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Fast Degradable Polycaprolactone for Drug Delivery Seo Hee Chang, Hyun Jung Lee, Sohee Park, Yelin Kim, and Byeongmoon Jeong Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00266 • Publication Date (Web): 09 May 2018 Downloaded from http://pubs.acs.org on May 9, 2018

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Fast Degradable Polycaprolactone for Drug Delivery Seo Hee Chang, Hyun Jung Lee, Sohee Park, Yelin Kim, and Byeongmoon Jeong* Department of Chemistry and Nanoscience, Ewha Womans University, 52 Ewhayeodae-gil, Seodaemun-gu, Seoul, 03760, Korea

ABSTRACT: Polycaprolactone (PCL) was reported a long time ago; however, its biomedical applications has not been extensively investigated in comparison with poly(lactide-co-glycolide) (PLGA) due to its too slow degradation profile. Here, we are reporting an oxalate-connected oligocaprolactone multiblock copolymer (PCL-OX) as a fast degradable PCL while maintaining its crystalline properties and low melting point of PCL. The in-vivo application of the paclitaxelloaded PCL-OX microspheres provided a steady plasma drug concentration of 6-9 µg/ml over 28 days, similar to that of the PLGA microspheres. Both PCL and PLGA microspheres were completely cleared two months after in-vivo implantation. The PCL-OX microspheres showed a similar tissue compatibility to that of PLGA microspheres in the subcutaneous layer of rats. These findings suggest that PCL-OX is a useful biomaterial that solves the slow degradation problems of PCL, and thus may find uses in other biomedical applications as an alternative to PLGA.

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INTRODUCTION

ACS Paragon Plus Environment

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Aliphatic polyesters, including polyglycolide, polylactide, poly(lactide-co-glycolide) (PLGA), poly(hydroxyl butyrate), and polydioxanone have been extensively investigated as biodegradable materials for uses in suturing and drug delivery.1,2 In particular, PLGA has been most extensively investigated due to its biocompatibility and controllable degradation profile of one to several months.3 Since its successful application for luteinizing hormone releasing hormone (LHRH) microsphere technology by the Lupron Depot® system, buserelin acetate, triptorelin pamoate, octreotide acetate, lanreotide, and risperidone sustained release systems have been commercialized by using PLGA.4 On the other hand, polycaprolactone (PCL) has remained almost forgotten in the last few decades as a biomaterial and has only returned recently as a tissue engineering scaffold and three-dimensional printing material due to its low melting point and facile processibility into specific shapes.5,6 PCL is a material that degrades very slowly, with up to two to three years required for complete degradation under in-vivo conditions.5 One successful example of the use of PCL as a drug delivery carrier is the Capronor® implant for contraceptive drug delivery for two years.7 As compared with PLGA, PCL also has the advantages of an inexpensive production cost. PCL exhibits a two-phase of degradation profile under in-vivo conditions.8,9 First, hydrolysis of the polymer occurs for its transformation to a low-molecular-weight oligocaprolactone (OCL). During degradation, the crystallinity increases because OCL tends to have higher crystallinity than high molecular weight PCL. Then, the OCL with a molecular weight of less than 3,000 Daltons undergoes phagocytosis by phagosomes of macrophage and giant cells, followed by intracellular degradation by esterases.10,11 Oxalate ester bonds are degraded by hydrogen peroxide via dioxetanedione as a reactive intermediate.12 The reactive intermediate instantaneously decomposes into carbon dioxide.


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Recently, polyoxalate was reported as a novel polymeric prodrug for ischemia/reperfusion treatments and as an anticancer drug delivery system which involve the accumulation of reactive oxygen species (ROS) including hydrogen peroxide.13-17 The aromatic oxalate polymers were degraded by ROS, and antioxidants which are the components of the polymers were released from the polymers. ROS can also build up by the subcutaneous injection of microspheres through the acute and chronic inflammation.18,19 Based on the above considerations, we synthesized a PCL multiblock copolymer, PCL-OX as a fast degradable PCL, wherein OCLs are connected with oxalate bonds. OCL with a molecular weight of 2,000 Daltons was used. Microspheres encapsulating a model drug, paclitaxel, were prepared, and the degradation behaviors of the PCL-OX and the relevant drug release profile were investigated under in-vitro and in-vivo conditions. Paclitaxel is commonly administered by intravenous constant infusion into the blood stream over 3 or 24 hours after dilution of a 1/1 mixture of ethanol and Cremophor El in water every three weeks.20 Due to its narrow therapeutic window and poor water solubility, PLGA-based paclitaxel encapsulating drug delivery systems including micelles, nanoparticles and microspheres are actively being researched.21-24 PLGA microspheres were also prepared by use of the same method as a solely comparative system with PCL-OX microspheres for degradation, drug release behaviors, and tissue compatibility.

