Biodegradable Copolymers Obtained by Solution Polymerization

Various aliphatic polyesters, such as poly (lactic acid) (PLA) and poly (ε-caprolactone) (PCL), have been of great interest due to their biocompatibility, biodegradability and mechanical strength1-5. PLA can be obtained from renewable resources, however, it is a brittle material which represents an important handicap for further applications in biomedical area, packaging, industrial and agricultural fields6. On the other hand, PCL is characterized by a relatively low glass transition temperature (~60 °C); thus, at room temperature it behaves as a tough plastic7. PLA and PCL copolymers have been developed in order to take advantage of synergistic improvement in properties offered by both polymers8. The PLLA and PCL can be obtained by ring-opening polymerization of L-lactide (LLA) and ε-caprolactone (CL), respectively, using Stannous octoate as the initiator and low molecular weight alcohols. The most used technique for that is mass polymerization, which employs only monomer and catalysts9,10. The advantages of mass polymerization are the high polymer purity and the easy production. On the other hand, the reaction control becomes difficult due to the formation of hot spots during the synthesis, which causes heat dissipation problems. The solution polymerization technique can reduce this difficulty since a solvent is added to the reaction medium, despite the few papers developed in this area used toxic organic solvents11. However, when it is interesting to modify the properties of these polymers by adding nanoparticles in situ, the presence of a solvent can make the process easier. In the last two decades, there is growing interest in the modification of the properties of PLLA, which is rigid and brittle, either by obtaining copolymers with other monomers to provide flexibility, or by adding nanoparticles to improve its tensile strength and lower its gas permeability12-14. The objective of this study was to synthesize a copolymer of PLLA and PCL by solution polymerization, using toluene as solvent. Evidence of copolymer formation is discussed in terms of carbon and hydrogen nuclear magnetic resonance analysis, while the molar masses were evaluated by gel permeation chromatography.


Introduction
Various aliphatic polyesters, such as poly (lactic acid) (PLA) and poly (ε-caprolactone) (PCL), have been of great interest due to their biocompatibility, biodegradability and mechanical strength [1][2][3][4][5] .PLA can be obtained from renewable resources, however, it is a brittle material which represents an important handicap for further applications in biomedical area, packaging, industrial and agricultural fields 6 .On the other hand, PCL is characterized by a relatively low glass transition temperature (~60 °C); thus, at room temperature it behaves as a tough plastic 7 .PLA and PCL copolymers have been developed in order to take advantage of synergistic improvement in properties offered by both polymers 8 .
The PLLA and PCL can be obtained by ring-opening polymerization of L-lactide (LLA) and ε-caprolactone (CL), respectively, using Stannous octoate as the initiator and low molecular weight alcohols.The most used technique for that is mass polymerization, which employs only monomer and catalysts 9,10 .
The advantages of mass polymerization are the high polymer purity and the easy production.On the other hand, the reaction control becomes difficult due to the formation of hot spots during the synthesis, which causes heat dissipation problems.The solution polymerization technique can reduce this difficulty since a solvent is added to the reaction medium, despite the few papers developed in this area used toxic organic solvents 11 .However, when it is interesting to modify the properties of these polymers by adding nanoparticles in situ, the presence of a solvent can make the process easier.
In the last two decades, there is growing interest in the modification of the properties of PLLA, which is rigid and brittle, either by obtaining copolymers with other monomers to provide flexibility, or by adding nanoparticles to improve its tensile strength and lower its gas permeability [12][13][14] .
The objective of this study was to synthesize a copolymer of PLLA and PCL by solution polymerization, using toluene as solvent.Evidence of copolymer formation is discussed in terms of carbon and hydrogen nuclear magnetic resonance analysis, while the molar masses were evaluated by gel permeation chromatography.

