Synthesis and Characterization of Poly(DL-lactide) Containing Fluorene Structures

9,9-bis[4-(2-hydroxy-3-acryloyloxypropoxy)phenyl]fluorene (BPF) hydroxyl groups (-OH) were used as initiators in the ring-opening polymerization reaction with DL-lactide monomers at different molar ratios to synthesize a Poly(DL-lactide) polymer containing bisphenol fluorene structure and acrylate functional groups (DL-BPF). The polymer’s structure and molecular weight range were analyzed using NMR (1H, 13C) and gel permeation chromatography. DL-BPF was then subjected to photocrosslinking using the photoinitiator Omnirad 1173, resulting in the formation of an optically transparent crosslinked polymer. Characterization of the crosslinked polymer involved analyzing its gel content, refractive index, thermal stability (via differential scanning thermometry (DSC) and thermogravimetric analysis (TGA)), as well as conducting cytotoxicity tests. The crosslinked copolymer exhibited a maximum refractive index of 1.5276, a maximum glass transition temperature of 61.1 °C, and cell survival rates higher than 83% in the cytotoxicity tests.


Introduction
Polylactide (PLA) is a bio-based polyester known for its high biocompatibility and biodegradability. It possesses desirable mechanical properties, including a high elastic modulus, high tensile strength, and low elongation at break. PLA finds applications in various fields such as consumer goods, packaging, agriculture, and biomedicine, and it is currently gaining popularity [1][2][3][4]. However, PLA faces limitations in terms of brittleness, low toughness, poor heat resistance, slow crystallization rates, and hydrophobicity. To overcome these limitations, researchers have explored methods such as chemical modification, copolymerization, and polymer blending to enhance the crystallinity, thermal stability, mechanical properties, and hydrophobicity/hydrophilicity of PLA [5][6][7][8][9].
In general, the performance of polylactic acid (PLA) can be enhanced by functionalizing its end groups, enabling the utilization of specific end-functional compounds for polymerization. Branched structures of PLA possess numerous multifunctional groups, and the physical, chemical, and mechanical properties of PLA can be modified by altering its molecular structure. Control over the molecular branching structure can be achieved through various polymer synthesis methods and processing strategies [10][11][12].
The literature demonstrates an increasing interest in the synthesis of branched and starshaped PLA. The transition from linear PLA to branched and star-shaped structures has implications for biocompatibility, thermal properties, mechanical properties, rheological properties, degradation characteristics, drug release rate, crystallization nucleation, and the hydrophilicity/hydrophobicity of the polymer [13][14][15][16]. While copolymerization and

Synthesis of DL-BPF
The DL-lactide and the 9,9-bis[4-(2-hydroxy-3-acryloyloxypropoxy)phenyl]fluorene (BPF) in molar ratios of 5:1, 10:1,15:1, 20:1, respectively, were placed in a 250 mL flask with three necks and dried under vacuum for 1 h. In a flask filled with dry nitrogen gas, Sn(Oct) 2 (0.05 wt%) and 200 ppm 4-methoxyphenol were added. The mixture was heated at 150 • C using an oil bath for 12 h with stirring (Scheme 1). After the reaction, the reactor was cooled to room temperature. The resulting polymer was dissolved in acetone and then precipitated by adding it to an excess of deionized water while agitating the solution. The precipitated material was washed with deionized water and dried under reduced pressure [46][47][48].

Synthesis of DL-BPF
The DL-lactide and the 9,9-bis[4-(2-hydroxy-3-acryloyloxypropoxy)phenyl]fluorene (BPF) in molar ratios of 5:1, 10:1,15:1, 20:1, respectively, were placed in a 250 mL flask with three necks and dried under vacuum for 1 h. In a flask filled with dry nitrogen gas, Sn(Oct)2 (0.05 wt%) and 200 ppm 4-methoxyphenol were added. The mixture was heated at 150 °C using an oil bath for 12 h with stirring (Scheme 1). After the reaction, the reactor was cooled to room temperature. The resulting polymer was dissolved in acetone and then precipitated by adding it to an excess of deionized water while agitating the solution. The precipitated material was washed with deionized water and dried under reduced pressure [46][47][48]. Scheme 1. Synthesis of DL-BPF.

