Synthesis and Characterization of Novel Biodegradable Poly

Dec 8, 2007 - ... Chinese Academy of Sciences, Changchun 130022, Peopleʼs Republic of China, and Graduate School of Chinese Academy of Sciences, ...
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Biomacromolecules 2008, 9, 376–380

Synthesis and Characterization of Novel Biodegradable Poly(carbonate ester)s with Photolabile Protecting Groups Zhigang Xie,†,‡ Xiuli Hu,†,‡ Xuesi Chen,† Jing Sun,†,‡ Quan Shi,†,‡ and Xiabin Jing*,† State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Changchun 130022, People’s Republic of China, Chinese Academy of Sciences, Changchun 130022, People’s Republic of China, and Graduate School of Chinese Academy of Sciences, Beijing 100039, People’s Republic of China Received August 13, 2007; Revised Manuscript Received October 3, 2007

Novel biodegradable poly(carbonate ester)s with photolabile protecting groups were synthesized by ring-opening copolymerization of L-lactide (LA) with 5-methyl-5-(2-nitro-benzoxycarbonyl)-1,3-dioxan-2-one (MNC) with diethyl zinc (Et2Zn) as catalyst. The poly(L-lactide-co-5-methyl-5-carboxyl-1,3-dioxan-2-one) (P(LA-co-MCC)) was obtained by UV irradiation of poly(L-lactide acid-co-5-methyl-5-(2-nitro-benzoxycarbonyl)-1,3-dioxan-2one) (P(LA-co-MNC)) to remove the protective 2-nitrobenzyl group. The free carboxyl groups on the copolymers P(LA-co-MCC) were reacted with paclitaxel, a common antitumor drug. Gel permeation chromatography and NMR studies confirmed the copolymer structures and successful attachment of paclitaxel to the copolymer.

Introduction Aliphatic polyesters, such as poly(-caprolactone) (PCL), poly(L-lactide) (PLA), and polyglycolide (PGA), are of great interest as biomaterials due to their excellent biodegradability, bioresorbability, and mechanical properties.1–4 The potential applications of these polymers are further broadened when functional pendant groups are incorporated into the polymer backbones. Various chemical approaches have been developed to introduce amino,5,6 carboxyl,7–16 hydroxyl,17–20 and mercapto groups21,22 onto such polyesters via ring-opening polymerization (ROP) of lactones or lactides with the appropriate functional monomers, i.e., cyclic diesters, lactones of different sizes, morpholine-2,5-diones, and N-carboxyanhydride derivatives from amino acids. There are some good reviews about functionalization of the aliphatic polyesters.23–25 Recently, poly(carbonate ester)s composed of functionalized carbonates are also described.26–32 Compared to other approaches such as morpholine-2,5-dione derivatives, cyclic diesters, and lactones, functionalized cyclic carbonates are synthesized and polymerized more easily and more efficiently. In order to prevent inter- and intramolecular side reactions, the functional groups in the comonomers have to be protected before copolymerization. However, the deprotection usually is difficult and may lead to breakdown of the polymeric chains. For example, Pd/C-catalyzed hydrogenation is widely used in deprotection, but the residual Pd/C and damage to the polymeric chains are both troublesome.33,34 Herein, we provide an easy way to remove the protective groups only by UV irradiation. Photolabile protecting groups (PPG) have found many important applications in synthetic and biomedical chemistry. From a synthetic point of view, PPG are orthogonal to other protecting groups and do not require reagents and/or heating * Corresponding author: tel, +86-431-85262775; fax, +86-431-85685653; e-mail, [email protected]. † State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry. ‡ Chinese Academy of Sciences and Graduate School of Chinese Academy of Sciences.

for their removal. For biomedical applications, PPGs can provide spatial and temporal control of the release of bioactive substrates.35,36 To our knowledge, poly(carbonate ester)s with PPG remains relatively unexplored. In this work, a novel six-membered cyclic carbonate monomer that contains photolabile protecting groups, 5-methyl-5-(2-nitrobenzoxycarbonyl)-1,3-dioxan-2-one (MNC), was synthesized from 2,2-bis(hydroxymethyl)propionic acid. We further investigated the ring-opening copolymerization of MNC with Llactide (LA). By UV irradiation of P(LA-co-MNC) solution in THF, the pendant carboxyl groups were released and finally reacted with paclitaxel, a common antitumor drug.