Material and Methods
The monomers employed in this work were ε-caprolactone (CL) 97% (Sigma Aldrich) and the L-lactide (LLA) (Purac  ).The initiator stannous octoate, Sn(Oct) 2 , was purchased from Sigma Aldrich  .Toluene and methanol, both in spectrophotometric grade, were obtained from Merck  .

Synthesis of PLLA and copolymers of PLLA-co-PCL
The synthesis of copolymers from L-lactide (LLA) and ε-caprolactone (CL) was carried out in a three-necked bottom flask (250 mL) with the central neck adapted to a Graham condenser.The side necks were coupled to a thermometer (ETS IKA) connected to the heating plate with magnetic stirrer.The copolymerization was carried out in different feed ratios of LLA to CL (100/0, 95/5, 90/10, 85/15 and 80/20, w/w).Sn(Oct) 2 , toluene and methanol were added to  1.
After 24 hours, the polymer solution was precipitated in cold methanol, the filtered solvent was removed.Then, the polymer was dried under vacuum.

Characterization
The samples of PLLA and PLLA-co-PCL were subjected to carbon-13 nuclear magnetic resonance analysis ( 13 C-NMR) in a Varian Mercury Plus spectrometer operating at 500 MHz, 294 K, for 1 hour.In addition, 1 H nuclear magnetic resonance ( 1 H-NMR) was performed in a Bruker AC 200 at 294 K.The solvent used was deuterated chloroform (CDCl 3 ), and tetramethylsilane (TMS) was used as an internal standard.
TG curves to l-lactide and ε-caprolactone were obtained in TA Q20 equipment, operating from 20 to 400 °C, at 10 °C/min.The monomers samples were conditioned in a platinum sample holder and analyses were conducted under N 2 atmosphere with a flow of 40 mL/min.
Gel Permeation Chromatography (GPC) was performed using Waters 1525-detector ELSD 2424, Styragel HR3ETHF and Styragel HR5RTHF (4.6 × 300 mm) columns.All samples were dissolved in chloroform at a concentration of 1.0% (w/v) and analyzed at 40 °C.Chloroform was used as eluent (flow rate = 0.5 mL/min).Molecular weights (M n and M w ) and polydispersity (M w /M n ) were determined with respect to the polystyrene standard.Table 2 presents 13 C-NMR signals achieved by different researchers in studying copolymers of PLLA-co-PCL by bulk polymerization technique 10 , copolymers of PHB and PCL 15 and PLLA synthesis by direct condensation of lactic acid 16 , and in the synthesis of poly (L-D-L lactic acid) 4 .

Results and Discussion
The 13 C-NMR spectra for samples 100/0, 95/5, 90/10 and 85/15 showed signals relating to the chemical structure of l-lactide (LLA), with chemical shifts at 169 ppm relative to the It is observed that in the synthesis of the 85/15 copolymer, only 2.7% of the final product consists of CL units, whereas in the synthesis of 80/20 copolymer, 30.3% of the final product consists of CL units.
During solution polymerization, the temperature (120 °C) used for the synthesis is sufficient to volatilize the molecules of ε-caprolactone (CL) and L-lactide (LLA), which possibly has hindered the reactivity between the two monomers.Thermogravimetric analysis of monomers (Figure 3) showed that the starting temperature of mass loss (T onset ) is 64 °C for CL and 104 °C for the LLA, and the two monomers lose mass by volatilization.Even if the system is heated under reflux, monomers may remain in dynamic equilibrium, and as the temperature of volatilization of CL is lower, the gaseous state is richer in CL, decreasing the reactivity of this monomer.
Castro 10 studied random copolymers of poly(l-lactic acid) and polycaprolactone via mass polymerization without the addition of solvents.The researcher varied the L-lactide/ε-caprolactone ratios, and observed by C 13 -MNR that the synthesis fed with less than 20 wt% of ε-caprolactone produced structures with 100% of poly(l-lactic acid).The author also used a temperature of 120 °C and a closed vacuum system.Some studies on the synthesis of aliphatic polyesters have been carried out in recent years and the NMR technique has been used to confirm the formation of polymers.The 1 H-NMR signals for the pure PCL and PLLA based on these works are presented in Table 3.
Figure 4 shows the H 1 -MNR spectra of PLLA 100/0 samples.The H 1 -NMR spectra for samples with less than 20 wt% of CL showed only signals referring to the LLA units.For PLLA (100/0), a major intensity doublet at 1.62 and 1.59 ppm refers to the signals of methyl groups (-CH 3 ).Two doublets of lower intensity appeared at 1.54 and 1.53 ppm, and at 1.51 and 1.50 ppm, which can be assigned to methyl groups bonded to the terminal carbons.A quadruplet related to groups -(CH-CH 3 ) appeared at 5.14, 5.17, 5.21 and 5.24 ppm.A second quadruplet assigned to terminal groups -(CH-CH 3 ) was recorded at 4.34, 4.38, 4.41, 4.45 ppm.
Motta & Duek 4 studied the synthesis of PLLA using the same catalyst, but via mass polymerization.The researchers prepared polymers with molar ratio monomer/catalyst between 4,000 and 5,500, and obtained polymers with molecular Table 3. Signals of 1 H-NMR for PCL and PLLA, obtained from reactions of ring-opening using Sn(Oct) 2 [2,15,17]   .