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EXPERIMENTAL SECTION Materials. Oligocaprolactone diol (OCL) (average Mn = 2,000 Daltons) (Sigma-Aldrich,

USA) and PLGA (Evonik, Germany) were used as received. The PLGA consists of 50/50 DL-


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lactide/glycolide (by mole). The molecular weight and inherent viscosity of the PLGA (0.10 wt.% in chloroform) were 13,700 Daltons and 1.6 dl/g, respectively. Oxalyl chloride was used as received from Alfa Aesar (USA). Toluene (Daejung, Korea) was distilled over sodium before use and trimethylamine (Sigma-Aldrich, USA) was dried by potassium hydroxide before use. Polyvinyl alcohol (PVA) (86 to 89% hydrolyzed, average molecular weight 57,000–66,000) and paclitaxel were purchased from Alfa Aesar (USA) and were used as received. Synthesis of PCL-OX. PCL-OX was synthesized with a coupling reaction of OCL 2000 and oxalyl chloride. The OCL (20.00 g, 10 mmol; M.W.=2,000 Daltons) was dissolved in anhydrous toluene (100 ml) and the residual water was removed by azeotropic distillation. After the reaction mixture was cooled down to 20 oC, 100 ml of dry toluene was added as solvent. Oxalyl chloride (0.872 ml, 10 mmol) was added to the reaction flask. Subsequently, dry trimethylamine (2.788 ml, 20 mmol) in 30 ml of dry toluene was slowly dropped into the reaction flask. The mixture was stirred at 20 oC for 20 hours under dry nitrogen conditions. Then, triethylammonium chloride salt was removed by filtration, and the product was purified by precipitation into diethyl ether. The residual solvent was removed under high vacuum. The yield was about 85%. Characterization of PCL-OX. PCL-OX was characterized by 1H-NMR spectroscopy (500 MHz NMR spectrometer; Varian, USA) and gel permeation chromatography (GPC) (SP930D; Younglin, Korea). Chloroform-D and tetrahydrofuran was used for solvent for 1HNMR and GPC analysis. Intrinsic Viscosity of PCL-OX. The intrinsic viscosity of PCL-OX was obtained by extrapolating inherent viscosity versus concentration in a range of 0.01–0.10 g/dl.


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Differential Scanning Calorimetry. The differential scanning calorimeter (DSC) thermograms of PCL-OX and OCL were obtained by DSC (N-650; Scinco, Korea). The heating and cooling rate was 1.0 oC/min, and the second heating curves were recorded. Microsphere Preparation. Microspheres were prepared by the oil-in-water single emulsion method.25 PCL-OX (1.90 g) and paclitaxel (0.10 g) were dissolved in dichloromethane (20.0 ml). The solution was slowly injected into an aqueous polyvinyl alcohol solution (1.0 wt.%) and emulsified with a homogenizer (VCX750; Sonics & Materials Inc., USA) at 20 oC. The solution was then homogenized for three hours at 20 oC. Dichloromethane was evaporated and paclitaxel loaded microspheres were prepared. The microspheres were collected by centrifugation at 1,000 rpm for two minutes, washed with distilled water four times, and freezedried. All of the procedures were carried out at 5–10 oC to prevent aggregation of the microspheres. PLGA microspheres were prepared by the same method, except that the concentration of PLGA (2.85 g) and paclitaxel (0.15 g) in methylene chloride (20 ml) was higher to control the size of the microspheres. Encapsulation Efficiency and Drug Loading Content. Microspheres (1.0 mg) were accurately weighed and dissolved in 1.0 ml of methylene chloride. The amount of loaded paclitaxel was analysed by use of a high-performance liquid chromatography (HPLC) system (Waters 1525B, USA) with a photodiode detector (Waters 2998, USA). The paclitaxel concentration was determined at a wavelength of 227 nm. A Jupiter® 5 µm C18 300A LC column (5 µm, 4.6 mm x 250 mm) was used. Acetonitrile/water 1:1 (v/v) was used as a mobile phase and the flow rate was 1.0 ml/min. The drug-loading content (LC) and encapsulation efficiency (EE) were calculated by using the following equations; LC (%) = 100 x wt. of drug/(wt. of drug + wt. of polymer)

