Chapter 12
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L-Lysine-Modified
Poly(ester-urethane) Based on Polycaprolactone for Controlled Release of Hydrocortisone Karla A. Barrera-Rivera* and Antonio Martínez-Richa* Departamento de Química, División de Ciencias Naturales y Exactas, Universidad de Guanajuato, Noria Alta S/N, Guanajuato, Guanajuato 36050, México *E-mail:
[email protected]. *E-mail:
[email protected].
A linear L-lysine poly(ester-urethane) (PUR) was successfully prepared from polycaprolactone diol (DEG1), hexamethylenediisocyanate (HDI), and L-lysine ethyl ester dihydrochloride. Evidence from Fourier transform infrared (FT-IR) spectra indicates that intermolecular hydrogen-bonded species of PUR are notably influenced by the presence of L-lysine. Thermal and morphological properties of polyurethanes (PUs) were characterized by differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). It was found that L-lysine provokes profound changes in the thermal and crystalline properties of the polymer. The presence of L-lysine has a strong influence in the water-uptake behavior of the polymer. Films of PU containing 1% (w/w) hydrocortisone were placed in three different buffer systems: (1) citrate buffer (pH 4.0), (2) phosphate buffer (pH 7.0), and (3) borate buffer (pH 10.0) at room temperature and monitored in vitro up to 48 h for their release behavior. Amino acid-containing degradable PUR was tested for delivery of hydrocortisone under different pH conditions. The release profiles of hydrocortisone occurred by a two-stage process and followed a non-Fickian behavior.
© 2018 American Chemical Society Cheng et al.; Green Polymer Chemistry: New Products, Processes, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
Introduction In the field of biomedical research, there is great interest in the synthesis of degradable polymers with potential applications in drug delivery and tissue engineering. Based on their good mechanical properties, flexibility, and biocompatibility, some polyurethanes (PUs) have been used to fabricate biomedical devices. In particular, biodegradable PUs have been mainly employed in the design of drug-delivery systems (1). Enzyme-catalyzed polymerization may become a versatile method for the production of sustainable PUs: Lipases, for example, are renewable catalysts with high catalytic activities. The most prominent advantage of using hydrolytic enzymes for the production of polymers is the reversible polymerization–degradation reaction that allows chemical recycling. Polymer chains that contain enzymatically hydrolyzable moieties can be specifically cleaved by a hydrolytic enzyme to produce potentially re-polymerizable low-molecular-weight fragments that can be recycled (2). Yarrowia lipolytica lipases (YLL) are attracting the interest of scientists and industrial researchers due to their ability to catalyze important high-value applications in the food, pharmaceutical, fine-chemicals, and environmental industries (3). Hydrocortisone [11,17,21-trihydroxy-(11b)-pregn-4-ene-3,20-dione] is a hydrophobic corticosteroid drug widely used in the treatment of various skin condition symptoms, such as redness, swelling, itching, as well as certain types of arthritis, allergies, and asthma. It relieves symptoms related to certain hormone deficiencies and has immunosuppressive action. Hydrocortisone has also been used as a supportive-care medication for cancer (4). In this study, hydrocortisone was used as a model for drug-controlled release. We recently reported a study of the degradation behavior of two different PURs, each synthesized from (1) polycaprolactone (PCL) diol obtained by way of enzymatic polymerization and (2) hexamethylenediisocyanate (HDI). One sample is the neat linear PU; the other contains L-lysine as amino acid chain extender. Under composting conditions, half-maximal degradation time (t50) of both samples is approximately 25 days (5). In this work, we report a study to investigate the potential use as a drug-delivery system of these biodegradable, nontoxic PURs.