Photocrosslinking Conditions
Photocrosslinking was performed in a homemade UV vacuum oven (UV wavelength: 248-578 nm, intensity: 10-15 mW/cm 2 ). The DL-BPF polymer was first dissolved in acetone, then mixed with the photoinitiator, Omnirad 1173 (3 wt%). After the solvent was removed, irradiation was performed under vacuum at 60 °C, for a maximum of 10 min irradiation, which yielded a transparent crosslinked polymer ( Figure 1). Scheme 1. Synthesis of DL-BPF.

Photocrosslinking Conditions
Photocrosslinking was performed in a homemade UV vacuum oven (UV wavelength: 248-578 nm, intensity: 10-15 mW/cm 2 ). The DL-BPF polymer was first dissolved in acetone, then mixed with the photoinitiator, Omnirad 1173 (3 wt%). After the solvent was removed, irradiation was performed under vacuum at 60 • C, for a maximum of 10 min irradiation, which yielded a transparent crosslinked polymer ( Figure 1).

NMR Spectroscopy
The uncrosslinked DL-BPF polymer was dissolved using CDCl 3 as the solvent. The chemical structure of DL-BPF was determined using Bruker Avance III HD-600 MHz NMR spectrometer to obtain 13 C and 1 H-NMR spectra.

Gel Permeation Chromatography
Approximately 100-120 mg of uncrosslinked DL-BPF polymer was weighed and dissolved in tetrahydrofuran (THF). The molecular weight of the uncrosslinked DL-BPF polymer was measured using gel permeation chromatography (Jasco, RI-2031 Plus, Japan). The measurement conditions were as follows: the mobile phase was THF, the temperature was set at 35 • C, the flow rate of the mobile phase was 10 mL/min, and polystyrene was used as the calibration standard.

NMR Spectroscopy
The uncrosslinked DL-BPF polymer was dissolved using CDCl3 as the solvent. The chemical structure of DL-BPF was determined using Bruker Avance III HD-600 MHz NMR spectrometer to obtain 13 C and 1 H-NMR spectra.

Gel Permeation Chromatography
Approximately 100-120 mg of uncrosslinked DL-BPF polymer was weighed and dissolved in tetrahydrofuran (THF). The molecular weight of the uncrosslinked DL-BPF polymer was measured using gel permeation chromatography (Jasco, RI-2031 Plus, Japan). The measurement conditions were as follows: the mobile phase was THF, the temperature was set at 35 °C, the flow rate of the mobile phase was 10 mL/min, and polystyrene was used as the calibration standard.
The polystyrene standard was obtained from POLYMER STANDARDS SERVICE (PSS). The GPC column setup consisted of three columns in a series: Jordi Gel DVB Mixed Bed, Waters Styragel ® HR4E THF, and Waters Styragel ® HR3 THF. The molecular weight was calculated based on the calibration curve obtained using the polystyrene standard, with an R-value of 0.9986.

Differential Scanning Calorimetry (DSC)
The uncrosslinked and photocrosslinked DL-BPF polymer samples weighing approximately 5-8 mg were placed in dedicated aluminum pans for DSC (differential scanning calorimetry) analysis. The glass transition temperature (Tg) of the polymer was measured using a HITACHI DSC7000X instrument, Japen. The measurement conditions were as follows: under a nitrogen atmosphere, heating rate of 10 °C/min, heating range from −10 °C to 110 °C, and the heating cycle was repeated twice. The glass transition temperature (Tg) of the DL-BPF polymer was determined from the midpoint of the DSC curve obtained during the second heating cycle. The polystyrene standard was obtained from POLYMER STANDARDS SERVICE (PSS). The GPC column setup consisted of three columns in a series: Jordi Gel DVB Mixed Bed, Waters Styragel ® HR4E THF, and Waters Styragel ® HR3 THF. The molecular weight was calculated based on the calibration curve obtained using the polystyrene standard, with an R-value of 0.9986.