(1)


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EE (%) = 100 x actual drug loaded/theoretical drug loaded

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(2)

Scanning Electron Microscopy. The scanning electron microscopy (SEM) images of the drug-encapsulating microspheres were obtained by using a scanning electron microscope (JSM-6700F; JEOL, Japan) at an accelerating voltage of 10 kV. In-Vitro Drug Release. Paclitaxel-loaded microspheres (1.0 mg) were suspended in phosphate buffered saline (PBS; 0.3 ml, pH=7.4, 150 mM). The aqueous suspension was injected into a dialysis bag (cutoff molecular weight: 10 KDaltons). The bag was placed in a vial, and PBS (3.0 ml) containing tween 80 (0.10 % w/v) was added as a release medium, where the membrane bag is fully immersed in the release medium. Triplicate experiments were done for each system. The vials were shaken at a rate of 30 strokes/minute at 37℃. The released medium (3.0 ml) was collected and the whole medium was replaced by a fresh one at the specific sampling time interval. The drug concentration was analyzed by HPLC. In-Vitro Degradation of Polymers. Paclitaxel-loaded microspheres (1.0 mg) were suspended in PBS (0.3 ml, pH=7.4, 150 mM) without tween 80. All of the procedures conducted were the same as in the in-vitro drug release experiment except that PBS without tween 80 was used as a medium. The polymers were collected at 7 and 28 days of incubation, and were analyzed by GPC. In addition, microsphere degradation was investigated as a function of hydrogen peroxide concentration (0, 10, and 100 mM) in the PBS over three days. In-Vivo Drug Release. Four-week old male Sprague-Dawley rats were purchased from Central Lab (Animal Inc., Korea) and stabilized for five days. Paclitaxel-loaded microspheres (1.0 mg) were suspended in PBS (0.3 ml, pH=7.4, 150 mM). The aqueous suspension was then injected into the subcutaneous layer of the rats. Triplicate experiments was done for each system.


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All rats were freely given for food and water. The plasma concentration of paclitaxel was analyzed by HPLC. In-Vivo Degradation of Polymers and Histocompatibility. The degradation of the polymers was traced by using the GPC profile of the polymers recovered from the subcutaneous layer of the rats. The histocompatibility of the microspheres was analysed by using the haematoxylin and eosin (H&E) staining method around the implant site of the microspheres in the rats. Animal Procedure. In accordance with the Institutional Animal Care and Use Committees (IACUC) guidelines, all the procedures using animals were approved by the Committee of Ewha Womans University.

■ RESULTS AND DISCUSSION

PCL-OX, a multiblock copolymer of OCL oxalate, was synthesized by reacting α,ω-dihydroxy OCL and oxalyl chloride.

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H-NMR spectra (Figure 1) exhibited the appearance of a

characteristic ɛ-methylene peak (4.2− 4.3 ppm; ɛ” in Figure 1) of caprolactone next to the oxalate group and the disappearance of ɛ-methylene peak (3.6− 3.7 ppm; ɛ’ in Figure 1) of OCL next to the hydroxyl end groups. The δ-methylene peak of terminal caprolactone of OCL also showed a similar change in the chemical shift from 1.5− 1.6 ppm to 1.7− 1.8 ppm.


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Figure 1. 1H-NMR spectrum (CDCl3) of PCL-OX. Oligocaprolactone (OCL) spectrum is also shown for comparison. δ‘ and ɛ’ come from the end repeating units of OCL. δ” and ɛ” come from the end repeating units of OCL connected to oxalate bonds.

