3.1. Structural Characterization
We monitored the progress of the polymerization reaction using FTIR. The most prominent changes were observed on isocyanate groups and ether linkage. The isocyanate groups of intermediate I () at 2250 cm−1 disappeared after PEGylation, whereas the ether linkage of PEG block in ESHU () appeared at 1100 cm−1 indicating PEGylation occurred to both isocyanate end groups of intermediate I. The strong peak at 1680 cm−1 was assigned to carbonyl groups of amide bonds. Moreover, other characteristic peaks such as amide N-H and urethane C-O-C were observed at 1520 and 1250 cm−1, respectively.
FTIR spectra of (A) intermediate I and (B) ESHU. The reactive isocyanate groups of intermediate I at 2250 cm−1 (*) completely disappeared while ether linkage corresponding to PEG in ESHU at 1100cm−1 (#) appeared after the PEGylation.
To further examine the chemical structures of ESHU, we performed 1H FTNMR analysis (). The methylene protons in PEG (a) and N-BOC-serinol (d) were confirmed at 3.65 and 4.0–4.2 ppm, respectively. The methylene protons adjacent to nitrogen in HDI unit (e) were observed at 3.17 ppm. The protons bound to nitrogen in HDI and N-BOC-serinol (b,c) were confirmed at 4.85–5.25 ppm. The signal at 1.42 ppm was assigned to methyl protons in the BOC groups.
1H FTNMR spectra of ESHU in CDCl3. The presence of a, e and g protons indicate the presence of PEG, polyurethane and BOC-protected amine groups in ESHU.
The hydrophobic:hydrophilic ratio of reverse thermal gels is an important factor to determine thermal behaviors such as gelling rate and phase transition temperature. To estimate the hydrophobic:hydrophilic ratio, we measured molecular weights (Mw
) of ESHU. The Mw
of ESHU was 3,955 with relatively narrow PDI (1.66). We designated the value, calculated by the Mw
of PEG subtracted from Mw
of ESHU, as the Mw
of hydrophobic block. The ratio was calculated by the comparison of Mw
of PEG and hydrophobic block. Thus, the ratio of hydrophobic:hydrophilic was 2.6 which was in the range (1.2 – 6) as reported previously [21
3.2. Sol-gel phase transition
Reverse thermal gels undergo sol-gel phase transitions caused by rapid changes in solubility upon heating or cooling. They are fluidic at low temperatures and gel at elevated temperatures. Further heating beyond the gelation temperature can result in phase separation. To use reverse thermal gels as injectable biomaterials, it should gel at body temperature. We investigated the phase transition phenomenon of ESHU rheologically by measuring elastic modulus upon temperature sweep and time sweep (). No significant changes in elastic modulus were observed below 30°C indicating it remained fluidic (). A sharp increase in elastic modulus was observed between 30–39°C corresponding to the sol to gel phase transition. Further heating over 39°C led to a decrease in elastic modulus corresponding to phase separation. Although the gelling and phase separation temperature depended on the polymer concentrations, all the polymer solutions remained gel at body temperature (see supplementary data Fig. S1
). To estimate how fast ESHU can gel in the human body, we measured the elastic modulus at 37°C (). Regardless of concentration, the rapid increase in elastic modulus completed within 3 min suggesting that ESHU solution can form a gel quickly upon injection. These observations indicate that ESHU possesses appropriate thermal properties for biomedical applications.
Fig. 4 The elastic modulus of ESHU. (A) Temperature sweep was recorded in the temperature range of 25–45°C (0.5°C/min) at concentrations of 20 and 30% (wt). (B) Time sweep was recorded at 37°C at concentrations of 20 and 30% (wt) (more ...)
3.3. In vitro degradation
Degradation rate is an important parameter of biomaterials, especially those intended for applications in tissue engineering and drug delivery. To determine the degradability of ESHU, we measured the changes of Mw
in PBS and CE solution at 37°C. The degradation rate was expressed as the ratio of the Mw
upon degradation and that of the new polymer (). No significant changes of Mw
were observed in 14 days in PBS and approximately 1.9% decrease was observed at day 45. The presence of CE greatly accelerated the degradation, which reached 6.6, 12.0 and 20.2% in 7, 14 and 45 days respectively. There are many evidences of in vitro
degradation of polyurethanes by enzymes such as chymotrypsin [39
] and CE [34
]. Polyurethanes can also be degraded by oxidation. An in vivo
oxidative environment can be formed by immune system via macrophages and foreign body giant cells. Several researches have revealed that polyurethanes were degraded by oxidation mimicking an in vivo
biodegradation mechanism of immune system [40
]. Thus the biodegradation rate of ESHU is expected to be faster in vivo
In vitro degradation of ESHU in PBS and CE solution. The degradation of ESHU was much faster in the presence of CE. Data are presented as means ± S.D (n=3).