Experimental Materials Before use, 97% ε-polactone (ε-CL) 97% was dried over calcium hydride and distilled under reduced pressure. Diethylene glycol (DEG) 99%, Lewatit VP OC 1026 beads, stannous 2-ethylhexanoate ~95%, HDI ≥98%, L-lysine ethyl ester dihydrochloride (L-Lys; purity ≥99%), triethylamine 99%, 1,2-dichloroethane anhydrous 99.8%, and hydrocortisone 98% were purchased from Sigma Aldrich and used as received. Citrate buffer (pH 4.0), phosphate buffer (pH 7.0), borate buffer (pH 10.0), and ethanol were purchased from Karal and used as received. YLL was obtained and immobilized according to a procedure described in the literature (6). 164 Cheng et al.; Green Polymer Chemistry: New Products, Processes, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
Lipase Isolation and Immobilization Lipase production by YLL was made as previously reported by Barrera et al. (6). Before immobilization, Lewatit VPOC 1026 beads were activated with ethanol (1:10 beads:ethanol) for 5 h, washed with distilled water, and dried under vacuum for 24 h at room temperature. The beads (1 g) were shaken in a rotatory shaker in 15 mL of YLL-lipase solution with 0.2 mg/mL at 4 °C for 14 h. After incubation, the carrier was filtered off, washed with distilled water, and then dried under vacuum for 24 h at room temperature. Synthesis of α,ω-Telechelic poly(ε-caprolactone)diol PCL diol (DEG1) was prepared as described in (7), from 10 mmol of ε-CL and 1 mmol of DEG in the presence of 12 mg YLL-1026 (immobilized lipase). Vials were stoppered with a Teflon silicon septum and placed in a thermostated bath at 120 ºC for 6 h. No inert atmosphere was used. After the reaction was stopped, the enzyme was filtered off, and the PCL diol was dried at room temperature for 12 h and stored at ambient temperature in a desiccator until used. Synthesis of PURs Bearing Amino Acids PURs were prepared according to the literature (8). Dry PCL diol (2.5 g), HDI in the appropriate amount (OH:NCO ratio = 1:1), and 15 mL of 1,2-dichloroethane were charged in a round bottom flask. The catalyst, stannous 2-ethylhexanoate (1% mol by PCL diol moles) was added and stirred for 1 h and 30 min at 80 ºC; after that time, 0.3498 mmol of amino acid and 1.046 mmol of triethylamine were added to the reaction mixture and allowed 5 h for reaction. The resulting solution was poured over a Teflon petri dish (d-10 cm). The solution was allowed to stand at ambient temperature for 24 h for solvent evaporation. The film was then released and dried at room temperature (Scheme 1). Using 1H-NMR, it was determined that approximately 65% of PU chains contain L-lysine as end-group (5).
Scheme 1. Chemo-enzymatic synthesis of PUR bearing L-lys. 165 Cheng et al.; Green Polymer Chemistry: New Products, Processes, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
Preparation of Films Containing 1% Hydrocortisone (w/w) PUR films were mixed in solution (1,2-dichloroethane as solvent) with 1% (w/ w) of hydrocortisone and stirred at room temperature until they were completely dissolved; the solution was then poured over a Teflon dish (d-10 cm) and allowed to stand at room temperature for solvent evaporation. The film was then released and dried at room temperature. In Vitro Hydrocortisone-Release Studies Hydrocortisone release was evaluated in vitro by ultraviolet–visible light (UV-VIS) spectroscopy. A PUR film (1 × 1 cm) was placed in 3 mL of buffer at different pH values in a quartz cell. The hydrocortisone concentration was determined using a Perkin-Elmer UV-VIS spectrometer lamba 25 at 248 nm during 48 h. Samples were analyzed by duplicate. Swelling Degree Film specimen with a dimension of 3 × 3 cm was dried to a constant weight (Wd) at room temperature. Subsequently, the dried specimen was placed in a flask with 50 mL of the different pH solutions (pH 4, 7, and 10) for 24 h. The swollen specimen was then taken out of the flask, wiped with a filter paper, and weighed (Ww). The swelling degree is calculated according to Equation 1.
where Wd is the weight of the dried film, and Ww is the weight of the swollen film.
Characterization of the PURs Fourier Transform Infrared Spectra were obtained at room temperature using attenuated total reflection technique on films deposited over a diamond crystal on a Perkin-Elmer Spectrum Two™ spectrometer with an average of four scans at 4 cm-1 resolution. Peak analysis was performed using the Origin 6.1 computer program for PC. Thermal Analysis PU samples were conducted on a Mettler Toledo differential scanning calorimeter (DSC)-820e using heating and cooling rates of 10 °C/min. Thermal scans were performed from 25 to 80 ºC, 80 to −90 °C, and −90 to 80 °C in air atmosphere. 166 Cheng et al.; Green Polymer Chemistry: New Products, Processes, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
Thermogravimetric Analysis Thermogravimetric analysis (TGA) scans were recorded on a TA Instruments simultaneous TGA-DSC SDT Q600 at 10 °C/min with dynamic atmosphere of high-purity air at 100 mL/min.
