Polyoxalate Nanoparticles as a Biodegradable and Biocompatible

Jan 29, 2010 - One of major challenges in the drug delivery lies in the development of nanoparticles that are effectively delivered to targeted cells ...
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Biomacromolecules 2010, 11, 555–560

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Polyoxalate Nanoparticles as a Biodegradable and Biocompatible Drug Delivery Vehicle Seho Kim,†,‡ Kyeongyeol Seong,†,‡ Onyou Kim,‡ Soojin Kim,‡ Hansol Seo,‡ Myunghoon Lee,‡ Gilson Khang,‡,§ and Dongwon Lee*,‡ Department of Polymer · Nano Science and Technology, Polymer Fusion Research Center, and Department of BIN Fusion Technology, Chonbuk National University, Dukjin, Jeonju, Korea, 561-756 Received August 17, 2009; Revised Manuscript Received January 18, 2010

One of major challenges in the drug delivery lies in the development of nanoparticles that are effectively delivered to targeted cells and release their payload over an extended period to achieve a clinical response. In this paper, we report a new family of biocompatible and biodegradable polymer, termed polyoxalate that degrades hydrolytically into nontoxic byproducts. Polyoxalate was synthesized from a simple one-step polymerization reaction of 1,4cyclohexanedimethanol and oxalyl chloride and had a MW of ∼11000 Da. This polymer was designed to degrade by water hydrolysis into 1,4-cyclohexanedimethanol and oxalic acid, which can be easily removed from a body. Polyoxalate had a hydrophobic backbone and was formulated into nanoparticles with a mean diameter of 600 nm, which is suitable for drug delivery involving phagocytosis by macrophages. Polyoxalate nanoparticles were readily taken up by RAW 264.7 macrophage cells and HEK (human embryonic kidney) 293 cells and exhibited a minimal cytotoxicity in a time- and dose-dependent manner. In comparison with PLGA nanoparticles, polyoxalate nanoparticles had a significantly higher cell viability. We anticipated that the ease of synthesis and excellent biocompatibility make polyoxalate highly potent for numerous applications in drug delivery.

Introduction Nanotechnology approaches where a constant dose of drugs is delivered directly to the diseased sites or cells for an extended period may result in alternative therapeutic opinion for patients. The challenge lies in the design of nanoparticles that are effectively delivered to the targeted cells and release their payload over an extended period to achieve a clinical response. Therefore, over the past two decades, there has been considerable interest in the development of biodegradable polymeric nanocarriers as effective drug delivery vehicles.1-3 Polymeric drug delivery vehicles include polymer nanoparticles, block copolymer micelles, dendrimers, and hydrogels.4-6 Numerous polymers have been extensively used in the pharmaceutical research because they are capable of delivering drugs to a target site and improve the pharmacological and therapeutic properties of drugs, while minimizing adverse side effects.7 Various drug delivery vehicles, composed of biodegradable polymers, have also been designed for enhanced serum stability, biocompatibility and in vivo circulation time. Polymer nanoparticles, primarily based on poly(D,L-lacticco-glycolic acid), (PLGA) and polycaprolactone have been widely utilized for delivery of diverse therapeutic agents such as proteins, peptides, nucleic acids, and water insoluble small molecular drugs.4,6,8-10 They are especially useful for delivering drugs requiring continuous and sustained release with a single bolus administration because of their excellent biocompatibility and biodegradability through natural pathways. These polyesterbased nanoparticles also exhibit an excellent shelf life, suitable physicochemical properties, and well-characterized degradation products. However, their applications are potentially problematic * To whom correspondence should be addressed. Tel.: 82-63-270-2344. Fax: 82-63-270-2341. E-mail: [email protected]. † Both authors contributed to this work equally. ‡ Department of Polymer · Nano Science and Technology. § Department of BIN Fusion Technology.