Experimental Section Materials. L-Lactide (LA) was purchased from PURAC Biochem bv Gorinchem and recrystallized from ethyl acetate several times. Diethyl zinc (Et2Zn) was kindly supplied by Professor Xianhong Wang in Changchun Institute of Applied Chemistry. Tetrahydrofuran (THF) was purified by distillation from sodium with benzophenone. Triethylamine (TEA) was refluxed over phthalic anhydride and then distilled over CaH2. Dichloromethane was treated with hexamethylene diisocyanate for 5 h at 50 °C and distilled to remove any traces of amine and alcohol. 2-Nitrobenzyl bromide, ethyl chloroformate, dicyclohexycarbodiimide (DCC), dimethylaminopyridine (DMAP) and 2,2-bis(hydroxymethyl)propionic acid were purchased from Aldrich and used without further purification. Paclitaxel was purchased from Xi’an Baosai Biotechnology, Inc., in China. Other regents were commercially available and used as received. Measurements. 1H NMR spectra were recorded on a Bruker AV300 M in CDCl3 at 25 °C. Chemical shifts were given in parts per million from that of tetramethylsilane as an internal reference. Gel permeation chromatography (GPC) measurements were conducted with a Waters 410 GPC instrument equipped with a Waters Styragel HT6E column and a differential refractometer detector. THF was used as eluent at a flow rate of 1 mL · min-1 at 35 °C. The molecular weights were calibrated with polystyrene standards. Differential scanning calorimetry (DSC) analyses were carried out at a heating rate of 10 °C/min on a Perkin-Elmer Pyris 1.

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Scheme 1. Synthesis of 5-Methyl-5-(2-nitro-benzoxycarbonyl)1,3-dioxan-2-one (MNC)

Synthesis of 5-Methyl-5-(2-nitro-benzoxycarbonyl)-1,3-dioxan2-one (MNC). Five grams (37 mmol) of 2,2-bis(hydroxymethyl)propionic acid and 2.4 g (43 mmol) of KOH were dissolved in 50 mL of DMF, and the potassium salt was allowed to form by stirring the reaction mixture at 100 °C for 1 h. Eight grams (37 mol) of 2-nitrobenzyl bromide was then added dropwise to a solution, and the mixture was stirred and allowed to react with maintaining temperature for 15 h. After completion of the reaction, the solvent was evaporated; the residue was dissolved in 200 mL of diethyl ether and extracted with distilled water. The crude product was then purified by recrystallization from toluene to give 2-nitrobenzyl 2,2-bis(hydroxymethyl)propionate, as white crystals. Yield: 60%. A 3.9 g portion of (38 mmol) TEA was added dropwise to a mixture of 2-nitrobenzyl 2,2-bis(hydroxymethyl)propionate (5 g, 19 mol) and ethyl chloroformate (4.1 g, 38 mmol) dissolved in 200 mL of THF at 0 °C over a period of 30 min. The reaction mixture was stirred at room temperature for 2 h. Precipitated TEA hydrochloride was filtered off, and the filtrate was concentrated under reduced pressure. The residue was recrystallized from THF and diethyl ether. White crystals were obtained. Yield: 80%. Copolymerization of MNC with LA. Under the protection of argon, prescribed amounts of LA, MNC, and Et2Zn were added to a dried polymerization vessel. The vessel was degassed by several vacuum-argon purging cycles to remove oxygen and trace moisture. The vessel was then sealed under vacuum and placed in an oil bath at 120 °C for 20 h. The reaction was terminated by cooling the vessel to room temperature. The copolymer P(LA-co-MNC) was dissolved in dichloromethane and precipitated into an amount of cold methanol, isolated by filtration, and dried under vacuum at room temperature. Deprotection by UV Irradiation. The protected copolymer P(LAco-MNC) (0.4 g) was dissolved in 40 mL of THF. The solution was stirred in glass beaker under UV lamp (365 nm, 20 mW/cm2) for 5 h. Then the system was evaporated and dropped into a large amount of diethyl ether to precipitate the deprotected copolymer P(LA-co-MCC) with free pendant carboxyl groups. It was dried under vacuum at room temperature. Conjugate with Paclitaxel. In a dried flask, 0.10 g of P(LA-co5%MCC) (carboxyl groups, 0.036 mmol) was dissolved in 10 mL of anhydrous dichloromethane, and then 31 mg (0.036 mmol) of paclitaxel, 11 mg (0.054 mmol) of DCC, and 6.6 mg (0.054 mmol) of DMAP were added into the above solution at 0 °C. The reaction was carried out under stirring for 48 h at 0 °C. The byproduct dicyclohexylurea was filtered out. The filtrate was condensed under a reduced pressure and poured into an excess amount of methanol with stirring. The P(LAco-MCC)/paclitaxel was collected by filtration, washed with methanol three times, and finally dried in vacuo at room temperature overnight. Purification of the conjugate from unreacted paclitaxel was done by dialysis in chloroform with a cellulose membrane (cutoff Mn 5000) for 2 days.