Differential Scanning Calorimetry (DSC)
The uncrosslinked and photocrosslinked DL-BPF polymer samples weighing approximately 5-8 mg were placed in dedicated aluminum pans for DSC (differential scanning calorimetry) analysis. The glass transition temperature (T g ) of the polymer was measured using a HITACHI DSC7000X instrument, Japen. The measurement conditions were as follows: under a nitrogen atmosphere, heating rate of 10 • C/min, heating range from −10 • C to 110 • C, and the heating cycle was repeated twice. The glass transition temperature (T g ) of the DL-BPF polymer was determined from the midpoint of the DSC curve obtained during the second heating cycle.

Thermogravimetric Analysis (TGA)
The photocrosslinked DL-BPF polymer samples weighing between 4 and 6 mg were weighed and subjected to thermogravimetric analysis (TGA) using a Perkin-Elmer Pyris 1 TGA instrument. The measurement conditions were as follows: the samples were heated under a nitrogen flow of 20 cm 3 /min at a heating rate of 10 • C/min, reaching a temperature of 830 • C. The TGA analysis observed the thermal degradation behavior of the DL-BPF polymer during the heating process, tracking any changes in weight as a function of temperature.

Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR)
The infrared spectra of DL-BPF polymer before and after photocrosslinking were measured using an attenuated total reflection Fourier transform infrared spectrometer (ATR-FTIR) equipped with a germanium crystal (PerkinElmer, Spectrum 100, USA). The instrument was set to scan the wavelength range of 550-4000 cm −1 with a resolution of

Derivation of Gel Content
Briefly, 400 mg of photocrosslinked DL-BPF polymer is mixed with 4 g of acetone, ensuring complete infiltration of the polymer by acetone. The mixture is left to stand at room temperature for 24 h. After 24 h, the solution containing the polymer is filtered to separate the insoluble polymer. The insoluble polymer is then placed in a vacuum oven at 50 • C and subjected to vacuum-drying for 12 h to ensure complete removal of acetone. The gel residue of DL-BPF polymer, which is insoluble in acetone, can be calculated using the following formula: Gel residue (%) = (W B /W A ) × 100% W A : Initial weight of photocrosslinked DL-BPF polymer W B : Weight of photocrosslinked DL-BPF polymer after soaking in acetone for 24 h, filtration, and vacuum-drying.

Measurement of Refractive Index
A sample of photocrosslinked DL-BPF polymer with dimensions of 20 × 10 × 1 mm was prepared. The refractive index of the sample was measured using the ATAGO Abbe refractometer (DR-A1-Plus, ATAGO, Japan).

Cytotoxicity Test
The cytotoxicity of photocrosslinked 10DL-BPF polymer was assessed according to the ISO 10993-5:2009 standard. Four sets of samples were prepared, including blank samples, negative control samples, positive control samples, and test samples. All of the tested materials were extracted using culture medium with 10% fetal bovine serum to prepare liquid extracts of the materials, which were then cultured with L929 mouse fibroblast cells for 24 h (at 5 ± 1% CO 2 and 37 ± 1 • C). The cytotoxicity of the tested materials was qualitatively evaluated by monitoring the morphology of the L929 cells in accordance with the grading criteria of the ISO 10993-5:2009 standard, and the results were recorded. The MTT assay (cell viability analysis) was performed to determine the cell survival rates, using two-fold serial dilutions of the liquid extract (100, 50, 25, and 12.5%). The cells were incubated in these dilutions for 24 h in an incubator, with five replicate measurements for each dilution. All test materials were prepared according to the methods described in ISO 10993-12 standard. Figure 2 shows the nuclear magnetic resonance (NMR) spectrum of DL-BPF. In the 1 H-NMR spectrum, the -CH absorption peak of Poly(DL-lactide) was (δH4 = 5.14-5.23 ppm [ 1 H, CH]), the methyl-CH 3 absorption peak of Poly(DL-lactide) was (δH1, δH2 = 1.46-1.61 ppm [ 3 H, CH 3 ]), the absorption peak of CH at one end of DL-BPF polymer was (δH3 = 4.31-4.39 ppm [ 1 H, -CH]), the -CH absorption peak of BPF was (δH4 = 5.40-5. 48 ppm [ 1 H, CH]), the CH 2 absorption peak of BPF was (δH6 = 4.05-4.10 ppm, δH7 = 4.40-4.56 ppm [ 2 H, CH 2 ]), the vinyl hydrogen (-CH=CH 2 ) absorption peak of BPF was (δH8, δH9, δH10 = 5. 75-6.42 ppm [ 3 H, CH=CH 2 ]), and the absorption of protons of benzene ring in BPF structure was (δH11, δH12, δH13, δH14, δH15, δH16 = 6.75-7.73 ppm [ 1 H, CH]). The residual solvent peak is at 2.13 ppm. The polymers with DL-lactide and BPF structures were obtained by ring-opening polymerization as determined by 1 H-NMR.