3.4. In vitro cytotoxicity
The in vitro cytotoxicity of ESHU was examined according to ISO 10993-5 guideline on extraction methods to evaluate the toxicity of biomaterials. The morphologies of cells exposed to the extracts () resembled that on the control () and most cells are viable after exposure to the extracts as indicated by green fluorescence micrographs captured in the live/dead assay (). These images are consistent with the high percentage of live cells observed in live/dead assay (). All numbers were calculated by comparing the fluorescence of live and dead cells collected by the microplate reader. The percentage of live cells was statistically the same as the control based on a two-tailed Student’s t-test. We did not perform viability test with diluted solutions because the original extracts showed good cytocompatibility.
Fig. 6 In vitro cytotoxicity of ESHU toward baboon smooth muscle cells by MCDB extracts for 24 h at 37°C. Phase contrast of cell morphologies of control (A) and extract (B). No differences were observed between control and extract. Fluorescence images (more ...)
3.5. In vivo biocompatibility
In order to investigate host response of ESHU, in vivo biocompatibility test was performed. We examined the highest possible concentration of ESHU to test the most severe host response. All the animals survived throughout the study with no malignant infection and abscess at the injection sites. Tissues adjacent to the polymer showed native histological structure with regular muscle alignment and cell morphology at 3 days (). H&E staining revealed the presence of significant inflammatory infiltrates around the polymer. MTS staining showed a loose collagen layer (blue) surrounding the polymer and appeared to be newly formed and immature. Two weeks post-injection (), the tissues surrounding polymer showed regular cell morphology and alignment. The amount of inflammatory infiltrate decreased significantly and MTS staining showed collagen depositions around the polymer appeared to be more mature. Generally, the tissues surrounding the polymer showed characters of fibrous tissues, and host cells infiltrated into the polymer and began to remodel the gel. Four weeks after injection (), the inflammation was largely resolved with tissue architecture surrounding the polymer mostly returned to normal. The polymers and the host tissue appeared to be well integrated. Cellular remodeling occurred in most part of the gel. Tissues within the gel contained smaller amount of collagen and had less organized structures than the surrounding tissues.
Fig. 7 Photomicrographs of H&E and MTS stained sections of the tissues adjacent to ESHU injection site (marked by ***). The tissues were harvested after: 3 days (A, D, and G), 14 days (B, E, and H), and 28 days (C, F, and I). (A–C) Low magnification (more ...)
ED1 staining was used to estimate macrophage activities triggered by the injection of the gel. At 3 days (), a large number of newly recruited (ED1-positive) macrophages aggregated around the polymers and presented red-stained band around the implant, which indicated an acute inflammatory reaction. We attributed this to a non-specific inflammatory reaction because it was widely observed in many implanted biomaterials [47
]. Two weeks post-injetion (), the density of ED1-positive cells decreased slightly. However, there was no difference statistically. Four weeks after injection (), the density of ED1-positive cells significantly decreased and the band of newly-recruited macrophages disappeared indicating the infiltration of macrophages into the gel and the cellular remodeling of the material accompanied by mild inflammatory reactions. To quantify ED1+ macrophages, 5 images were chosen randomly around tissue-ESHU interface and quantified using Nikon NIS-Elements software. The sequential and significant decrease in the number of macrophages per square millimeter from 1589 at 3 days to 1329 at 14 days and 194 at 28 days indicating a mild inflammation that was mostly resolved by 4 weeks (). Thus, ESHU showed biocompatibility in SC implantation even at the highest dose.
Fig. 8 Representative photomicrographs (200×, scale bar = 60 μm) of injection sites immunohistochemically stained for ED1+ macrophages. Tissues were harvested after: (A) 3 days; (B) 14 days, and (C) 28 days. (D) The number of ED1+ macrophages (more ...)
3.6. Functionalization of ESHU with IKVAVS
Typical components of injectable reverse thermal gels are poly(L-lactic acid) [48
], poly[(lactic acid)-co-(glycolic acid)] [18
], poly(ε-caprolactone) [51
] and poly(N-isopropylacrylamide) [53
]. These polymers show good biocompatibility and sol-gel phase transitions around body temperature. There is a significant emphasis on functionalized injectable gels for biomedical applications. Recent reports include tertiary amines [55
], a RGD conjugated poly(organophosphazene) [58
], and a dopamine conjugated hyaluronic acid [59
]. We created a serine-based reverse thermal gel by simple chemistry without catalyst. The polymer is easy to purify and contains a primary amine group in each repeating unit that leads to versatile bio-functionalization. To verify the ability of bio-functionalization using the ESHU platform, we investigated a reaction between ESHU and IKVAVS. IKVAV is a laminin epitope that is known as a promotor of cell adhesion and neurite outgrowth [60
]. To reserve the bioactivity of IKVAV, we needed a spacer amino acid that directly reacts with the primary amine of NH2
-ESHU. We chose Ser
because IKVAVS is the natural sequence in laminin. To our knowledge, this is the first report of an IKVAVS-containing thermal gel.
