Temperature-responsive biocompatible copolymers incorporating hyperbranched polyglycerols for adjustable functionality

Abstract

Temperature-triggered copolymers are proposed for a number of bio-applications but there is no ideal material platform, especially for injectable drug delivery. Options are needed for degradable biomaterials that not only respond to temperature but also easily accommodate linkage of active molecules. A first step toward realizing this goal is the design and synthesis of the novel materials reported herein. A multifunctional macromer, methacrylated hyperbranched polyglycerol (HPG-MA) with an average of one acrylate unit per copolymer, was synthesized and copolymerized with N-isopropylacrylamide (NIPAAm), hydroxyethyl methacrylate-polylactide (HEMAPLA) and acrylic acid (AAc). The potential to fully exploit the copolymers by modification of the multiple HPG hydroxyl groups will not be discussed here. Instead, this report focuses on the thermoresponsive, biocompatible, and degradation properties of the material. Poly(NIPAAm-co-HEMAPLA-co-AAc-co-HPG-MA) displayed increasing lower critical solution temperatures (LCST) as the HPG content increased over a range of macromer ratios. For the copolymer with the maximum HPG incorporation (17%), the LCST was ~30 °C. In addition, this sample showed no toxicity when human uterine fibroid cells were co-cultured with the copolymer for up to 72 h. This copolymer lost approximately 92% of its mass after 17 hours at 37 °C. Thus, the reported biomaterials offer attractive properties for the design of drug delivery systems where orthogonally triggered mechanisms of therapeutic release in relatively short time periods would be attractive.

Scheme 1

Thermogelling Biomaterials from the Acrylic Macromers Methacryalted-Hyperbranched Polyglycerol (HPG-MA) ^a and 2-Hydroxyethyl Methacrylate-poly(lactic acid) (HEMAPLA) ^b Copolymerized with Monomers N-Isopropylacrylamide (NIPAAm) and Acrylic Acid (AAc) by Radical Polymerization. ^a Methacrylate moieties that enable incorporation of HPG into the copolymer are introduced in the first step. The schematic illustration of this reaction is simplified, recognizing that on average one out of 29 pendant HPG hydroxyl groups reacted in the methylation step; ^b The HEMAPLA macromer is prepared as a standalone reaction. The resulting four component copolymer is a branched statistical copolymer.

Figure 1

¹H NMR spectra (CD₃OD) of hyperbranched polyglycerol (HPG) obtained from anionic polymerization initiated with 1,1,1-tris(hydroxymethyl)propane (A). ¹H NMR spectra (DMSO-d₆) of methacrylated HPG (DS = 0.16) (B); the insert shows the magnified region where the acylate peaks of HPGMA appear (C).

Figure 2

ESI-TOF of (A) HPG-MA and (B) HPG macromer precursor.

Figure 3

¹H NMR spectra (DMSO-d₆) of HEMAPLA.

Figure 4

(a) ¹H NMR spectra (DMSO-d₆) of copolymers of poly(NIPAAm-co-HEMAPLA-co-AAc-co-HPG-MA), where the spectra represent (A) Control; (B) HPG Low; (C) HPG Med; and (D) HPG High.

Figure 5

¹³C NMR spectra for poly(NIPAAm-co-HEMAPLA-co-AAc-co-HPG-MA) where the spectrum represents polymer sample HPG High.

Figure 6

MALDI of copolymers (A) HPG-High and (B) Control.

Figure 7

LCST determination by DSC analysis for all solutions of poly(NIPAAm-co-HEMAPLA-co-AAc-co-HPG-MA). The gray box focuses on the temperature range where all transitions were observed as indicated by the minimum in the endotherm trace.

Figure 8

LCST determination by measurement of copolymer solution optical absorption as a function of temperature.

Figure 9

MTS assay to measure the cytotoxicity of HPG or copolymer HPG High at various concentrations. The materials were incubated with cultured fibroid cells for a total of 72 h before assessing cell viability in each group (n = 2). No statistically significant difference was noted relative to the control.

Figure 10

Degradation studies of 16.7 wt% copolymer gel HPG High at 37 °C showing GPC curves (A) and change in the molecular weight with time (B).

Figure 10

Degradation studies of 16.7 wt% copolymer gel HPG High at 37 °C showing GPC curves (A) and change in the molecular weight with time (B).

Figure 11

¹H-NMR (D₂O) spectral change during hydrolytic degradation of a representative poly(NIPAAm-co-HEMAPLA-co-AAc-co-HPG-MA) sample: (A) after 0 hours; (B) 15 hours; (C) 21 hours; and (D) 56 hours of degradation.

Figure A1

NMR spectra of hyperbranched polyglycerol (HPG-1 in Table S1) obtained in the anionic polymerization initiated with 1,1,1-tris(hydroxylmethyl)propane (TMP): (A) ¹³DEPT spectrum, (B)¹³C inverse gated spectrum.

Figure A2

Representative MALDI-TOF mass spectra of HPG. Peaks are separated by 74 g/mole (the mass of the monomer glycidol).