The retention time in the GPC chromatogram changed from 7.4 minute (OCL) to 4.5 minute (PCL-OX) (Supporting Information: Figure S1). Uniform distribution of the molecular weights suggests that the PCL-OX is well-synthesized. The molecular weight (Mw) against poly(ethylene glycol) standards and the polydispersity index of PCL-OX were 97,450 Daltons and 2.0, respectively. The molecular weight of PCL-OX was also determined by solution viscometry by using tetrahydrofuran as a solvent. Intrinsic viscosity determined from an extrapolated line of inherent viscosity versus concentration was 1.26 dl/g (Supporting Information: Figure S2). From the Mark-Houwink-Sakurada equation (Equation 3), the viscosity average molecular weight of the PCL-OX can be determined.


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[η] = kMva

(3)

where k=14.1 x 10-5 and a=0.79 in tetrahydrofuran for PCL.26 Viscosity average molecular weight of PCL-OX was determined to be 100,300 Daltons, which is similar to the weight average molecular weight (Mw) determined by GPC. The DSC thermogram shows that the melting of the PCL-OX and OCL are observed in 32.2− 50.2 oC (Tpeak = 44.7 oC) and 33.1− 55.7 oC (Tpeak = 50.7 oC), respectively. Melting point is important in practical applications because it affects the biodegradability of microspheres as well as the permeability of incorporated drugs. In addition, microspheres can be fused by interface diffusion when the application or formulation temperatures are higher than the melting point. The enthalpy of melting for PCL-OX and OCL was determined to be 61.0 J/g and 94.6 J/g, respectively. The enthalpy of melting for 100% crystalline PCL (ΔHm,0) was reported to be 139.5 J/g.27 The crystallinity (χc) of PCL-OX can be calculated by using Equation 4.28 χc = ΔHm/ΔHm,0

(4)

Therefore, the crystallinity (χc) of PCL-OX was calculated to be 43.7 %, which is similar to that of PCL with 100,000 Daltons. The crystallinity of PCL is reported to decrease from 80% to 40 % as their molecular weight increases from 5000 to 100,000 Daltons.26


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Figure 2. DSC thermograms of PCL-OX and OCL. The second heating curves are shown. The heating rate was 1.0 oC/min.

Paclitaxel-encapsulating PCL-OX microspheres were prepared by the oil-in-water emulsion method.25 For comparison, paclitaxel-encapsulating PLGA microspheres with a similar size were also prepared. SEM images showed the microspheres with a size of 10–130 µm (Figure 3). The loading contents of the PCL-OX and PLGA microspheres were 4.4 % and 4.9 %, respectively. The drug encapsulation efficiencies of the PCL-OX and PLGA microspheres were 81.4 % and 94.3 %, respectively.


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Figure 3. a) SEM images of PCL-OX and PLGA microspheres encapsulating paclitaxel. The scale bar is 100 µm. b) Size distribution of the microspheres.

In-vitro experiments revealed that 47 % and 85 % of the paclitaxel was released from the PCL-OX and PLGA microspheres, respectively, over 28 days without a significant initial burst release (Figure 4a). A faster release profile for the PLGA microspheres was observed in the invitro experiment by using PBS containing tween 80 (0.10 % wt/v) as a drug release medium. The in-vivo experiments were carried out by injecting the paclitaxel-containing microspheres into the


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subcutaneous layer of rats. A steady plasma drug concentration (Cp) of 6-9 µg/ml was observed for the PCL-OX and PLGA microspheres over 28 days (Figure 4b). The constant plasma drug concentration is very important for the drug with a narrow therapeutic window like paclitaxel. To realize the constant Cp value of a drug, microsphere size and polymer structure should be carefully controlled because the size of the microsphere and chemical structure of PLGA affect the drug release profile.29,30 In current study, a constant plasma drug concentration of paclitaxel was realized over 28 days by using PCL-OX microspheres as well as PLGA microspheres with a 10–130 µm in size. pH can decrease around the implant site due to ROS production, which might increase the degradation of polyesters, and thus increases drug release rate from the microspheres.31-33 Oxalate bonds are reported to be preferentially cleaved by ROS.12-17 Due to these reasons, the in vivo release of paclitaxel from the PCL-OX might be accelerated, and thus increased a plasma concentration of the paclitaxel from the PCL-OX microspheres.


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Figure 4. a) In-vitro drug release profiles from PCL-OX and PLGA microsphere. N=3. b) Plasma drug concentration of the paclitaxel after subcutaneous injection of the PCL-OX and PLGA microspheres into the subcutaneous layer of rats. N=3.