PCL
Poly(lactide acid) Signal Hydrogens Signal Hydrogens weights between 75,000 and 150,000 g/mol.The 1 H-NMR spectra show chemical shifts (δ) at 5.15 ppm for the CH groups and at 1.58 ppm for CH 3 groups, both singlet signals 4 .The 95/5, 90/10 and 85/15 samples showed similar spectra and none of them indicate any evidence of the presence of CL units.For the 85/15 sample (Figure 5), a major intensity doublet at 1.58 and 1.55 ppm refers to groups (-CH 3 ) and another of lower intensity at 1.48 and 1.44 ppm is assigned to terminal groups (-CH 3 ).Quadruplets were recorded at 5.10, 5.13, 5.17 and 5.20 ppm, and at 4.05, 4.08, 4.12 and 4.15 ppm, corresponding to the groups (-CH-CH 3 ) and terminals (-CH-CH 3 ), respectively.All obtained data are in accordance to the literature 2,4 .
The 80/20 sample, however, presents several peaks such as singlet and duplets in the region between 1.71 and 1.45 ppm.The duplets at 1.57 and 1.56, and at 1.49 ppm and 1.48 ppm can be assigned to the group (-CH 3 ) of LLA units, suggesting that copolymers with different sequences of two monomers can be obtained (Figure 6).The peaks of highest intensity at 1.53 and 1.16 ppm can be assigned to methylene hydrogens (-CH 2 -) bonded to the β carbon and to the γ carbon of CL unit, respectively.A peak around 2.34 ppm can be assigned to α methylene hydrogens of CL unit.The peak related to hydrogens bonded to the δ carbon of CL units arises at 4.96 ppm.Two quadruplets between 5.13 and 5.25 ppm refer to the hydrogens of the groups (-CH-CH 3 ) of LLA units.Once again, the signal split can indicate two different configurations for the copolymer of PLLA-co-PCL.
However, the 1 H-NMR spectrum for the obtained PLLA has a quadruplet with chemical shifts (δ) at 5.14, 5.17, 5.21 and 5.24 ppm for the CH group, and a doublet at 1.59 and 1.62 for CH 3 groups.It is noteworthy that the polymerization technique employed in this study is the solution polymerization and the molar ratio of monomer/catalyst is 1825, which can lead to the formation of polymers of low molecular weight.
Table 4 presents the molecular weight (M n and M w ) of PLLA and copolymers as well as polydispersity (M w /M n ).Except from sample 80/20 (1,6.10 3 g/mol), the molecular weights ranged from 1,0.10 4 to 1,3.10 4 g/mol, showing that the increase CL concentration affects the molecular weight of copolymers, difficult to obtain poly(LLA-co-CL) with desired high molecular weight 10,18 .Samples 100/0, 95/5,     Low molar ratio monomer / initiator, humidity and the presence of oxygen are factors that may have contributed to the reduction in molecular weight 19,20 .