Results and Discussion Two different PUs were synthesized from the enzymatically synthesized PCLdiol: the neat PURDEG1HDI and the PUR modified with L-lysine amino acid as end-group (PURDEG1 L-lys). The reaction was followed by FT-IR by checking the disappearance of the stretching vibration of the isocyanate group (-N=C=O at 2270 cm−1), which confirms the completion of the reaction between the hydroxyl group from the enzymatically synthesized PCL-diol and the isocyanate group from HDI after 90 min. The FTIR analysis of PURDEG1 and the PURDEG1 L-lys films (containing hydrocortisone) revealed the typical bands of PCL-based PUs: (1) a peak in the region of 3350 cm-1 due to N-H stretching; (2) -CH2- stretching (from 3100 to 2700 cm-1); (3) C=O stretching (1724 cm-1); (4) N-H bending and C-N stretching from the urethane group (1530 and 1242 cm-1); and (5) C-O-C stretching (1166 cm-1). After analyzing the peak pattern of both spectra, some differences were observed between them. A sharper peak in the PURDEG1 HDI spectrum centered at 1530 (urethane group) was observed. In addition, small differences in shape for the broad peak in the 1200−1120 region [due to C-O and C-N(C=O) stretching band] were apparent. The main difference observed in the spectra was for the carbonyl stretching band (C=O), which contains information on intermolecular and self-associated species through hydrogen bonding (Figure 1).
Figure 1. FT-IR spectra (carbonyl zone) of the synthesized PURs. To estimate the content of the inter-associated and the self-associated carbonyl bonds by way of hydrogen bonds, peak deconvolution analysis was performed. The degree of the carbonyl groups participating in hydrogen bonding 167 Cheng et al.; Green Polymer Chemistry: New Products, Processes, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
can be recorded by the carbonyl hydrogen bonding index, R (9), derived by using Equation 2:
The results are reported in Table 1.
Table 1. FT-IR Data for the Synthesized PURs
It is evident that the content of inter-associated species in the PUR decreases in the presence of L-lys. This is expected because L-lys moieties compete with the N-H bonds of the urethane functional group to create associated species. In the hydroxyl zone (Figure 2), a shoulder at 3520 cm-1 (“free” OH) is more evident in the FT-IR spectrum of PURDEG1 L-lys (see arrow). In addition, the peak shape between the two spectra differs slightly in this zone.
Figure 2. FT-IR spectra (hydroxyl zone) of the synthesized PURs:PURDEG1 L-lys (top line) and PURDEG1 (bottom line). 168 Cheng et al.; Green Polymer Chemistry: New Products, Processes, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
Thermal Properties Thermal properties for PURs containing hydrocortisone were registered from 25–80 °C followed by cooling and reheating from −90 to 80 °C. The results from DSC measurements are listed in Table 2. In the first heating (25−80 °C), PURDEG1HDI L-lys showed a flat baseline, whereas PURDEG1HDI displayed a transition due to pure PCL-phase melting (Figure 3). This behavior indicates that melt transition for PCL is inhibited by the presence of amino acid in the polymeric matrix of PURDEG1 HDI. In the second heating, a difference in peak positions (by 3.4 °C) and the heat of fusion are apparent.
Figure 3. DSC curves of the synthesized PURs. A) First heating and B) second heating.
Figure 4. Crystallization DSC curves of the synthesized PURs. Figure 4 shows the thermograms for PCL crystallization (cooling from 80 to −90 °C) for synthesized PURs. Whereas PURDEG1 HDI L-Lys shows a crystallization peak at 3.9 °C, the other PUR has a Tc of −10.7 °C. In addition, greater enthalpy is recorded for the L-lysine containing PUR. Differences in crystalization temperatures and the heat of fusion indicates that more crystalline zones are induced during the second heating in the amino acid-containing sample. 169 Cheng et al.; Green Polymer Chemistry: New Products, Processes, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
Table 2. Thermal Properties Obtained by DSC for the Synthesized PURS-Containing Hydrocortisone Sample
PURDEG1HDI DEG1 L-LYS HDI
Tm (°C) First heating
Tm (°C) Second heating
ΔHm (J/mol) First heating
Tc (°C)
ΔHm (J/mol) Second heating
44.8
18.93
36.9
25.2
−10.7
−
−
40.3
29.9
3.9
TGA results for PURDEG1 and PURDEG1 L-lys containing hydrocortisone are shown in Figure 5 and Table 3. The onset decomposition temperatures (Tonset) for PURDEG1HDI, and PURDEG1 L-lys are 292 and 291 °C, respectively. Similarly, the decomposition temperature peaks (Tmax) for these compounds are at 325 and 329 °C, respectively. Between these two compounds, L-lysine chain extender unit is only present in PURDEG1 L-lys. Because the hard-segment content of the compound increases when chain-extender content is greater, the decomposition temperature or thermal resistance of the compound slightly increases.