The degradation characteristics of the PCL-OX and PLGA microspheres were compared from in-vitro and in-vivo experiments by gel permeation chromatography. The in-vivo degradation of PCL-OX was much more accelerated than the in-vitro degradation, with the appearance of new peaks at 5.0-7.0 minutes in chromatogram (Figure 5a). On the other hand, previous studies reported that degradation of PLGA under in-vivo conditions is similar to that under in-vitro conditions.34-36 Degradation of PLGA microspheres often results in core-first degradation due to the build-up of acidic conditions inside of the microspheres, which leads to the autocatalysis mechanism of the degradation.37 Oxalate bonds are reported to be degraded by hydrogen peroxide, as was discussed in the introduction section. In addition, ROS build up in the implanted site of the microspheres because the inflammatory cells release various as ROS as a host defense mechanism.19,38,39 To confirm the possibility of degradation of PCL-OX by ROS,


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in-vitro degradation of PCL-OX was investigated as a function of hydrogen peroxide concentration. The biological concentration of hydrogen peroxide are 50-100 µM.40,41 During inflammation, ROS level increases.42 In the current study, we investigate the degradation of PCL-OX at higher concentrations (0, 10, and 100 mM) over three days as an accelerated study to prove the involvement of the ROS in polymer degradation.43,44 The GPC profile clearly demonstrated that the degradation of PCL-OX is accelerated as the concentration of hydrogen peroxide increased (Figure 5b). However, the degradation of PLGA was not sensitive to the concentration of hydrogen peroxide in the same concentration range of 0–100 mM (Supporting Information: Figure S3). Therefore, we can conclude that the accelerated drug release from the PCL-OX microspheres in the in-vivo conditions than in-vitro conditions is related to the ROStriggered degradation of PCL-OX, as will be confirmed next section on the accumulation of inflammatory cells around PCL-OX implant.


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Figure 5. a) GPC chromatogram of PCL-OX 7 days (-7D) and 28 days (-28D) of incubation of the microspheres in PBS at 37 oC (in-vitro: blue curves) and in the subcutaneous layer of rats (invivo: red curves). b) GPC chromatogram of PCL-OX 3 days of incubation in PBS at 37 oC as a function of hydrogen peroxide. The legends are concentration of hydrogen peroxide in PBS.

Tissue compatibility of the PCL-OX microspheres was compared with that of the PLGA microspheres by H & E staining around the implant site in the subcutaneous layer of rats (Figure 6). The inflammatory cells (stained in dark purple) of lymphocytes and macrophages aggregated around the implant site on day 7. On day 28, the number of inflammatory cells was significantly decreased, whereas the interfaces between polymers and tissues were replaced by the collagen capsule (stained in red). Both PCL and PLGA systems were completely cleared two months after in-vivo implantation. The aggregation of inflammatory cells, collagen capsule formation, and clearance of polymers are typical mild inflammatory sequences of biodegradable polymers after in-vivo implanation.45 After implantation of PLGA microspheres by subcutaneous injections, the


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release of acidic oligomers with carboxylic acid end groups lead to the inflammation in the vicinity of the microspheres. The inflammatory cells generally increases in number to a maximal value and decreases with chronic responses.35 A similar collagen capsule was also observed around the PCL-OX and PLGA microsphere implants on day 28.

Figure 6. H&E stained images around the implanted microspheres. The images 7 (-7D) and 28 (28D) days after subcutaneous injection of the PCL and PLGA microspheres are shown.

■ CONCLUSIONS

An oxalate-connected multiblock copolymer, PCL-OX, was synthesized as a fast degradable PCL. PCL-OX exhibited a melting point of 44.7 oC and a crystallinity of 40 %. Paclitaxel-


