGA and BPLP-co-PLGA nanoparticle dispersions (insert: TEM images of corresponding nanoparticles). (c) Excitation and emission spectra of BPLP-co-PLGA 75/25-100 nanoparticles, 5-Fu loaded BPLP-co-PLGA 75/25-100 nanoparticles and control PLGA 75/25 nanoparticles with a concentration of 5 mg/mL in PBS solution. (d) BPLP-co-PLGA 75/25-50 nanoparticle (500µg/mL)-uptaken hMSCs observed under fluorescence microscope with (1) Monochrome filter (insert, TEM image of BPLP-co-PLGA 75/25-50 nanoparticles, particle size: 110nm), (2) GFP filter, (3) Cy3 filter, and (4) DAPI filter.

Fig. 7

Drug loading and release study of BPLP-co-PLGA nanoparticles. (a) SEM image of 5-fluorouracil (5-Fu) loaded BPLP-co-PLGA 75/25-100 nanoparticles. (b) Standard curve of 5-Fu obtained by high-performance liquid chromatography (HPLC) (insert, UV-vis curve of 5-Fu). (c) 5-Fu encapsulation efficiency and loaded contents in PLGA and BPLP-co-PLGA nanoparticles. (d) Complete release profiles of 5-Fu from 5-Fu loaded PLGA nanoparticles (as control) and 5-Fu loaded BPLP-co-PLGA nanoparticles in PBS (pH 7.4) at 37 °C.