for infection and inflammation-associated diseases because of their acidic degradation products, which can lead to the pH values and cause inflammation.7,11 Slow hydrolysis kinetics also limits their applications for the treatment of inflammatory diseases. Consequently, there is great interest in developing new strategies for the synthesis of noninflammatory and biodegradable polymers as a drug delivery vehicle, which can degrade into nontoxic compounds.2,12,13 Previously, hydrophobic polyoxalates have been suggested for the biodegradable medical devices such as absorbable sutures and controlled release. The polyoxalates were prepared by the ester interchange reaction of diols with ester of oxalic acid, preferably diethyl oxalate in the presence of catalyst such as stannous octoate.14 Copolyoxalates were also prepared using a mixture of cyclic and linear diols. Polyoxalate and copolyoxalate were synthesized from a complicate two step reaction, wherein the reactants were first heated with stirring under a nitrogen atmosphere to form a prepolymer with the removal of ethanol, followed by postpolymerization under heat and reduced pressure to obtain a final polymer. It was reported that polyoxalate and copolyoxalates show mild and slight tissue reactions during the first five days of in vivo implantation and could be used for controlled release of steroids and narcotic antagonists.15 Despite the potential of polyoxalates and copolyoxalates as biodegradable medical devices, there is no indication of nanoparticle formulation and actual release work. In this study, we developed new biodegradable hydrophobic polymer nanoparticles composed of polyoxalate that has peroxalate ester linkages in its backbone. Polyoxalate synthesized using hydrophobic cyclic diols was hydrophobic and therefore could be formulated into nanoparticles via an emulsion procedure. Peroxalate ester linkages on this polymer can be cleaved by water hydrolysis, leading to the degradation of polymers into a nontoxic low molecular weight substance that can be easily excreted. Herein, we report the synthesis and physicochemical

10.1021/bm901409k  2010 American Chemical Society Published on Web 01/29/2010

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Scheme 1. Synthesis and Degradation of Polyoxalate

properties of polyoxalate and demonstrate its potential as a new family of biodegradable drug delivery vehicles.

Experimental Section Polymer Synthesis. 1,4-Cyclohexanedimethanol (16.6 mmol) was dissolved in 14 mL of dry dichloromethane (DCM), under nitrogen, to which triethylamine (43 mmol) was added dropwise at 4 °C. Oxalyl chloride (16.6 mmol) in 4 mL of dry DCM was added to the mixture dropwise at 4 °C. The reaction was kept under a nitrogen atmosphere at room temperature for 8 h, quenched with a saturated brine solution, and extracted with additional DCM. The combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum. The obtained polymer was isolated by precipitation in cold hexane. The molecular weight was determined to be ∼11000 (polydispersity ) 1.8) by a gel permeation chromatography (GPC) using polystyrene standards. The chemical structure of polymers was identified with a 400 MHz 1H NMR spectrometer (JNM-EX400 JEOL). 1H NMR in deuterated chloroform on a 400 MHz spectrometer: 4.1-4.5 (m, CH2-OCO), 1.8-2.0 (m, CH2CH), 1.4-1.6 (m, CH2CH2CHCH2CH2CH), 1.0-1.1 (m, CH2CH2CHCH2CH2CH). 13C NMR in deuterated chloroform on a 100.53 MHz spectrometer: δ 25.5, 28.4, 34.0, 36.6, 69.4, 71.4, 157.9. Nanoparticle Preparation. A total of 50 mg of polymers dissolved in 500 µL of DCM was added to 5 mL of 10 (w/v)% poly(vinyl alcohol) solution. The mixture was sonicated using a sonicator (Fisher Scientific, Sonic Dismembrator 500) for 30 s and homogenized (PRO Scientific, PRO 200) for 1 min to form a fine oil/water emulsion. The emulsion was added into 20 mL PVA (1 w/w%) solution and homogenized for 1 min. The remaining solvent was removed by rapid stirring for at least 3 h. Nanoparticles were obtained by centrifuging at 11000 × g for 5 min at 4 °C, washing with deionized water twice, and lyophilizing the recovered pellet. The SEM images of peroxalate nanoparticles were made using a scanning electron microscope (S-3000N, Hitachi). Rhodamine Release Kinetics. Rhodamine was encapsulated in the polyoxalate nanoparticles using single emulsion procedures. A total of 20 mg of rhodamine-loaded nanoparticles was placed in a test tube containing 10 mL of PBS (phosphate buffer solution, pH 7.4). The tube was continuously shaken and incubated at 37 °C. At appropriate intervals, the tube was centrifuged at 5000 × g for 1 min. A 2 mL aliquot of supernatant was periodically taken and replaced with an equal