Conclusion
The technique of polymerization, employing toluene as solvent at 120 °C, is effective for obtaining PLLA and the copolymer of PLLA-co-PCL, if CL mass fractions are above 20%.These results were confirmed by 13 C-NMR and 1 H-NMR techniques, since the signals in chemical shifts related to CL units were identified only for the 80/20 sample.This can be related to the fact that the monomers volatilize at temperatures below 120 °C, generating a dynamic equilibrium between the gas and liquid phases in the reaction system, and as the CL presents a volatilization temperature lower than that of the LLA, the former can be in greater amount in the vapor phase.
The 13 C-NMR signal intensities of (-CH 3 ) and (-CHCH 3 ) groups of LLA units, and (-CH 2 -) groups of CL units are lower than the values mentioned by other authors, which suggests that the full area of these peaks is also smaller and therefore the molecular weights of both PLLA and PLLA-co-PCL (80/20) are low.This fact may be associated with the solvent chose for the synthesis 21 .The present study shows that higher CL concentrations and smaller headspace can facilitate obtaining copolymers.

Figure 1
Figure 1 shows the chemical reaction between the monomers of L-lactide and ε-caprolactone in order to obtain the copolymer of PLLA-co-PCL.The letters in the chemical structure of copolymer indicate potential signals emitted by carbons or hydrogens evidenced by C 13 -NMR and H 1 -NMR techniques, respectively.Table2presents13 C-NMR signals achieved by different researchers in studying copolymers of PLLA-co-PCL by bulk polymerization technique 10 , copolymers of PHB and PCL15 and PLLA synthesis by direct condensation of lactic acid16 , and in the synthesis of poly (L-D-L lactic acid)4 .The 13 C-NMR spectra for samples 100/0, 95/5, 90/10 and 85/15 showed signals relating to the chemical structure of l-lactide (LLA), with chemical shifts at 169 ppm relative to the

Figure 1 .
Figure 1.Chemical structure of PLLA-co-PCL copolymer with carbons marked for identification by 13 C-NMR.

Figure 6 .
Figure 6. 1 H-NMR spectrum of a PLLA 80/20 sample obtained by solution polymerization after 24 hours of reaction at 120 °C.

Figure 3 .
Figure 3. TG curves of LLA and CL monomers.

Figure 4 .
Figure 4. 1 H-NMR spectrum of a sample of PLLA 100/0 obtained by solution polymerization after 24 hours of reaction at 120 °C.

Figure 5 .
Figure 5. 1 H-NMR spectrum of a sample of PLLA 85/15 obtained by solution polymerization after 24 hours of reaction at 120 °C.

Table 1 .
Mass and molar composition of reaction media for the preparation of PLLA and its copolymers with PCL.

Table 2 .
13emical shifts (ppm) from13C-NMR spectra cited in literature and obtained experimentally at 68.9 ppm related to tertiary carbon, and at 16.6 ppm related to the methylene group -CH-.Chemical shifts of caprolactone unit (CL) were only presented in the 80/20 sample.In Figure2there is a comparison between the C 13 -NMR spectra for samples of PLLA (100/0) and PLLA-co-PCL (80/20).The areas of the 13 C-NMR signals related to ε-CL -CH 2 groups (20.2 ppm) and the -CH 3 groups of the LLA (16.2 ppm) were calculated, and the ratio between them expresses the percentage of ε-CL chemically bound to LLA, according to Equation 1.

Table 4 .
PLLA and copolymers molecular weight (M n and M w ), polydispersity (M w /M n ) obtained by GPC.