Results and Discussion Synthesis of MNC. The novel cyclic carbonate monomer MNC was synthesized by a two-step reaction from 2,2-

Figure 1. The 1H NMR spectrum of the carbonate monomer MNC. Scheme 2. Synthesis and Deprotection of the Poly(carbonate ester)s

Table 1. Related data on the Bulk Copolymerization of LA and MNCa mol% of MNC

sample P(LA-co-5%MNC) P(LA-co-10%MNC) P(LA-co-15%MNC) P(LA-co-20%MNC) P(LA-co-40%MNC)

Mnc Mwc yield feed productb (104) (104) Mw/Mnc (%) 5 10 15 20 40

5 10 14 19 37

1.01 1.27 1.67 1.13 1.43

1.70 2.38 2.86 2.09 2.49

1.69 1.87 1.71 1.85 1.74

98 95 85 88 89

a Copolymerization was carried out at 130 °C for 20 h. The molar ratio of Et2Zn to monomer (LA + MNC) was 1/200. b Determined by 1H NMR. c Apparent molecular weight determined by GPC (CHCl3 as eluent).

bis(hydroxymethyl)propionic acid. The synthetic pathway is shown in Scheme 1. First, 2-nitrobenzyl bromide was used to protect the carboxyl group, and then the 2-nitrobenzyl 2,2bis(hydroxymethyl)propionate obtained was reacted with ethyl chloroformate in THF by using TEA as a catalyst. The chemical structure of MNC was characterized by 1H NMR spectroscopy. As shown in Figure 1, the signals from 7.5 to 8.2 ppm are assigned to phenyl ring protons present in the MNC repeat units. The signals at 4.2 and 4.7 ppm are assigned to methylene protons in the carbonate ring. Copolymerization of MNC with LA. In the previous papers, Et2Zn is an efficient catalyst for the synthesis of biodegradable polycarbonates and poly(β-hydroxyl acid)s.37 Herein, we also used Et2Zn as catalyst to prepare the copolymer of LA and MNC through ring opening copolymerization. Scheme 2 shows the synthesis of the copolymer and subsequent deprotection. Table 1 summarizes some results of the copolymerization of LA and MNC. It can be seen that the polymerization yield is quite high, and compared to LA, MNC displays similar activity

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Figure 4.

13

C NMR spectrum of copolymer P(LA-co-10%MNC).

Figure 2. GPC curves of copolymer P(LA-co-10%MNC) (A) and P(LAco-10%MCC) (B).

Figure 5. Expanded carbonyl group region of the 13C NMR spectrum of the copolymer containing 20 mol % MNC.

