*2.2. The Preparation of P(OEGMA-co-MEO2MA) Thermosensitive Microgels-Based Hydrogel*

*2.2. The Preparation of P(OEGMA-co-MEO2MA) Thermosensitive Microgels-Based Hydrogel*  Next, we studied the feasibility of using P(OEGMA-co-MEO2MA) microgels to construct thermosensitive hydrogels through the thermal induction method. Referring to our previously reported method [29], we configured the PBS solutions of microgel samples with a concentration of 3.0 wt%, and placed them at 37° C, which was higher than their VPTT. Then, we observed the formation of hydrogels by the inverted method and photo-Next, we studied the feasibility of using P(OEGMA-co-MEO2MA) microgels to construct thermosensitive hydrogels through the thermal induction method. Referring to our previously reported method [29], we configured the PBS solutions of microgel samples with a concentration of 3.0 wt%, and placed them at 37 ◦C, which was higher than their VPTT. Then, we observed the formation of hydrogels by the inverted method and photographed the changes of these hydrogels.

graphed the changes of these hydrogels. As shown in Figure 3, after maintaining for 10 min at 37 °C, all three types of P(OEGMA-co-MEO2MA) microgels could gel and form white hydrogels. This result was consistent with that of the traditional PNIPAM thermosensitive polymer. When the temperature was higher than their VPTT, the microgels, in their hydrophobic contraction state, would aggregate under the synergistic influence of hydrophobicity and the shielding effect of ions from PBS, thus forming hydrogel macroscopically. The gelation times were about 5 min to 10 min. Meanwhile, we found that the hydrogels formed from M1 and M2 showed obvious shrinkage after 2 h, and the shrinkage degree increased with time. After 48 h, the volume of the hydrogels formed from M1 and M2 was less than 1/2 As shown in Figure 3, after maintaining for 10 min at 37 ◦C, all three types of P(OEGMA-co-MEO2MA) microgels could gel and form white hydrogels. This result was consistent with that of the traditional PNIPAM thermosensitive polymer. When the temperature was higher than their VPTT, the microgels, in their hydrophobic contraction state, would aggregate under the synergistic influence of hydrophobicity and the shielding effect of ions from PBS, thus forming hydrogel macroscopically. The gelation times were about 5 min to 10 min. Meanwhile, we found that the hydrogels formed from M1 and M2 showed obvious shrinkage after 2 h, and the shrinkage degree increased with time. After 48 h, the volume of the hydrogels formed from M1 and M2 was less than 1/2 of the initial value. This phenomenon of volume shrinkage has been reported several times regarding traditional PNIPAMs and other thermosensitive hydrogels, because of the dehydration of thermosensitive materials at higher temperature [19,20]. As an ideal scaffold material, the volume contraction should be avoided as far as possible in order to maintain sufficient cell growth space and ensure the circulation of nutrients and metabolic waste [4,6].

Encouragingly, we found that the volume change of the hydrogel formed by the prepared sample M3 with a higher VPTT was less than 5% and no significant shrinkage was observed. So, we also observed and recorded the formation of hydrogels from M4 and M5 microgels with the same VPTT and larger sizes. It can be seen from Figure 4 that the hydrogels formed from M4 and M5 also showed obvious shrinkage after 2 h, and the shrinkage degree increased with time, which was hardly surprising.

waste [4,6].

waste [4,6].

different times.

different times.

different times.

of the initial value. This phenomenon of volume shrinkage has been reported several times regarding traditional PNIPAMs and other thermosensitive hydrogels, because of the dehydration of thermosensitive materials at higher temperature [19,20]. As an ideal scaffold material, the volume contraction should be avoided as far as possible in order to maintain sufficient cell growth space and ensure the circulation of nutrients and metabolic

of the initial value. This phenomenon of volume shrinkage has been reported several times regarding traditional PNIPAMs and other thermosensitive hydrogels, because of the dehydration of thermosensitive materials at higher temperature [19,20]. As an ideal scaffold material, the volume contraction should be avoided as far as possible in order to maintain sufficient cell growth space and ensure the circulation of nutrients and metabolic

*Gels* **2022**, *8*, x FOR PEER REVIEW 6 of 13

**Figure 3.** Photographs of the P(OEGMA-co-MEO2MA) hydrogels formed from M1–M3 microgels at **Figure 3.** Photographs of the P(OEGMA-co-MEO2MA) hydrogels formed from M1–M3 microgels at different times. drogels formed from M4 and M5 also showed obvious shrinkage after 2 h, and the shrinkage degree increased with time, which was hardly surprising.