volume of fresh PBS. The concentration of rhodamine in the supernatant was measured using a fluorospectrometer (Jasco, FP6000) by comparison with the standard calibration curve prepared with known concentrations of rhodamine solutions. Cell Toxicity of Nanoparticles. The cytotoxicity of polyoxalate nanoparticles was investigated using a 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) reduction assay. RAW 264.7 macrophage cells were seeded at a density of 1 × 106 cells/well in a 24 well plate and incubated for 24 h to reach ∼80% confluency. Cells were treated with various amounts of nanoparticles (10 µg/mL to 100 µg/mL) and incubated for 1, 2, and 3 days. Each well was given 100 µL of MTT solution and was incubated for 4 h. Dimethyl sulfoxide (DMSO, 1000 µL) was added to cells to dissolve the resulting formazan crystals. After 10 min of incubation, the absorbance at 570 nm was measured using a microplate reader (Thermolex, Molecular Device Co.). The cell viability was obtained by comparing the absorbance of nanoparticles-treated cells to that of control cells. Cellular Internalization. The uptake of rhodamine-loaded nanoparticles was studied in living RAW264.7 cells and HEK 293 cells using a confocal laser scanning microscope. Freshly prepared nanoparticles were diluted to 1 mg/mL with PBS. Cells were treated with 50 µL of nanoparticle suspension and incubated at 37 °C for 15 min. Nanoparticles containing medium were removed and cells were washed with fresh media twice. Fluorescent images were made using a confocal laser scanning microscope (Carl Zeiss Inc.) at 3 h postincubation.

Results and Discussion Polyoxalate was synthesized from a one-step condensation polymerization reaction between oxalyl chloride and 1,4-cyclohexanedimethanol. It has been reported that aromatic peroxalate ester derivatives are highly labile in water, and peroxalate polymers with aromatic rings adjacent to peroxalate ester linkages are highly reactive to hydrogen peroxide but are rapidly hydrolyzed by water.16-19 These polymers containing a highly labile aromatic peroxalate ester are not suitable as a drug delivery vehicle. In this work, polyoxalate was synthesized using an aliphatic diol, 1,4cyclohexanedimethanol, which has poor electron-withdrawing properties, thus, increasing their stability in the aqueous environ-

Figure 1. Characterization of polyoxalate synthesized from the reaction of 1,4-cyclohexanedimethanol and oxalyl chloride. (A)1H NMR spectrum of peroxalate polymer; (B) 13C NMR spectrum of peroxalate polymer.

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Figure 2. GPC trace of polyoxalate in THF.

Figure 4. SEM image of polyoxalate nanoparticles.

Figure 3. Hydrolysis kinetics of polyoxalate.

Figure 5. Release kinetics of rhodamine from polyoxalate nanoparticles.