Gel Formation and Gel Permeation Chromatography of DL-BPF
According to the GPC measurement results in Figure 4, DL-BPF exhibits a single peak, confirming its status as a single polymer. The molecular weight of DL-BPF polymers can be controlled by adjusting the molar ratio of DL-lactide to BPF. The GPC measure-

Gel Formation and Gel Permeation Chromatography of DL-BPF
According to the GPC measurement results in Figure 4, DL-BPF exhibits a single peak, confirming its status as a single polymer. The molecular weight of DL-BPF polymers can be controlled by adjusting the molar ratio of DL-lactide to BPF. The GPC measurements indicate that the number-average molecular weight of DL-BPF polymers increases with higher DL-lactide molar ratios. Calculations based on the DL-lactide molecular weight (144 g/mol) multiplied by its molar ratio and adding the BPF molecular weight (606 g/mol) provide an estimated molecular weight range for DL-BPF, which closely aligns with the measured number-average molecular weight obtained from GPC.  Table 1. DL-BPF samples of molecular weight and state of matter gel fraction (%).   Therefore, it can be inferred that DL-lactide undergoes ring-opening polymerization reactions with the alkyl groups on the BPF molecular chain, leading to the extension of the polymer chains. This process is controlled by the molar ratio of DL-lactide.

Polymer DL:BPF a Mn c (g/mol) MnGPC b (g/mol) MwGPC b (g/mol) Mw/MnGPC b Gel Residue (%)
During the polymer crosslinking process, once the length of the polymer chains exceeds a certain threshold, glass transition occurs. This limits the participation of some free radicals and double bonds in the crosslinking reaction, preventing them from reaching 100% conversion. Consequently, the gel content test results demonstrate that DL-BPF polymers, regardless of their molar ratios and molecular weights, exhibit gel contents of 90% or higher (see Table 1). In the attenuated total reflection Fourier transform infrared (ATR-FTIR) spectrum shown in Figure 5, we observed the changes in the characteristic peak of the C=C double bond of the acrylate functional group in DL-BPF polymer. At the wavelength of 1635 cm −1 , there is a distinct small peak, which represents the characteristic peak of the C=C double bond in the uncrosslinked DL-BPF polymer. After UV irradiation, the characteristic peak of the C=C double bond disappears, indicating that under the presence of the photoinitiator Omnirad 1173, the acrylate functional groups (C=C) at both ends of the DL-BPF polymer undergo free radical polymerization upon UV irradiation and form a crosslinked network structure in the DL-BPF polymer.

Refractive Index of Photocrosslinked DL-BPF
The measurement data in Table 2 show that the refractive index of DL-BPF polymers decreases with an increase in the molar ratio of DL-lactide monomers. BPF monomers,

