3.1. Analysis of Polymer Architecture
To the best of our knowledge, there were no reports on the ATRP of NIPAM with glucosamine derivatives as the initiator. The GA initiator and GA-PNIPAM were characterized by 1
HNMR, GPC, FT-IR. Using an NIPAM/GA initiator/CuBr/Me6
TREN feed ratio of 50:1:1:1, we could obtain different conversion percents and products with different
values. The data in Table 1
show that the polymers had a narrow molecular weight distribution, the
of the GA-PNIPAM (i) and (ii) remained narrow with values in the range of 1.14 to 1.18 and 1.09 to 1.17, respectively. Compared with the GA-PNIPAM (i) system, the conversion rate and molecular weight of the C-1 initiator of GA were low at the initial reaction; this may be caused by the stereospecific blockade of the phthalimide. As the reaction proceeded, the polymer chain extended in an equatorial bond position of the chair conformation of β
-glucosamine to overcome the resistance.
After a series of purifications, GPC traces of polymers (shown in Figure 1
) were relatively symmetric and showed no tailing at either side, suggesting the absence of any small molecular residues in the final product, such as the initiator, monomer or other byproducts.
To prepare a well-defined polymer, synthesizing the two pure precursors as an initiator is necessary. Figure 2
shows the 1
HNMR spectra of the initiator and the product for (i). In Figure 2
a, the peaks located at 8.05 ppm and in the range of 5.52 to 4.06 ppm were ascribed to the protons adjacent to GA, whereas the signals at 1.93 ppm in the range of 2.15 to 1.98 ppm corresponded to the methyl and acetyl groups, respectively; these peaks revealed that bromide was successfully introduced into the GA, protected by acetyl groups. The signals at 6.55, 4.0, 2.93, 1.60 and 1.02 ppm in Figure 2
b were attributed to the protons of the repeating units of NIPAM, and the signal at 8.50 ppm was the characteristic signal of protons adjacent to nitrogen atoms.
showed the 1
HNMR spectra of the initiator and the product for (ii). In Figure 3
a, the peaks located at 8.10 ppm in the range of 6.12 to 4.05 ppm were ascribed to the protons adjacent to GA, those at 7.90 to 7.74 ppm were due to the phthalimide, and the signals at 1.93 ppm in the range of 2.15 to 1.98 ppm corresponded to the methyl and acetyl groups, respectively; these peaks revealed that the GA initiator (ii) was synthesized successfully by 1-hydroxy-3,4,6-tri-O
-glucose and 2-Bromopropionyl bromide. The signals at 6.55, 4.0, 2.93, 1.60 and 1.02 ppm in Figure 3
b were attributed to the protons of the repeating units of NIPAM, and the signal at 8.50 ppm was the characteristic signal of protons adjacent to nitrogen atoms. We can clearly observe the peaks for each hydroxyl of the GA. Since the hydrogen atoms of the amino acid are very active, we cannot observe the amino signal peaks in deuterated reagents, and the peaks at 1.7 ppm to 1.4 ppm may be the superposition of the amino acid with water or NIPAM repeat units. However, we measured the aqueous solution of the polymer which was alkaline in this experiment.
The structure of GA-PNIPAM (i) and (ii) were also characterized by the FT-IR spectrum (as shown in Figure 4
). The absorption peaks characteristic of PNIPAM can be clearly observed, as evidenced by the presence of the band at 3446 cm−1
in Figure 4
b, which was assigned to the stretching vibration (νN–H
) of the acylamino group. The band at 1653 cm−1
was ascribed to amide I (mainly the carbonyl stretching vibration (νC=O
)) and the band at 1558 cm−1
was ascribed to amide II (mainly the N–H bending vibration (δN–H
)), and in Figure 4
c, these peaks appeared at 3436 cm−1
, 1632 cm−1
and 1544 cm−1
, respectively. As seen in Figure 4
b, the peak at 1750 cm−1
which corresponds to ester group absorption disappeared in Figure 4
c, and at 3304 cm−1
in the Figure 4
b the typical bands of the amino groups of GA-PNIPAM (ii) are visible. Thus, it was suggested that the well-defined GA-PNIPAM (i) and (ii) were successfully prepared through ATRP using a different initiator.
3.3. Assessment of Cell Viability
In our current study, by introducing the hydrophilic monomer GA into the PNIPAM chain, the LCST of the resultant polymers could be tuned to near body temperature for better biological application. So P4 of the GA-PNIPAM (i) and (ii) was used to evaluate the cytotoxicity. A cytotoxicity study on L-929 and HepG2 was conducted to investigate the biocompatibility of the PNIPAM homopolymer, GA, GA-PNIPAM (i) and (ii). The percent of cell viability was determined by comparison with cells that were not exposed to samples, which were used as the control group. No statistically significant cytotoxicity of the PNIPAM homopolymer was observed in cells, as shown in Figure 6
A,B. Neither the P4 GA-PNIPAM polymer (i) nor (ii) was toxic in either cell line over a broad concentration range from 0.10 to 1000.00 μg/mL, and no clear difference between polymers was observed with regard to cytotoxicity to L-929. The viability of the HepG2 cells decreased to approximately 90% for GA-PNIPAM (i), whereas the value was only 80% for GA-PNIPAM (ii). It showed that GA-PNIPAM (ii) had suppressing activity for cancer cells; Figure 6
C also shows evidence of this, which may be due to the amino groups present in GA-PNIPAM (ii). From Figure 6
B,C), GA (reduction by sodium methylate) could effectively kill tumor cells, but the suppressing activity of GA-PNIPAM (ii) was weaker than that of GA. This might be due to the GA content of the polymer that was synthesized by ATRP being lower than that of the pure GA. Furthermore, at each concentration level of the dispersions of the GA and GA-PNIPAM (ii), cell survival decreased considerably as the time increased, as shown in Figure 6
C, and cell activity was decreased along with the concentration. GA and GA-PNIPAM (ii) could change the growth state of HepG2 cells at the concentration of 1000.00 μg/mL after feeding 24 has shown in Figure 6
D. HepG2 cells had a good condition and grew vigorously at the initial experiment (Figure 6
D-1); when the GA and GA-PNIPAM (ii) were incubated in the cells, a large number of necrotic cells appeared, especially in the GA group. These indicated that GA-PNIPAM (ii) had the inhibition of tumor cell growth, and it could be a new smart material for antitumor effects and it may enhance the biocompatibility of GA for biomedicine.