Figure 3. 1H NMR spectrum of copolymer P(LA-co-10%MNC).

so that the MNC content in the copolymer is close to that in the monomer feed. The GPC curves of the protected copolymers with different contents of MNC all show a unimodal peak. One typical GPC trace is shown in Figure 2A for P(LA-co10%MNC) in which the molar content of MNC is 10%. Its molecular weight and molecular weight distribution are 12700 and 1.87, respectively. These data imply that the copolymerization was completed successfully, and no homopolymerization of LA or MNC took place. 1 H and 13C NMR spectral data were used to characterize the copolymer of LA and MNC. Figure 3 displays the 1H NMR spectrum of copolymer P(LA-co-10%MNC). According to previously published spectra of PLA,38 the signals at 5.2 and 1.6 ppm are due to the CH and CH3 protons in LA repeat unit, respectively. The signals from 7.5 to 8.2 ppm are assigned to phenyl ring protons present in MNC repeat units. The signal at 5.5 ppm is assigned to methylene protons close to the phenyl ring. Figure 4 displays the 13C NMR spectrum of copolymer P(LA-co-10%MNC). The assignments in Figure 4 are based on the 13C spectra of the PLA and PMNC homopolymers. In short, the NMR spectra of the copolymer contain all signals of the LA and MNC components. This is the powerful evidence for the successful copolymerization of LA and MNC. Sequence Distribution. The sequence of distribution of the copolymer was determined from 13C NMR spectral analysis.

As reported in previous articles,19,33 the 13C NMR spectrum may be correlated with eight possible triads for the two repeating units, LLA (L) and MNC (M), that is, LLL, LLM, MLL, LML, LMM, MLM, MML, and MMM. The expanded carbonyl group region of the 13C NMR spectrum of the copolymer containing 20 mol % MNC is shown in Figure 5. Signal at 169.98 ppm is assigned to the triad LLL, which is the most prevalent sequence in the copolymer. The neighboring signals that appear are shoulders of the 169.98 ppm resonance that are likely due to sequence effects beyond triads. The signals at 169.84 ppm were assigned to the central carbonyl in the triad LLM. The carbonyl carbons of carbonates MNC units showed two signals at 154.38 and 154.35 ppm. The signals at 154.38 and 154.35 ppm are assigned to the central carbonyl in the triad MMM and MML. The expanded carbonyl group region of the 13C NMR spectrum of the copolymer containing 40 mol % MNC shown in Figure 6 also proved the occurrence of signal splitting of carbonyl resonance. Moreover, changing the MNC molar content to 40% in copolymers increased the signal intensity of LLM and MMM while diminishing the intensity of LLL and MML. These results indicate that the copolymers P(LA-co-MNC) are statistical copolymers. Thermal Properties. The thermal properties of copolymers P(LA-co-MNC) were obtained from DSC measurements. The DSC curves of P(LA-co-MNC) are shown in Figure 7. The homopolymer PLA is a highly crystalline material and has a melting temperature (Tm) at 174 °C and glass transition temperature (Tg) at 60 °C. The fact that only one Tg was detected for every sample in the examined temperature range from 0 to 180 °C demonstrates that the polymer is a random copolymer

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Figure 6. Expanded carbonyl group region of the 13C NMR spectrum of the copolymer containing 40 mol % MNC. DSC thermograms of P(LA-co-MNC) (second heating run).