Refractive Index of Photocrosslinked DL-BPF
The measurement data in Table 2 show that the refractive index of DL-BPF polymers decreases with an increase in the molar ratio of DL-lactide monomers. BPF monomers, due to the presence of bisphenol fluorene structures, have a higher refractive index. According to previous literature, the refractive index of PLA is reported to be 1.4557 [49]. Therefore, incorporating the bisphenol fluorene structures of BPF monomers into the Poly(DL-lactide) chains can indeed increase the refractive index of DL-BPF polymers. Depending on the molar ratio, the refractive index of DL-BPF polymers can range from 1.4852 to 1.5276. However, it was also observed that the refractive index decreases as the molar ratio of DL-lactide monomers increases. This suggests that after the completion of the ring-opening polymerization reaction, the molecular chains of DL-BPF polymers also elongate. Upon photocrosslinking, the Poly(DL-lactide) segments occupy a larger proportion of the DL-BPF molecular chains, thereby limiting the increase in refractive index.  Table 3 presents the thermal coefficients of DL-BPF, which were determined through differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). The initial DL-BPF polymer, prior to photocrosslinking, consists of short chains. However, after undergoing photocrosslinking, it transforms into an optically transparent polymer with a network-like structure. This structural transformation leads to an increase in the glass transition temperature (T g ) by 13.6-30.3 • C, as shown in Figure 6. The extent of this increase depends on the molar ratio of the reactants. The inherent thermal stability of the bisphenol fluorene structure in BPF contributes to the overall thermal stability of the polymer. The T g and thermal degradation temperature of the DL-BPF polymer are influenced by factors such as chain structure, molecular weight, and degree of crosslinking. The densely crosslinked acrylate side chains and bisphenol fluorene structures in the polymer restrict the movement of polymer chains and reduce the free volume within the polymer matrix. Among the different DL-lactide-to-BPF molar ratios studied, a ratio of 5:1 was found to optimize the T g , resulting in a significant increase of 30.3 • C. Table 3. Summary of DSC and TGA results.   As shown in Figure 7, the maximum thermal decomposition temperature of Poly(DLlactide) is 319.37 • C. BPF exhibits a two-step thermal decomposition curve, with maximum decomposition temperatures at 420.95 • C and 626.0 • C. DL-BPF shows a three-step thermal decomposition curve, corresponding to the thermal decomposition curves of Poly(DLlactide) and BPF. Additionally, we observed that as the molecular weight of DL-BPF polymer increased, the peak areas of BPF's thermal decomposition (T max2 and T max3 ) decreased. This can be attributed to the proportion of BPF within the overall molecular chain, which decreases with an increase in the molar ratio of DL-lactide monomers.

Thermal Analysis
Regarding the thermal decomposition temperature, although the DL-BPF polymer formed a crosslinked network structure after UV irradiation and contained bisphenol fluorene structures in its molecular chain, the thermal decomposition temperature did not show a significant increase. This suggests that the thermal properties of DL-BPF polymer, influenced by the heat resistance of bisphenol fluorene structures, were not significantly enhanced due to the predominant presence of Poly(DL-lactide) chains in the overall molecular structure. Consequently, the T 5% (temperature at which 5% weight loss occurs) of the photocrosslinked DL-BPF polymer and the thermal decomposition temperature of the Poly(DL-lactide) segments only slightly increased with an increase in the molecular weight.

The Cytotoxicity Testing of Photocrosslinked DL-BPF
The cytotoxicity of photocrosslinked 10DL-BPF was evaluated according to the ISO 10993-5:2009 standard. An inverted microscope was used to observe the morphology of the L929 cells for qualitative morphological grading of the cytotoxicity. The cells exposed to 10DL-BPF were slightly lysed or altered, which corresponds to grade 1 cytotoxicity (see Figure 8). Quantitative evaluation of the cytotoxicity was performed by using the MTT assay to measure the cell survival rate, where the 100% 10DL-BPF extract contained 16.6% fewer cells than the control, corresponding to a cell survival rate of 83.4% (see Figure 9). According to the ISO 10993-5:2009 grading criteria, a material is deemed cytotoxic if it

The Cytotoxicity Testing of Photocrosslinked DL-BPF
The cytotoxicity of photocrosslinked 10DL-BPF was evaluated according to the ISO 10993-5:2009 standard. An inverted microscope was used to observe the morphology of the L929 cells for qualitative morphological grading of the cytotoxicity. The cells exposed to 10DL-BPF were slightly lysed or altered, which corresponds to grade 1 cytotoxicity (see Figure 8). Quantitative evaluation of the cytotoxicity was performed by using the MTT assay to measure the cell survival rate, where the 100% 10DL-BPF extract contained 16.6% fewer cells than the control, corresponding to a cell survival rate of 83.4% (see Figure 9). According to the ISO 10993-5:2009 grading criteria, a material is deemed cytotoxic if it reduces the cell viability by more than 30% or achieves a numerical cytotoxicity grade greater than 2. Therefore, these results indicate that photocrosslinked 10DL-BPF is not cytotoxic to L929 cells.