Figure 5. TGA curves of the synthesized PURs. 170 Cheng et al.; Green Polymer Chemistry: New Products, Processes, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
Table 3. Thermal Properties Obtained by TGA of the PURs PUR
Tonset (°C)
Decomposition (%)
Tmax (°C)
Decomposition (%)
PURDEG1HDI
292
12
325
85
PURDEG1 L-Lys HDI
291
15
329
83
Swelling Degree L-Lysine is an α-amino acid and is an essential building block for all proteins in the body. It contains an ε-amino group in the aliphatic side chain (R group), which gives the molecule a hydrophilic character (10). Swelling degrees of the synthesized PURs containing hydrocortisone PURDEG1HDI in different buffers at pH 4, pH 7, and pH 10 for 24 h were 0.3%, 0.3%, and 1.2%, respectively, and for PURDEG1 L-lys were 2.5%, 7.1%, and 1.1% (see Table 4), respectively. The swelling degree depends on the presence of L-lysine and the amount on PCL content in the PUR. Three factors affect swelling behavior. One factor is the presence of L-lysine because this moiety is hydrophilic and favors the absorption of water. Another important factor is the relative amount of PCL in the PURs, which influences swelling behavior because of its hydrophobic nature. Lower content of PCL in the PUR reflects in a higher swelling degree. The third factor is the physical cross-link effect of the hard segment. The presence of hard segment acts as physical cross-links in the PUs and subsequently enhances their physical properties as mechanical strength, hardness, and solvent resistance. In that regard, the presence of L-lysine and the lower relative amount of PCL chains in the PUR explain the higher values of water uptake recorded for the PURDEG1 L-lys sample at pH = 4 and 7. At greater pH (more basic) values, a decrease in the swelling degree is observed for the PURDEG1 L-lys at pH = 10 after 24 h of being in contact with the buffer solution; values for both polymers are approximately the same. After 48 h, degradation of PURDEG1 L-lys occurred. The release profiles for hydrocortisone at three different pH values for the two samples are shown in Figures 6 and 7. The hydrocortisone release consists of a two-stage process: an initial rapid-release stage followed by a second slowerrelease stage. After 48 h, the amount of hydrocortisone released from PURDEG1HDI without L-lysine at pH 4 was approximately 16%; at pH 7 it was 25%; whereas at pH 10 a maximum at 15% was observed after 35 h. For this sample, after 35 h a sharp decrease in the release was observed, which can be attributed to the degradation of the PU under basic conditions at pH 10.
171 Cheng et al.; Green Polymer Chemistry: New Products, Processes, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
Table 4. Swelling Degree of the Synthesized PUs at Room Temperature PUR DEG1 HDI
DEG1 L-lys
pH
Swelling degree (%) at 24 h
Swelling degree (%) at 48 h
4
0.3
−0.2
7
0.3
0.2
10
1.2
0.6
4
2.5
2.1
7
7.1
1.8
10
1.1
−12.6
Figure 6. In vitro drug-release profile for hydrocortisone under different hydrolytic conditions for PURDEG1HDI.
The amount of hydrocortisone released from the PUR containing L-lysine after 48 h at pH 4 was approximately 14%; at pH 7 it was 18%; and at pH 10 it was 50%. Under the conditions studied, PURDEG1 L-lys shows a better performance as a delivery system than PURDEG1 because more amount of drug is delivered if the same amount of time is considered. There is no evidence of decomposition for this sample under basic conditions at this stage. 172 Cheng et al.; Green Polymer Chemistry: New Products, Processes, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
Figure 7. In vitro drug-release profile for hydrocortisone under different hydrolytic conditions for PURDEG1 L-lys.
