Thermally responsive injectable hydrogel incorporating methacrylate-polylactide for hydrolytic lability

Abstract

Injectable thermoresponsive hydrogels are of interest for a variety of biomedical applications, including regional tissue mechanical support as well as drug and cell delivery. Within this class of materials there is a need to provide options for gels with stronger mechanical properties as well as variable degradation profiles. To address this need, the hydrolytically labile monomer, methacrylate-polylactide (MAPLA), with an average 2.8 lactic acid units, was synthesized and copolymerized with N-isopropylacrylamide (NIPAAm) and 2-hydroxyethyl methacrylate (HEMA) to obtain bioabsorbable thermally responsive hydrogels. Poly(NIPAAm-co-HEMA-co-MAPLA) with three monomer feed ratios (84/10/6, 82/10/8, and 80/10/10) was synthesized and characterized with NMR, FTIR, and GPC. The copolymers were soluble in saline at reduced temperature (<10 degrees C), forming clear solutions that increased in viscosity with the MAPLA feed ratio. The copolymers underwent sol-gel transition at lower critical solution temperatures of 12.4, 14.0, and 16.2 degrees C, respectively, and solidified immediately upon being placed in a 37 degrees C water bath. The warmed hydrogels gradually excluded water to reach final water contents of approximately 45%. The hydrogels as formed were mechanically strong, with tensile strengths as high as 100 kPa and shear moduli of 60 kPa. All three hydrogels were completely degraded (solubilized) in PBS over a 6-7 month period at 37 degrees C, with a higher MAPLA feed ratio resulting in a faster degradation period. Culture of primary vascular smooth muscle cells with degradation solutions demonstrated a lack of cytotoxicity. The synthesized hydrogels provide new options for biomaterial injection therapy where increased mechanical strength and relatively slow resorption rates would be attractive.

Figure 1

(a) ¹H-NMR and (b) ¹³C-NMR spectra for MAPLA.

Figure 1

(a) ¹H-NMR and (b) ¹³C-NMR spectra for MAPLA.

Figure 3

(a) ¹H-NMR and (b) ¹³C-NMR spectra for poly(NIPAAm-co-HEMA-co-MAPLA) (80/10/10).

Figure 3

(a) ¹H-NMR and (b) ¹³C-NMR spectra for poly(NIPAAm-co-HEMA-co-MAPLA) (80/10/10).

Figure 4

LCST determination by (a) measurement of copolymer solution optical absorption; (b) measurement of shear modulus on a rheometer, 1 Hz, 2% strain; (c) DSC analysis, 5°C/min.

Figure 4

LCST determination by (a) measurement of copolymer solution optical absorption; (b) measurement of shear modulus on a rheometer, 1 Hz, 2% strain; (c) DSC analysis, 5°C/min.

Figure 4

LCST determination by (a) measurement of copolymer solution optical absorption; (b) measurement of shear modulus on a rheometer, 1 Hz, 2% strain; (c) DSC analysis, 5°C/min.

Figure 5

Gelation process of the poly(NIPAAm-co-HEMA-co-MAPLA) (80/10/10) hydrogel (PBS, 16.7 wt %) and the microstructure of the hydrogel formed at 37°C after 30 sec and 1 day. Samples were quenched with liquid nitrogen and freeze dried at −40°C.

Figure 6

Gelation of the poly(NIPAAm-co-HEMA-co-MAPLA) hydrogels (PBS, 16.7 wt %). Water contents were plotted against the incubation time of the hydrogels in a 37°C water bath.

Figure 7

Viscosity of the poly(NIPAAm-co-HEMA-co-MAPLA) hydrogel solutions (PBS, 16.7 wt %) at 10°C.

Figure 8

Tensile curves (a) and maximum tensile strength (b) of the poly(NIPAAm-co-HEMA-co-MAPLA) hydrogels at 37°C. Hydrogels were formed in 37°C water bath for 24h.

Figure 9

Dynamic shear modulus of the poly(NIPAAm-co-HEMA-co-MAPLA) hydrogels at 37°C. Strain, 5%. Hydrogels were formed in a 37°C water bath for 24 hrs.

Figure 10

(a) ¹H-NMR of poly(NIPAAm-co-HEMA-co-MAPLA) (80/10/10) before (a) and after (b) the removal of PLA by hydrolysis in 1M NaOH.

Figure 11

Mass loss curves of poly(NIPAAm-co-HEMA-co-MAPLA) hydrogels in PBS at 37°C.

Figure 12

Cytotoxity assay of poly(NIPAAm-co-HEMA-co-MAPLA) (80/10/10) hydrogel degradation products by cell metabolic activity assessment (MTS assay) for RSMCs cultured on tissue culture polystyrene. Hydrolyzed hydrogel solution was supplemented into cell culture medium at a final concentration of 5.0 mg/mL.

Figure 13

Live/dead staining of RSMC cultured on tissue culture polystyrene using (a) untreated culture medium and (b) culture medium containing 5.0 mg/mL hydrolyzed poly(NIPAAm-co-HEMA-co-MAPLA) (80/10/10). Observations were recorded at a culture time of 3 days. Scale bar: 100 μm.

Figure 13

Live/dead staining of RSMC cultured on tissue culture polystyrene using (a) untreated culture medium and (b) culture medium containing 5.0 mg/mL hydrolyzed poly(NIPAAm-co-HEMA-co-MAPLA) (80/10/10). Observations were recorded at a culture time of 3 days. Scale bar: 100 μm.