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encapsulating PCL-OX and PLGA microspheres with 10-130 µm in size were prepared and their drug release and degradation behaviours were compared. Paclitaxel was released in a slower rate from the PCL-OX microspheres than from the PLGA microspheres under in-vitro conditions by using PBS as a medium. However, the in-vivo release profiles or plasma drug concentrations from the PCL-OX microspheres and the PLGA microspheres were similar over 28 days. PCLOX degradation was accelerated more under in-vivo conditions than under in-vitro conditions. Both PCL and PLGA systems were completely cleared two months after in-vivo implantation. Hydrogen peroxide triggered the degradation of oxalate bonds in the PCL-OX, and is supposed to play a role in the process of degradation of and drug release from the PCL-OX under in-vivo conditions. Due to its less expensive production cost and viable potential in tissue engineering applications, PCL has begun to draw attention as a biomaterial. This paper supports that the slow degradation problems of PCL can be solved by incorporation of bioactive functional groups of oxalate bonds in the polymer backbone. In particular, the biosignals of ROS-triggered degradation of current PCL provides additional advantages of storage stability of the material as well as degradability after in-vivo application. The oxalate-connected PCL is expected to be an alternative to PLGA as a biodegradable polymer.

■ ASSOCIATED CONTENTS

Supporting Information The Supporting Information is available free of charge via the Internet at http://pubs.acs.org/. Gel permeation chromatogram of PCL-OX and OCL. Inherent viscosity versus concentration of


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PCL-OX. GPC profile of PLGA after 3 days of incubation in PBS at 37 oC as a function of hydrogen peroxide.

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AUTHOR INFOEMATION

Corresponding Author *Email: [email protected], Fax: +82 2 3277 2384. Author Contributions

SH Chang and Y Kim performed polymer synthesis and characterization. SH Chang did microsphere preparation, drug delivery, and degradation study. HJ Lee did the in-vivo assay. B Jeong designed the concept and wrote the paper.

Notes The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (2017R1A2B2007356, 2017R1A5A1015365, 2014M3A9B6034223).

■ REFERENCES


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(1) Nair, L. S.; Laurencin, C. T. Biodegradable Polymers as Biomaterials. Prog. Polym. Sci. 2007, 32, 762–798. (2) Dash, T. K.; Konkimalla, V. B. Poly-є-caprolactone Based Formulations for Drug Delivery and Tissue Engineering: A Review. J. Controlled Release 2012, 158, 15–33. (3) Shrama, S.; Parmar, A.; Kori, S.; Sandhir, R. PLGA-based Nanoparticles: A New Paradigm in Biomedical Applications. Trac-Trends Anal. Chem. 2016, 80, 30-40. (4) Mundargi, R. C.; Babu, V. R.; Rangaswamy, V.; Patel, P.; Aminabhavi, T. M. Nano/micro Technologies for Delivering Macromolecular Therapeutics Using Poly(D,L-lactide-co-glycolide) and Its Derivatives. J. Controlled Release 2008, 125, 193–209. (5) Woodruff, M. A.; Hutmacher, D. W. The Return of a Forgotten Polymer—Polycaprolactone in the 21st Century. Prog. Polym. Sci. 2010, 35, 1217–1256. (6) Williams, J. M.; Adewunmi, A.; Schek, R. M.; Flanagan, C. L.; Krebsbach, P. H.; Feinberg, S. E.; Hollister, S. J.; Das, S. Bone Tissue Engineering Using Polycaprolactone Scaffolds Fabricated via Selective Laser Sintering. Biomaterials 2005, 26, 4817–4827. (7) Ma, G.; Song, C.; Sun, H.; Yang, J.; Leng, X. A Biodegradable Levonorgestrel-Releasing Implant Made of PCL/F68 Compound As Tested in Rats and Dogs. Contraception 2006, 74, 141–147. (8) Woodward, S. C.; Brewer, P. S.; Moatamed, F.; Schindler, A. Pitt, C. G. The Intracellular Degradation of Poly(epsilon-caprolactone). J. Biomed. Mater. Res. 1985, 19, 437–444. (9) Sun, H., Mei, L.; Song, C.; Cui, X.; Wang, P. The In Vivo Degradation, Absorption and Excretion of PCL-based Implant. Biomaterials 2006, 27, 1735–1740.


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P.

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Nanoparticles

as

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Fast Degradable Polycaprolactone for Drug Delivery

Seo Hee Chang, Hyun Jung Lee, Sohee Park, Yelin Kim, and Byeongmoon Jeong*

Department of Chemistry and Nanoscience, Ewha Womans University, 52 Ewhayeodae-gil, Seodaemun-gu, Seoul, 03760, Korea


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Fast Degradable Polycaprolactone for Drug Delivery - ACS Publications

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