We monitored the incorporation of IKVAVS using FTIR. The most prominent change was observed on carbonyl peaks between 1600–1700 cm−1. The NH2-ESHU showed one carbonyl peak from amide bonds of ESHU backbone at 1680 cm−1 (). However, IKVAVS-ESHU showed two carbonyl groups at 1680 and 1630 cm−1. The peak at 1680 cm−1 was same as the one in NH2-ESHU. The peak at 1630 cm−1 was assigned to carbonyl groups from peptide bonds in IKVAVS-ESHU which was a strong evidence that IKVAVS was incorporated well (). Other characteristic peaks such as ether linkage of PEG block, amide N-H, and urethane C-O-C were observed at 1100, 1520, and 1250 cm−1, respectively.
FTIR spectra of (A) NH2-ESHU and (B) IKVAVS-ESHU. The carbonyl groups of the peptide bonds in IKVAVS-ESHU was observed at 1630 cm−1 (*) after IKVAVS incorporation.
We further examined the chemical structure of IKVAVS-ESHU using 1H FTNMR analysis (). The inset displaying the spectrum of NH2-ESHU showed no BOC groups at 1.42 ppm indicating a clean de-protection. The most prominent difference between NH2-ESHU and IKVAVS-ESHU was observed in the range of 0.8–2.2 ppm (dotted area). The methyl protons in Ile (a), Ala (c), Val (e) were observed at 0.93, 1.62, and 1.17 ppm, respectively. The methylene protons in Lys (b) were observed at 1.75 and 1.87 ppm. The hydroxyl proton in Ser (d) appeared at 2.04 ppm. The degree of substitution of IKVAVS was 41.3% as calculated by the ratio of the methylene proton in Lys and the methylene protons “f” in the polymer backbone (same designation as in ).
Fig. 10 1H FTNMR spectra of IKVAVS-ESHU in D2O. The incorporation of IKVAVS was confirmed by appearance of new peaks between 0.8–2.2 ppm. The presence of a, b, c, d, and e protons indicates the presence of Ile, Lys, Ala, Ser, and Val, respectively. The (more ...)
In order to investigate the potential as an injectable biomaterial, the elastic modulus of IKVAVS-ESHU was studied rheologically as described previously. In our previous works (data not shown), the solution of IKVAVS-ESHUs synthesized from low Mw of ESHU exhibited no thermal gelling properties regardless of concentrations. We deduced that the IKVAVS-ESHUs from low Mw were too hydrophilic to gel. Indeed an increase in the MW of the hydrophobic block of ESHU led to successful thermal gelation of IKVAVS-ESHU. The elastic modulus of this polymer solution increased with increasing temperature and formed gel at body temperature (). A dramatic increase in elastic modulus was observed between 28–36°C. The polymer completely gelled at 37°C () with no significant changes in elastic modulus until phase separation at 43°C. The measurement of the change of elastic modulus at 37°C mimicked the injection into a human body, which showed the polymer solution gelled completely in less than a minute (). These demonstrated that the IKVAVS-functionalized ESHU was an injectable thermal gel.
Fig. 11 The elastic modulus of IKVAVS-ESHU. (A) Temperature sweep was recorded in the temperature range of 25–45°C (0.5°C/min) at concentrations of 15% (wt). The polymer solution showed rapid change of elastic modulus upon heating and (more ...)
We successfully functionalized ESHU with IKVAVS through the primary amine groups on the polymer. The conjugation of IKVAVS led to changes of physicochemical properties. We monitored the structural changes by FTIR. The overall spectra of ESHU (), NH2-ESHU (), and IKVAVS-ESHU () were the same except the appearance of two carbonyl peaks in the IKVAVS-ESHU spectrum. We observed single carbonyl peak in both ESHU and NH2-ESHU at 1680 cm−1 originated from the urethane bonds. In contrast, IKVAVS-ESHU exhibited an additional carbonyl stretch at 1630 cm−1 corresponding to amide groups of the peptide bonds in IKVAVS. We further monitored structural changes by 1H FTNMR. The general proton signals of ESHU () and NH2-ESHU ( inset) were the same except that the methyl protons in ESHU at 1.42 ppm disappeared in the spectra of NH2-ESHU indicating the removal of BOC protective groups. On the other hand, IKVAVS-ESHU displayed a vastly different 1H FTNMR spectrum () at 0.8–2.2 ppm: New proton signals appeared at a, b, c, d, and e that corresponded to Ile, Lys, Ala, Ser, and Val respectively. The appearance of an additional strong amide stretch in IR and the correlated additional proton signals in NMR are indicative of functionalization with IKVAVS. The functionalization also led to the change of elastic property upon heating. The dramatic increase in elastic modulus of ESHU was observed at 30–39°C, and it decreased right after reaching maximum elastic modulus (). Whereas the dramatic increase in elastic modulus of IKVAVS-ESHU was observed at 28–36°C and maintained its gel state until 43°C (), much longer than ESHU gel. The rate of sol to gel phase transition at body temperature was also changed. IKVAVS-ESHU formed a complete gel in 40 sec () which was much faster than ESHU (3 min, ). These observations correlated well with the hydrophobic nature of IKVAVS, and the functionalization resulted in faster phase transition and a more stable gel.