Figure 8. 1H NMR spectrum of copolymer P(LA-co-10%MCC). Scheme 3. Chemical Structure of Paclitaxel

Figure 7. DSC thermograms of P(LA-co-MNC) (second heating run).

but not a mixture of PLA and PMNC homopolymers. With increasing content of MNC incorporated into the copolymer, decreasing Tg is observed. For the copolymers synthesized, no melting peaks are observed when the MNC content exceeds 5 mol %. The copolymer P(LA-co-5%MNC) shows a Tm at 149 °C. These results indicated that crystallization of the PLA segments are hindered due to the presence of PMNC units or segments. Deprotection by UV Irradiation. The 2-nitrobenzyl group and its analogues have been intensively employed as photoremovable protecting groups in a variety of synthetic strategies.39–41 Zhao et al. prepared light-dissociable block copolymer micelles by using 2-nitrobenzyl groups.42 Herein, we synthesized a copolymer P(LA-co-10%MNC) with the same protecting group, then deprotected by UV irradiation. Figure 8 shows the 1H NMR spectrum of the deprotected copolymer P(LA-co-10%MCC). Compared with Figure 3, we can see that phenyl ring protons from 7.5 to 8.2 ppm and the methylene protons at 5.5 ppm in the MNC repeat units disappear whereas other proton signals are little changed. The GPC trace of P(LA-co-10%MCC) in Figure 2B also shows a unimodal peak, and the molecular weight and molecular weight distribution are 10900 and 1.61, respectively. Compared to Figure 2A for the protected one, they do not change very much, implying that the polymeric chains were kept unchanged and no degradation of the copolymer chains occurred during the process of deprotection. These data imply that the deprotection of 2-nitrobenzyl groups was completed successfully. Conjugate with Paclitaxel. The free carboxyl groups on the copolymer chains are capable of functionalization, and this

provides opportunities for covalent attachment of biological epitopes for cell recognition, photo-cross-linkable moieties for three-dimensional gel formation, or pharmaceutical agents for drug delivery. Paclitaxel is a microtubule stabilizing drug and a potent chemotherapeutic agent. In this work, we conjugate paclitaxel with the pendant carboxyl groups on P(LA-co-MCC). Scheme 3 shows the chemical structure of paclitaxel. According to the literature,43–45 the most suitable positions in paclitaxel for structure modification are the 2′-hydroxl groups. Therefore, esterification takes place at the 2′-hydroxyl in the presence of DCC and DMAP at 0 °C. Figure 9 shows the 1H NMR spectra of the paclitaxel and P(LA-co-MCC)/paclitaxel conjugate. It shows that the characteristic peaks of paclitaxel can all be found in P(LA-co-MCC)/paclitaxel conjugate. The signals from 7.3

Figure 9. 1H NMR spectrum of (A) paclitaxel and (B) P(LA-co-MCC)/ paclitaxel conjugate.

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to 8.2 ppm for phenyl ring protons can be obviously seen in the 1H NMR spectra of P(LA-co-MCC)/paclitaxel conjugate. Because the unreacted paclitaxel could be removed by dialysis, 1 H NMR spectrum indicates that paclitaxel had been conjugated successfully with copolymer P(LA-co-MCC). As reported in previous article, 45 the paclitaxel content in the conjugate calculated from the peak intensities of the phenyl proton signal (7.3–8.2 ppm) and methyl proton signal (1.5 ppm) of PLA in the 1H NMR spectra (Figure 1c) was 19% by weight. Compared with the paclitaxel weight in feed, we could get the efficiency of paclitaxel binding as 78%.

Conclusion In summary, poly(carbonate ester)s with photolabile protecting groups were synthesized by ring-opening copolymerization of LA with MNC. GPC and 1H NMR studies confirmed the copolymer structures. A Tg was detected but no melting temperature was observed when the MNC content exceeds 5 mol %. Moreover, Tg of the copolymer decreases with increasing MNC content. The 2-nitrobenzyl group of copolymers P(LAco-MNC) was removed by UV irradiation. Finally, the free carboxyl groups on the copolymers P(LA-co-MCC) were reacted with antitumor drug paclitaxel. The successful attachment of paclitaxel indicates the possibility of poly(carbonate ester)s used further in specific drug delivery and tissue engineering. Acknowledgment. Financial support was provided by the National Natural Science Foundation of China (Project No. 20274048 and 50373043), by the National Fund for the Distinguished Young Scholars (Grant No. 50425309), and by the Chinese Academy of Sciences (Project No. KJCX2-SWH07).

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