Conclusions
In this study, we employed the -OH groups on the molecular chain of 9,9-bis[4-(2hydroxy-3-acryloyloxypropoxy)phenyl]fluorene (BPF) as initiators and adjusted the molar ratios of DL-lactide monomers in the ring-opening polymerization reaction. By conducting analysis using 13 C and 1 H-NMR spectroscopy, we successfully synthesized lowmolecular-weight polymers (DL-BPF) that contain both bisphenol fluorene structures and acrylate functional groups. Through careful control of the molar ratio of DL-lactide monomers, we achieved precise control over the molecular weight of DL-BPF within a specific range.
After the addition of the photoinitiator Omnirad 1173, the DL-BPF polymer, which contains acrylate functional groups at both ends, undergoes a free radical polymerization reaction initiated by UV light irradiation. The occurrence of photocrosslinking in the DL-BPF polymer was confirmed through ATR-FTIR spectroscopy, which showed a significant reduction and disappearance of the characteristic peak at 1635 cm −1 corresponding to the C=C double bond after UV light irradiation. This indicates the successful formation of crosslinks within the DL-BPF polymer.
Gel content measurements were performed to assess the degree of crosslinking in the DL-BPF polymer. The results revealed a gel content of 90%, indicating the formation of a highly crosslinked network structure when exposed to UV irradiation. This demonstrates the effectiveness of the photocrosslinking process in creating a stable and interconnected polymer network in the DL-BPF polymer.

Conclusions
In this study, we employed the -OH groups on the molecular chain of 9,9-bis[4-(2hydroxy-3-acryloyloxypropoxy)phenyl]fluorene (BPF) as initiators and adjusted the molar ratios of DL-lactide monomers in the ring-opening polymerization reaction. By conducting analysis using 13 C and 1 H-NMR spectroscopy, we successfully synthesized low-molecularweight polymers (DL-BPF) that contain both bisphenol fluorene structures and acrylate functional groups. Through careful control of the molar ratio of DL-lactide monomers, we achieved precise control over the molecular weight of DL-BPF within a specific range.
After the addition of the photoinitiator Omnirad 1173, the DL-BPF polymer, which contains acrylate functional groups at both ends, undergoes a free radical polymerization reaction initiated by UV light irradiation. The occurrence of photocrosslinking in the DL-BPF polymer was confirmed through ATR-FTIR spectroscopy, which showed a significant reduction and disappearance of the characteristic peak at 1635 cm −1 corresponding to the C=C double bond after UV light irradiation. This indicates the successful formation of crosslinks within the DL-BPF polymer.
Gel content measurements were performed to assess the degree of crosslinking in the DL-BPF polymer. The results revealed a gel content of 90%, indicating the formation of a highly crosslinked network structure when exposed to UV irradiation. This demonstrates the effectiveness of the photocrosslinking process in creating a stable and interconnected polymer network in the DL-BPF polymer.
After photocrosslinking, the DL-BPF polymer forms a tightly interconnected network structure due to its branched nature. The presence of bisphenol fluorene structures in the molecular chains restricts their rotational motion, resulting in an increased glass transition temperature (T g ) of the DL-BPF polymer. The DSC analysis showed that the T g can increase by up to 30.3 • C after photocrosslinking.
In terms of thermal decomposition analysis, although the DL-BPF polymer forms a network structure after photocrosslinking, the presence of Poly(DL-lactide) chains still occupies a significant portion of the overall molecular structure. Therefore, the thermal decomposition temperature of the DL-BPF polymer did not show a significant increase due to the formation of a crosslinked network and the presence of bisphenol fluorene structures.
Furthermore, the incorporation of bisphenol fluorene structures into the Poly(DLlactide) chains resulted in an increased refractive index of the DL-BPF polymer, with a maximum value of 1.5276. This enhancement in refractive index can be advantageous for optical applications. However, it should be noted that the overall refractive index decreases with an increase in the molar ratio of DL-lactide monomers.
In the qualitative assessment of cytotoxicity based on morphology, it was observed that the 10DL-BPF polymer exhibited a cytotoxicity grade of 1, indicating low cytotoxicity. Furthermore, quantitative evaluation using the MTT assay revealed that the 100% 10DL-BPF extract exhibited a cell survival rate of 83.4%. These results demonstrate the good biocompatibility of the photocrosslinked 10DL-BPF polymer.