**Figure 4.** Photographs of the P(OEGMA-co-MEO2MA) hydrogels formed from M3–M5 microgels at **Figure 4.** Photographs of the P(OEGMA-co-MEO2MA) hydrogels formed from M3–M5 microgels at different times.

**Figure 4.** Photographs of the P(OEGMA-co-MEO2MA) hydrogels formed from M3–M5 microgels at We further investigated the quantitative relation between the volume shrinkage degree Vt/V0 of the hydrogel (the ratio of the volume of the formed hydrogel at a certain time point to the initial volume) and time (Figure 5). We found that the hydrogels formed from M1 or M2 contracted rapidly in the first 8 h, when the volume was less than half of the initial volume. After 48 h, the volumes of the samples were about 20% and 30% of the initial volume, respectively. The shrinkage degree was very large. However, the volume of hydrogel formed from M3 still had a volume of more than 98% of the initial volume We further investigated the quantitative relation between the volume shrinkage degree Vt/V0 of the hydrogel (the ratio of the volume of the formed hydrogel at a certain time point to the initial volume) and time (Figure 5). We found that the hydrogels formed from M1 or M2 contracted rapidly in the first 8 h, when the volume was less than half of the initial volume. After 48 h, the volumes of the samples were about 20% and 30% of the initial volume, respectively. The shrinkage degree was very large. However, the volume of hydrogel formed from M3 still had a volume of more than 98% of the initial volume after 48 h without significant shrinkage. We speculated that the reason why the hydrogel formed by M3 did not shrink significantly may be related to its containing longer OEGMA chain fragments with excellent hydrophilic properties, which could hinder the shrinkage We further investigated the quantitative relation between the volume shrinkage degree Vt/V<sup>0</sup> of the hydrogel (the ratio of the volume of the formed hydrogel at a certain time point to the initial volume) and time (Figure 5). We found that the hydrogels formed from M1 or M2 contracted rapidly in the first 8 h, when the volume was less than half of the initial volume. After 48 h, the volumes of the samples were about 20% and 30% of the initial volume, respectively. The shrinkage degree was very large. However, the volume of hydrogel formed from M3 still had a volume of more than 98% of the initial volume after 48 h without significant shrinkage. We speculated that the reason why the hydrogel formed by M3 did not shrink significantly may be related to its containing longer OEGMA chain fragments with excellent hydrophilic properties, which could hinder the shrinkage of the hydrogel. In our previous report [29,34], we found that adding a small amount of PEG to the prepared PNIPAM microgel significantly reduced the shrinkage of the resulting hydrogel. Due to the presence of a large number of PEG fragments in OEGMA, we thought that OEGMA may have a similar effect to PEG in reducing hydrogel shrinkage.

after 48 h without significant shrinkage. We speculated that the reason why the hydrogel formed by M3 did not shrink significantly may be related to its containing longer OEGMA chain fragments with excellent hydrophilic properties, which could hinder the shrinkage We also compared the change of Vt/V<sup>0</sup> of the hydrogels formed from M3, M4, and M5 microgels. It was shown that, as the size of P(OEGMA-co-MEO2MA) microgel increased, the shrinkage of hydrogel also increased. For instance, after 48 h, the volume of the hydrogel samples formed from M3, M4, and M5 were about 98%, 19% and 17% of the initial volume, respectively. We think the reason was that the larger the particle size of the microgel particles with the same VPTT, the higher the water content. Therefore, when at a certain temperature higher than their VPTT, the shrinkage degree of microgels is greater, resulting in more solvents being extruded from the microgels, which would also cause more volume shrinkage of the macroscopic hydrogels.

of the hydrogel. In our previous report [29,34], we found that adding a small amount of PEG to the prepared PNIPAM microgel significantly reduced the shrinkage of the resulting hydrogel. Due to the presence of a large number of PEG fragments in OEGMA, we thought that OEGMA may have a similar effect to PEG in reducing hydrogel shrinkage.