Table 5. Values k and n (±95% Confidence Intervals) Obtained by Plotting the Logarithm of the Hydrocortisone Fraction Release Versus the Logarithm of Time at pH Values 4, 7, and 10a Matrix
k, (h)-n
n
R2
PURDEG1HDI (pH = 4)
2.5 × 10-2
0.51
0.98
PURDEG1HDI (pH = 7)
1.7 ×
10-2
0.70
0.97
3.2 ×
10-2
0.60
0.99
0.59
0.99
1.0
1.0
0.62
0.99
PURDEG1HDI (pH = 10) PURDEG1 L-Lys HDI (pH = 4) PURDEG1 L-Lys HDI (pH =7) PURDEG1 L-Lys HDI (pH =10) a
R2
2.13 × 1×
10-2
10-2
6.0 ×
10-2
values are also reported.
Kinetics of Drug Release The drug-transport mechanism was modeled using Equation 3:
where Mt/M∞ describes the portion of drug released at time t (M∞ is considered the same as the amount total drug loaded in each polymer); k is the constant of release rate and n corresponds to an important exponent value, which can be used to define the release mechanism. Normal Fickian diffusion for a thin polymer membrane is defined by n < 0.5, whereas case II transport is characterized by n = 1.0. 173 Cheng et al.; Green Polymer Chemistry: New Products, Processes, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
Using linear regression, the intercept and slope of the plot log (Mt/M∞) against time were determined. From these values, k and n were determined. All release profiles presented n values between 0.5 and 1 as listed in Table 5, which suggests that the release of hydrocortisone follows a non-Fickian behavior (11, 12).
Conclusions Biodegradable PURs have been successfully synthesized from PCL-diol, DEG, and HDI; one of the samples contains L-lys as chain extender/end-group. Chemical structure and molecular features were determined using FT-IR. DSC analysis reveals striking differences in crystallinity and transition temperatures. High temperature stability (up to ~290 °C) for the PURs was observed from the TGA thermograms. As expected, the hydrophilicity of PURs is increased with the presence of L-lysine as revealed by water-swelling analysis at different pH values. Hydrocortisone-release curves depend on pH, and release is more effective for PURDEG1 L-lys sample. Drug release occurs by a non-Fickian mechanism.
Acknowledgments The authors acknowledge financial support from Consejo Nacional de Ciencia y Tecnología (CONACyT), Grant 153922.
References 1.
2. 3. 4. 5. 6. 7.
8.
9.
Basu, A.; Farah, S.; Kunduru, K. R.; Doppalapudi, S.; Khan, W.; Domb A. J. Advances in Polyurethane Biomaterials. Series in Biomaterials 108; Woodhead Publishing: Cambridge, MA, 2016; pp 217–246. Yanagishita, Y.; Kato, M.; Toshima, K.; Matsumura, S. ChemSusChem. 2008, 1, 133–142. Brígida, A. I. S.; Amaral, P. F. F.; Coelho, M. A. Z.; Gonçalves, L. R. B. J. Mol. Catal. B: Enzym. 2014, 101, 148–158. Golbert-Gist, A. Chem. Eng. News 2005, 83, 25. Arrieta, M. P.; Barrera-Rivera, K. A.; Salgado, C.; Martínez Richa, A.; López, D.; Peponi, L. Poly. Degrad. Stabil. 2018, 152, 139–146. Barrera-Rivera, K. A.; Flores-Carreón, A.; Martínez-Richa, A. J. Appl. Polym. Sci. 2008, 2, 708–719. Barrera-Rivera, K. A.; Marcos-Fernández, A.; Martínez-Richa, A. Green Polymer Chemistry: Biocatalysis and Biomaterials;Cheng, H. N.,Gross, R. A., Eds.; ACS Symposium Series 1043; American Chemical Society: Washington, DC, 2010; pp 227–235. Barrera-Rivera, K. A.; Martínez-Richa, A. Green Polymer Chemistry: Biobased Materials and Biocatalysis; ACS Symposium Series 1192; American Chemical Society: Washington, DC, 2015; pp 27–40. Seymour, R. W.; Estes, G. M.; Cooper, S. L. Macromolecules 1970, 3, 579–583. 174 Cheng et al.; Green Polymer Chemistry: New Products, Processes, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
10. Nelson, D. L.; Cox, M. M. Lehninger Principles of Biochemistry, 4th ed.; Worth Publishers: Conway, AR, 2017; pp 76–79. 11. Ritger, P. L.; Peppas, N. A. J. Controlled Release 1987, 5, 23–36. 12. Ritger, P. L.; Peppas, N. A. J. Controlled Release 1987, 5, 37–42.
175 Cheng et al.; Green Polymer Chemistry: New Products, Processes, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