ment.18 Therefore, we reasoned that polyoxalate synthesized using aliphatic 1,4-cyclohexanedimethanol is hydrophobic and stable against water hydrolysis but can degrade by hydrogen peroxide and water on a time scale that is faster than PLGA. 1,4Cyclohexanedimethanol is widely used food packing materials and has approval for human consumption as an indirect food additive.11,20 Moreover, this compound has an excellent toxicity profile and does not undergo significant enzymatic transformations in vivo. Here, we demonstrate the potential of hydrophobic polyoxalate nanoparticles as a new family of biodegradable polymers for drug delivery applications. Scheme 1 illustrates the synthetic route of representative condensation polymerization of 1,4-cyclohexanedimethanol and oxalyl chloride. The polymerization of these monomers proceeded in dry DCM under nitrogen atmosphere to generate the corresponding polymers in ∼60% yield. The polymers were purified through repeated precipitation into cold hexane. Polyoxalate was obtained as pale yellow solids after drying under high vacuum. The chemical structure of polyoxalate was confirmed by the 1H NMR. Methylene protons next to hydroxyl groups of 1,4-cyclohexanedimethanol appear at 3.5 ppm (data not shown). The large peaks at ∼4.1 ppm correspond to the methylene protons adjacent to oxalate ester linkages, demonstrating the condensation reaction between 1,4cyclohexanedimethanol and oxalyl chloride to generate polyoxalate containing peroxalate ester linkages in the backbone (Figure 1). The chemical structure of polyoxalate can be further confirmed by the 13C NMR spectrum showing oxalate carbons at 157 ppm and methylene carbons next to oxalate esters at ∼70 ppm. Polyoxalate obtained from this reaction had a MW of ∼11000, corresponding to a degree of polymerization of ∼50 repeating units (Figure 2), with a polydispersity index of 1.8 and, therefore, has potential for formulation into nanoparticles. Polyoxalate is expected to readily degrade by water hydrolysis into oxalic acid and the monomeric compound, 1,4-cyclohexanedimethanol, which can be easily removed from a body. It

has been reported that peroxalate ester linkages in the polyoxalate and oxalic acid readily react with hydrogen peroxide to generate carbon dioxide.16,19,21,22 Hydrogen peroxide is known to be overproduced in inflammatory cells, in particular, macrophages and neutrophiles in inflammation associated diseases such as asthma, arthrosclerosis, cancer, and neurodegenerative diseases.16,23 Therefore, it is reasonable to expect that synthesized polyoxalate degrades into cyclohexanedimethanol, oxalic acid, and carbon dioxide in the inflammatory macrophages. DiVincenzo et al. reported that 97.5% of 1,4-cyclohexanedimethanol in rats was excreted intact after administration by gavage in doses of 40 or 400 mg/kg.13,24 This attribute also makes polyoxalate highly useful for drug delivery applications. The hydrolysis kinetics of polyoxalate was measured under physiological conditions. The degradation of polyoxalate by water hydrolysis was investigated by grinding the polymers into a fine powder and measuring the molecular weight change with time using GPC. The kinetics of polyoxalate degradation was investigated under the range of conditions likely to be encountered by polyoxalate nanoparticles upon phagocytosis. Degradation of polyoxalate was monitored at 37 °C at buffered pH values of 5.0 and 7.4 to appropriate the pH of the environments within the endosomal vesicles and the cytoplasm, respectively.25 Figure 3 demonstrates that polyoxalate degrades hydrolytically, having a half-life of ∼6.5 days at both pH 7.4 and pH 5.0. The hydrolysis of polyoxalate was pH-independent. Polyoxalate seems to degrade faster than PLGA, which has a half-life of a couple of weeks.26,27 The result demonstrates that aliphatic polyoxalate exhibited sufficient hydrophobicity and stability for nanoparticle formation and drug encapsulation. Polyoxalate nanoparticles were prepared using an oil-in-water emulsion method. The particle size and morphology were investigated using a scanning electron microscope (SEM). The SEM image of polyoxalate nanoparticles demonstrates that they are round spheres with a smooth surface with an average diameter of ∼600

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Figure 6. Cytotoxicity polyoxalate nanoparticles by an MTT assay. Cell viability was normalized by the value determined in untreated cells. **P < 0.01 (relative to PLGA at the same dose, n ) 6).