We also compared the change of Vt/V0 of the hydrogels formed from M3, M4, and M5 microgels. It was shown that, as the size of P(OEGMA-co-MEO2MA) microgel increased, the shrinkage of hydrogel also increased. For instance, after 48 h, the volume of the hydrogel samples formed from M3, M4, and M5 were about 98%, 19% and 17% of the initial volume, respectively. We think the reason was that the larger the particle size of the microgel particles with the same VPTT, the higher the water content. Therefore, when at a certain temperature higher than their VPTT, the shrinkage degree of microgels is greater, resulting in more solvents being extruded from the microgels, which would also cause more volume shrinkage of the macroscopic hydrogels. These results indicated that, by adjusting the thermosensitivity (VPTT) and particle size of these synthesized P(OEGMA-co-MEO2MA) microgels, the properties of the ob-These results indicated that, by adjusting the thermosensitivity (VPTT) and particle size of these synthesized P(OEGMA-co-MEO2MA) microgels, the properties of the obtained thermosensitive hydrogels can be adjusted, especially volume shrinkage. We also found that the obtained P(OEGMA-co-MEO2MA) thermosensitive hydrogels showed a small shrinkage degree only by controlling the synthesis conditions of P(OEGMA-co-MEO2MA) microgels; while for the commonly used PNIPAM hydrogels, complex operations, such as physical blending or copolymerization, were required to achieve the same effect. Therefore, the P(OEGMA-co-MEO2MA) thermosensitive hydrogel showed better application prospects in the biomedical field. In following work, we will continue to conduct in-depth studies on P(OEGMA-co-MEO2MA) microgel- based hydrogels, especially the gelation mechanism, gelation conditions and rheological behavior.

### tained thermosensitive hydrogels can be adjusted, especially volume shrinkage. We also *2.3. P(OEGMA-co-MEO2MA) Thermosensitive Hydrogels for 3D Cell Culture*

found that the obtained P(OEGMA-co-MEO2MA) thermosensitive hydrogels showed a small shrinkage degree only by controlling the synthesis conditions of P(OEGMA-co-MEO2MA) microgels; while for the commonly used PNIPAM hydrogels, complex operations, such as physical blending or copolymerization, were required to achieve the same effect. Therefore, the P(OEGMA-co-MEO2MA) thermosensitive hydrogel showed better application prospects in the biomedical field. In following work, we will continue to conduct in-depth studies on P(OEGMA-co-MEO2MA) microgel- based hydrogels, especially the gelation mechanism, gelation conditions and rheological behavior. A hydrogel without shrinkage is more suitable for cell embedding and 3D growth. Therefore, we selected microgel sample M3 as the scaffold material for subsequent 3D cell culture. A type of tumor cell, MCF-7 human breast cancer cell, was chosen as the model cell to explore the feasibility of using this hydrogel to construct a 3D cell model [29,30,33–35]. After a certain concentration of microgel dispersion, cells were evenly mixed at room temperature, and then transferred to 48-well culture plates and placed in a cell incubator at 37 ◦C for culturing. About 1 h later, a stable hydrogel was formed and the cells were encapsulated in situ in it. Part of the DMEM cell culture medium was added to continue this culture process.

*2.3. P(OEGMA-co-MEO2MA) Thermosensitive Hydrogels for 3D Cell Culture*  A hydrogel without shrinkage is more suitable for cell embedding and 3D growth. Therefore, we selected microgel sample M3 as the scaffold material for subsequent 3D cell culture. A type of tumor cell, MCF-7 human breast cancer cell, was chosen as the model cell to explore the feasibility of using this hydrogel to construct a 3D cell model [29,30,33– 35]. After a certain concentration of microgel dispersion, cells were evenly mixed at room temperature, and then transferred to 48-well culture plates and placed in a cell incubator at 37 °C for culturing. About 1 h later, a stable hydrogel was formed and the cells were Firstly, we investigated the viability of cells in the P(OEGMA-co-MEO2MA) hydrogel scaffold using MTT assay. As shown in Figure 6, the absorbance values measured by the MTT method increased significantly in the first two days of cell culture, which may be related to a rapid increase in the number of living cells in this hydrogel scaffold. This result also indirectly reflected the favorable biocompatibility of this hydrogel scaffold material. The increase of cell viability slowed down after the third day, which may be related to the accumulation of metabolic waste, and the reduction of nutrients and relative living space caused by the increase in the number of cells.

