ARTICLE pubs.acs.org/Biomac
Enzymatically Degradable Thermogelling Poly(alanine-co-leucine)poloxamer-poly(alanine-co-leucine) Hyo Jung Moon, Bo Gyu Choi, Min Hee Park, Min Kyung Joo, and Byeongmoon Jeong* Department of Chemistry and Nano Science, Department of Bioinspired Science (WCU), Ewha Womans University, Daehyun-Dong, Seodaemun-Ku, Seoul, 120-750, Korea
bS Supporting Information ABSTRACT: In the search for an enzymatically degradable thermogelling system, we are reporting poly(alanine-co-leucine)-poloxamer-poly(alanine-co-leucine) (PAL-PLX-PAL) aqueous solution. As the temperature increased, the polymer aqueous solution underwent sol-to-gel transition at 20-40 °C in a polymer concentration range of 3.0-10.0 wt %. The amphiphilic polymers of PAL-PLX-PAL form micelles in water, where the hydrophobic PALs form a core and the hydrophilic PLXs form a shell of the micelle. FTIR, circular dichroism, and 13C NMR spectra suggest that the R-helical secondary structure of PAL is preserved; however, the molecular motion of the PLX significantly decreases in the sol-to-gel transition range of 20-50 °C. The polymer was degraded by proteolytic enzymes such as matrix metalloproteinase and elastase, whereas it was quite stable against cathepsin B, cathepsin C, and chymotrypsin or in phosphate-buffered saline (control). The in situ formed gel in the subcutaneous layer of rats showed a duration of ∼47 days, and H&E staining study suggests the histocompatibility of the gel in vivo with a marginal inflammation response of capsule formation. A model drug of bovine serum albumin was released over 1 month by the preset-gel injection method. The thermogelling PAL-PLX-PAL can be a promising biocompatible material for minimally invasive injectable drug delivery.
’ INTRODUCTION Aqueous solutions of thermogelling polymers undergo sol-togel transition as the temperature increases. Therefore, the drug or cells are mixed in a sol state, followed by injection at a target site to make a depot system. Without the need of surgical procedure, a conventional syringe injection of the thermogelling polymer aqueous solution containing drug makes it a minimally invasive delivery system. Homo- and copolymers of lactic acid/glycolic acid/caprolactone, chitosan, polyphosphazenes, poloxamer derivatives, polycarbonates, polycyanoacrylates, β-lactoglobulin, elastin-like polypeptide (ELP)-based polymers, and silk-elastinlike polymers have been developed as biodegradable thermogelling polymer aqueous solutions.1-10 However, most thermogelling polymers suffer from reconstitution of the formulation because of the slow dissolution kinetics of the polymer in water. As the temperature increases, the aqueous solubility of the thermogelling polymer decreases. Therefore, the traditional method of preparing the aqueous solution of the thermogelling polymer is to dissolve the polymer in cold water, requiring a long time for reconstitution of the drug formulation as a solution. Thermogelling polymers with enzymatic degradability can solve the solubility problem by storing the polymer as an enzyme-free buffer solution. The polymer begins to be degraded by the enzymes only after in vivo application, suggesting the storage stability and ready-to-use formulation of the thermogelling polymer as an aqueous solution, rendering a significant improvement of the convenience for medical application. Recently, we r 2011 American Chemical Society
reported a poly(ethylene glycol)-poly(alanine-co-phenylalanine) (PEG-PAF) that underwent degradation by cathepsin B, cathepsin C, and elastase.11,12 The polymer showed stability in phosphate-buffered saline (in vitro) without any significant mass loss (10% decrease in the Rhelical content as the temperature increases from 20 to 50 °C.31 Another interesting observation in the FTIR spectra of PALPLX-PAL is the temperature-sensitive changes in N-H
Figure 4. (a) Mean residue ellipticity of PAL-PLX-PAL aqueous solution (0.01 wt %) as a function of temperature. (b) FTIR spectra of PAL-PLX-PAL aqueous solution (10.0 wt % in D2O) as a function of temperature. (c) 13C NMR spectra of the PAL-PLX-PAL (10.0 wt % in D2O) as a function of temperature.
stretching and bending modes of PAL-PLX-PAL at 30003700 and 1370-1540 cm-1, respectively.32 The PLX did not show a significant change in the FTIR spectra in this region, suggesting that the dipole moment of R-helical polypeptide of N-H is sensitive to the temperature change. 13 C NMR showed the significant collapse of PLX peak at 72.2-72.5 ppm and a slight upfield shift, whereas the PAL peak at 18-20 ppm showed a slight broadening as the temperature increased from 20 to 50 °C, suggesting that conformational changes in hydrophilic PLX are the major driving factor in the sol-to-gel transition of PAL-PLX-PAL while keeping the Rhelical secondary structure of the polypeptide (Figure 4c) By varying the composition of PAL at a fixed molecular weight of PLX at 2000 Da, not only the secondary structure of the polypeptide but also the sol-gel transition temperature of the PAL-PLX-PAL could be controlled. The secondary structures of the PAL in PAL-PLX-PAL aqueous solutions were analyzed by amide I band of FTIR spectra.19 The amide I band 1238
dx.doi.org/10.1021/bm101518c |Biomacromolecules 2011, 12, 1234–1242
Biomacromolecules
ARTICLE
Figure 6. Enzymatic degradation of the PAL-PLX-PAL. (a) Mass loss of the gel in the presence of enzymes (n = 3). (b) Gel permeation chromatogram of the remaining gel 6 days after the incubation. Control is the PAL-PLX-PAL incubated in phosphate-buffered saline (pH 7.4).
Figure 5. Deconvolution of amide I band in FTIR spectra of (a)A7.0L1.8-PLX-A7.0L1.8 and (b) A5.5L2.1-PLX-A5.5L2.1 aqueous solutions (10.0 wt % in D2O) at 20 °C. (c) Structure-property relationship of phase diagram of the PAL-PLX-PAL aqueous solutions determined by the test tube inverting method (n = 3).
at 1600-1700 cm-1 was deconvoluted into R-helix (1659 cm1 ), random coil (1646 cm-1), and parallel β-sheet (1628 and 1623 cm-1) (Figure 5a,b, fifth column in Table 1). The PI (A7.0L1.8-PLX-A7.0L1.8) with dominantly R-helical secondary structure showed a lower sol-to-gel transition temperature than PII (A5.5L2.1-PLX-A5.5L2.1) with a dominantly β-sheet structure (Figure 5c). The control of secondary structure polypeptide by varying the molecular weight of PEG-PA diblock copolymers has also been reported.20 As the molecular weight of PEG or PA increased, the transition of secondary structure from β-sheet to R-helix was observed. Current thermogelling polymers emphasize that the composition of the polypeptide controls the secondary structure of the polypeptide and also affects the sol-gel transition temperature of the polymer aqueous solution. To investigate the feasibility of biomedical applications of the PAL-PLX-PAL, we carried out a degradation study in in vitro buffered solution containing enzymes as well as in in vivo. Aqueous solution containing proteinase K was used as a
positive control, and phosphate-buffered solution without any enzymes was used as a negative control. The significant mass loss of the in situ formed gel was observed in aqueous solutions containing MMP-12, elastase, and proteinase K, whereas