Figure 7. Confocal fluorescence micrographs of RAW 264.7 and HEK 293 cells incubated with Rhodamine-loaded polyoxalate nanoparticles. Cells of 1 × 106 were treated with 100 µL of rhodamine-loaded nanoparticles (1 mg/mL). Free rhodamine was removed by centrifuging rhodamineloaded polyoxalate nanoparticles before the addition to cells. Before the images were captured, cells were washed with fresh culture media. Images were captured 3 h after the addition of nanoparticles.

nm (Figure 4). Polyoxalate nanoparticles in this range may be suitable for intracellular and extracellular drug delivery applications. In particular, they are suitable for drug delivery involving phagocytosis by macrophages, as macrophages readily phagocytose foreign matter in the range of 0.5-3 µm.20

To determine whether polyoxalate has suitable drug release profiles for treating inflammatory diseases, a release study was performed using rhodamine-loaded polyoxalate nanoparticles. Rhodamine-loaded nanoparticles were formulated using the same procedure as stated above. Rhodamine encapsulation

Polyoxalate Nanoparticles as a Drug Delivery Vehicle

efficiency was determined to be 12% by a fluorospectrometer. Rhodamine release characteristic of polyoxalate nanoparticles is presented in Figure 5. Rhodamine release began with an initial burst, as high as ∼60% on the first day. Half the rhodamine was released at approximately 12 h. The initial burst release represented the release of rhodamine that was loosely adsorbed on the surface or poorly entrapped in the microparticles. Then, rhodamine was released in a slow and continuous manner, reaching 75% release at day 3, as the physically entrapped rhodamine slowly diffused out through tortuous channels in the particles for release. We also evaluated the cytotoxicity of polyoxalate nanoparticles by an MTT assay. RAW 264.7 cells were incubated with a various amount of polyoxalate nanoparticles for 1, 2, and 3 days. For comparison purposes, PLGA (MW 8000) nanoparticles were also prepared and their cytotoxicity was compared to polyoxalate nanoparticles. Figure 6 illustrates the comparison of cytotoxicity between polyoxalate nanoparticles and PLGA nanoparticles at various time points. Both polyoxalate and PLGA nanoparticles exhibited a cytotoxicity in a time- and dosedependent manner. Interestingly, it was found that polyoxalate nanoparticles exhibited a significantly higher cell viability than PLGA nanoparticles, demonstrating polyoxalate has an excellent toxicity profile. The cellular uptake of polyoxalate nanoparticles was observed using rhodamine-loaded nanoparticles under a confocal laser scanning microscope. Figure 7 shows the confocal fluorescent micrographs of RAW 264.7 and HEK 293 cells treated with rhodamine-loaded polyoxalate nanoparticles. After 15 min of incubation, red fluorescence was observed inside the cells, suggesting the phagocytosis by polyoxalate nanoparticles. More red fluorescence from scattered nanoparticles was observed in the cytosol region at 3 h postincubation. Removing of out of focus fluorescent light provided the evidence that fluorescent foci were indeed inside the cells. The results demonstrate that hydrophobic polyoxalate nanoparticles exhibit great potential as a drug delivery vehicle.

Conclusions We have developed polyoxalate nanoparticles as a biodegradable and biocompatible drug delivery vehicle. Polyoxalate was designed to degrade by water hydrolysis into nontoxic small molecular weight compounds, 1,4-cyclohexanedimethanol and oxalic acid that can be easily excreted from a body. Polyoxalate had hydrophobic nature and was formulated into nanoparticles. Polyoxalate nanoparticles had a mean diameter of ∼600 nm and exhibited a minimal cytotoxicity to RAW 264.7 cells. Confocal fluorescent micrographs revealed that rhodamineloaded polyoxalate nanoparticles were readily taken up by RAW 264.7 cells and HEK 293 cells. Given their appealing features, such as the ease of synthesis and biocompatibility, polyoxalate nanoparticles have great potential for drug delivery applications. Acknowledgment. This work is supported by a grant from the Korea Healthcare Technology R&D Project, Ministry of Health, Wealth and Family Affairs, Republic of Korea (A084304). D.L. also acknowledges a New Faculty Support Program of Chonbuk National University.

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