We further observed the growth and morphology of cells in P(OEGMA-co-MEO2MA) thermosensitive hydrogel scaffolds by AO/EB staining, as shown in Figure 7. On the day of cell embedding (day 0), we found that dispersed MCF-7 cells did not adhere to the hydrogel scaffold, but grew uniformly in a single spherical shape in the scaffold. It is well known that cells can only stick to surfaces that are hydrophobic enough, so this cell behavior in the scaffold may be caused by the hydrophilic environment inside the scaffold. After 1 day

of culture, some smaller cell clusters could be observed, which may have formed by cell division or cell aggregation. As the culture time was further extended, cells grew faster and the number of cells increased, and most of them showed a green color in the observed field of view, indicating that the cells were still alive and the hydrogel scaffold could maintain the cell growth. When cultured for 7 days, we observed that most of the cells were able to form small multicellular spheres, which may be due to interaction between cells in adjacent cell clusters as the cells continued to spread and grow within the hydrogel scaffold, thus aggregating to form multicellular spheres. Therefore, we can speculate that this type of thermosensitive P(OEGMA-co-MEO2MA) hydrogel scaffold can be used to construct tumor multicellular sphere models and may have wide application prospects in drug screening and tumor research. Besides, based on our previous research experience [29], this type of hydrogel scaffold should also be able to be used for 3D culture of many other types of cells, which may extend its range of applications. encapsulated in situ in it. Part of the DMEM cell culture medium was added to continue this culture process. Firstly, we investigated the viability of cells in the P(OEGMA-co-MEO2MA) hydrogel scaffold using MTT assay. As shown in Figure 6, the absorbance values measured by the MTT method increased significantly in the first two days of cell culture, which may be related to a rapid increase in the number of living cells in this hydrogel scaffold. This result also indirectly reflected the favorable biocompatibility of this hydrogel scaffold material. The increase of cell viability slowed down after the third day, which may be related to the accumulation of metabolic waste, and the reduction of nutrients and relative living space caused by the increase in the number of cells.

*Gels* **2022**, *8*, x FOR PEER REVIEW 8 of 13

**Figure 6.** Viability of MCF-7 cells in formed thermosensitive P(OEGMA-co-MEO2MA) hydrogel as assessed by MTT assay. The data shown are the mean of three independent experiments. Error bars indicate the standard deviations. An asterisk (\*) indicates a significant difference between this group of data and the previous group (*p* < 0.05). **Figure 6.** Viability of MCF-7 cells in formed thermosensitive P(OEGMA-co-MEO2MA) hydrogel as assessed by MTT assay. The data shown are the mean of three independent experiments. Error bars indicate the standard deviations. An asterisk (\*) indicates a significant difference between this group of data and the previous group (*p* < 0.05). *Gels* **2022**, *8*, x FOR PEER REVIEW 9 of 13

used to construct tumor multicellular sphere models and may have wide application prospects in drug screening and tumor research. Besides, based on our previous research experience [29], this type of hydrogel scaffold should also be able to be used for 3D culture of many other types of cells, which may extend its range of applications. **Figure 7.** Fluorescence images of MCF-7 cells cultured in the formed thermosensitive P(OEGMAco-MEO2MA) hydrogel. The cells were stained with acridine orange (AO) and ethidium bromide (EB) beforehand. **Figure 7.** Fluorescence images of MCF-7 cells cultured in the formed thermosensitive P(OEGMAco-MEO2MA) hydrogel. The cells were stained with acridine orange (AO) and ethidium bromide (EB) beforehand.

Here, by using MEO2MA and OEGMA, having excellent biocompatibility and biodegradability as monomers, we synthesized five types of P(OEGMA-co-MEO2MA) thermosensitive microgel samples with different VPTTs or particle sizes by free radical

OEGMA, the VPTT of the obtained microgels increased. With decrease of the dosage of SDS, the sizes of the obtained microgels increased. All the types of prepared microgels could aggregate to form 3D hydrogels by thermal induction. All these hydrogels still showed characteristic shrinkage of thermosensitive hydrogels. However, we found that with the increase of the VPTT, or the decrease of the particle size, the less obvious the contraction. Among them, the volume of hydrogel formed from M3 still had a volume of more than 98% of the initial volume after 48 h without significant shrinkage. These hydrogels could be used as a scaffold for 3D culture of MCF-7 model cells and showed excellent biocompatibility. Cells can grow in this scaffold and tend to form multicellular sphere models. This is a simple method for the preparation of thermosensitive hydrogel 3D cell scaffolds, which shows enormous application prospects in the biomedical field, such as

Oligo(ethylene glycol) methacrylate (OEGMA, Mn= 300), 2-(2-methoxyethoxy) ethyl acrylate (MEO2MA), N,N′-methylene diacrylamide (BIS), sodium dodecyl sulfate (SDS), were obtained from Tianjin Heowns Biochemical Technology Co, LTD; potassium persulfate (KPS) was obtained from Tianjin Damao Reagent Factory; Phosphate buffer (PBS), DMEM medium, fetal bovine serum, streptomycin mixture, trypsin, thiazole blue (MTT), acridine orange (AO), ethidium bromide (EB) were obtained from Wuhan Rutgers Biotechnology Co; MCF-7 breast cancer cells were purchased from the Stem Cell Bank of the

*4.2. Synthesis and Characterization of P(OEGMA-co-MEO2MA) Thermosensitive Microgel* 

In this manuscript, we first used MEO2MA and OEGMA as polymerization monomers, BIS as crosslinker, SDS as surfactant, and KPS as initiator to generate P(OEGMA-

tumor research, drug screening, and tissue engineering.

**4. Materials and Methods** 

Chinese Academy of Sciences.

*4.1. Materials* 

**3. Conclusions** 

### **3. Conclusions**

Here, by using MEO2MA and OEGMA, having excellent biocompatibility and biodegradability as monomers, we synthesized five types of P(OEGMA-co-MEO2MA) thermosensitive microgel samples with different VPTTs or particle sizes by free radical polymerization. Their VPTTs and particle sizes were investigated by means of a nanometer particle size meter and ultraviolet spectrophotometer. With increase of the content of OEGMA, the VPTT of the obtained microgels increased. With decrease of the dosage of SDS, the sizes of the obtained microgels increased. All the types of prepared microgels could aggregate to form 3D hydrogels by thermal induction. All these hydrogels still showed characteristic shrinkage of thermosensitive hydrogels. However, we found that with the increase of the VPTT, or the decrease of the particle size, the less obvious the contraction. Among them, the volume of hydrogel formed from M3 still had a volume of more than 98% of the initial volume after 48 h without significant shrinkage. These hydrogels could be used as a scaffold for 3D culture of MCF-7 model cells and showed excellent biocompatibility. Cells can grow in this scaffold and tend to form multicellular sphere models. This is a simple method for the preparation of thermosensitive hydrogel 3D cell scaffolds, which shows enormous application prospects in the biomedical field, such as tumor research, drug screening, and tissue engineering.

### **4. Materials and Methods**

### *4.1. Materials*

Oligo(ethylene glycol) methacrylate (OEGMA, Mn = 300), 2-(2-methoxyethoxy) ethyl acrylate (MEO2MA), N,N<sup>0</sup> -methylene diacrylamide (BIS), sodium dodecyl sulfate (SDS), were obtained from Tianjin Heowns Biochemical Technology Co, LTD; potassium persulfate (KPS) was obtained from Tianjin Damao Reagent Factory; Phosphate buffer (PBS), DMEM medium, fetal bovine serum, streptomycin mixture, trypsin, thiazole blue (MTT), acridine orange (AO), ethidium bromide (EB) were obtained from Wuhan Rutgers Biotechnology Co; MCF-7 breast cancer cells were purchased from the Stem Cell Bank of the Chinese Academy of Sciences.

### *4.2. Synthesis and Characterization of P(OEGMA-co-MEO2MA) Thermosensitive Microgel Gels* **2022**, *8*, x FOR PEER REVIEW 10 of 13

In this manuscript, we first used MEO2MA and OEGMA as polymerization monomers, BIS as crosslinker, SDS as surfactant, and KPS as initiator to generate P(OEGMA-co-MEO2MA) thermosensitive microgels by free radical polymerization at 70 ◦C [26] (Scheme 1). The content of BIS was 2%, the mass of KPS were 0.0812 g, respectively. Three types of microgel samples M1, M2 and M3 were synthesized by changing the molar ratios of MEO2MA and OEGMA as 95 : 3, 90 : 8, and 85 : 13, respectively. In contrast to the synthesis of M3, another two types of microgel samples, M4 and M5, were synthesized by reducing the SDS dosage, while keeping the other substances constant. The detail feeding amount of each sample is in Table 1. co-MEO2MA) thermosensitive microgels by free radical polymerization at 70 °C [26] (Scheme 1). The content of BIS was 2%, the mass of KPS were 0.0812 g, respectively. Three types of microgel samples M1, M2 and M3 were synthesized by changing the molar ratios of MEO2MA and OEGMA as 95: 3, 90: 8, and 85: 13, respectively. In contrast to the synthesis of M3, another two types of microgel samples, M4 and M5, were synthesized by reducing the SDS dosage, while keeping the other substances constant. The detail feeding amount of each sample is in Table 1.

**Scheme 1.** The synthesis of P(OEGMA-co-MEO2MA) thermosensitive microgel. **Scheme 1.** The synthesis of P(OEGMA-co-MEO2MA) thermosensitive microgel.

The typical preparation process for sample M1 was as follows: 2.5002 g MEO2MA, 0.1259 g OEGMA, 0.0431 g BIS and 0.0284 g SDS were weighed accurately, dissolved in 100 mL deionized water, and transferred to a three-necked flask. After being passed into The typical preparation process for sample M1 was as follows: 2.5002 g MEO2MA, 0.1259 g OEGMA, 0.0431 g BIS and 0.0284 g SDS were weighed accurately, dissolved in 100 mL deionized water, and transferred to a three-necked flask. After being passed into N<sup>2</sup>

N2 for 1 h, KPS (dissolved in 5 mL of deionized water) was added to initiate polymerization under magnetic stirring. After 5 h, the reaction was terminated and transferred to a

and absorbance (turbidity) with temperature were measured by a nanometer particle size

The thermosensitive P(OEGMA-co-MEO2MA) hydrogels were prepared through the

A certain amount of P(OEGMA-co-MEO2MA) microgel samples were weighed and dissolved in phosphate buffered saline (PBS) to obtain a final solution with mass fraction of 3.0 wt%. Then, they were placed in a physiological temperature environment, which was higher than their VPTTs. The formation process of thermosensitive hydrogel from microgels and its stability and shrinkage were observed by the inverted method and recorded by camera. The gelation time was defined as the time point at which the inverted

To quantitatively analyze the shrinkage degree of the obtained hydrogels, we calculated the ratio of the volume of the obtained hydrogel at certain time point (Vt) to the initial volume (V0) by measuring with a ruler. The thermosensitive hydrogel sample with

The model cells used in this experiment were human breast cancer cells (MCF-7), which were cultured in a cell culture incubator using DMEM medium. The main components of the culture medium were 90% DMEM high glucose medium, 10% fetal bovine serum, and 100 unit/mL penicillin/streptomycin double antibodies. The culture tempera-

*4.3. Preparation and Characterization of P(OEGMA-co-MEO2MA) Microgels-Based* 

minimum shrinkage was screened out for the next cell experiments.

ture was 37 °C and the carbon dioxide concentration was 5%.

4.4.2. Cells Embedded and Cultured in Hydrogel Scaffold

*4.4. P(OEGMA-co-MEO2MA) Thermosensitive Hydrogels for 3D Cell Culture* 

meter and an ultraviolet spectrophotometer, respectively.

*Thermosensitive Hydrogel* 

4.4.1. Cell Culture

thermal induction method [29].

hydrogel no longer flowed visually.

for 1 h, KPS (dissolved in 5 mL of deionized water) was added to initiate polymerization under magnetic stirring. After 5 h, the reaction was terminated and transferred to a dialysis belt and dialysis for 7 days. Then, the sample was freeze-dried and preserved.

In order to test the thermosensitivities of these samples, the changes of particle size and absorbance (turbidity) with temperature were measured by a nanometer particle size meter and an ultraviolet spectrophotometer, respectively.
