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Cisplatin‐incorporated hyaluronic acid nanoparticles based on ion‐complex formation
PHARMACEUTICAL NANOTECHNOLOGY Cisplatin-Incorporated Hyaluronic Acid Nanoparticles Based on Ion-Complex Formation YOUNG-IL JEONG,1 SEONG-TAEK KIM,2 SHU-GUANG JIN,1 HYANG-HWA RYU,1 YONG-HAO JIN,1 TAE-YOUNG JUNG,2 IN-YOUNG KIM,2 SHIN JUNG1,2 1 Brain Tumor Research Laboratory, Research Institute of Medical Science, Medical School, Chonnam National University, Republic of Korea 2
Department of Neurosurgery, Chonnam National University Hwasun Hospital, Jeollanam-do 519-809, Republic of Korea
Received 13 June 2006; revised 30 March 2007; accepted 23 May 2007 Published online 2 August 2007 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21103
ABSTRACT: The aim of this study is to prepare cisplatin-incorporated nanoparticles based on ion complex formation between hyaluronic acid (HA) and cisplatin for antitumor drug delivery. To prepare nanoparticles using HA, bulk HA was degraded by hyaluronidases (HAses). Cisplatin-incorporated HA nanoparticles were prepared by mixing cisplatin with an aqueous solution of HA and then the nanoparticle solution was dialyzed to remove trace elements. Since glioma tumor cell lines are able to secrete HAse, extracts from U343MG and U87MG cell lines were used to test the release of cisplatin from the nanoparticles. The morphological observation of the cisplatin-incorporated nanoparticles showed that they had spherical shapes with a particle size around 100– 200 nm. The loading efficiency of cisplatin in the nanoparticles was about 67–81% (w/w) and cisplatin was continuously released from the nanoparticles for 4 days. Especially, the release rate of cisplatin from the nanoparticles increased when HAse was added to the release medium. In the results of the HA zymography, the U343MG cell line secreted HAse, while the U87MG cell line did not. When the extracts from U343MG were added to the release medium, the release rate of cisplatin was slightly increased, while the extracts from U87MG did not significantly affect the release rate of cisplatin. In conclusion, cisplatin-incorporated nanoparticles have sufficiently small particle sizes to use as a drug targeting system. The release of cisplatin from the nanoparticles was responsive to the secretion of HAse. These nanoparticles are suitable vehicles for an antitumor drug targeting system. ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:1268–1276, 2008
Keywords:
nanoparticles; cisplatin; hyaluronic acid; hyaluronidase; glioma cell
INTRODUCTION Hyaluronic acid (HA), a linear polysaccharide composed of alternating D-glucuronic acid and Nacetyl-D-glucosamine units, is one of the glycoCorrespondence to: Shin Jung (Telephone: þ82-61-3797666; Fax: þ82-61-379-7673; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 97, 1268–1276 (2008) ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association
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saminoglycan components of the extracellular matrix (ECM), the synovial fluid of joints, and the scaffolding comprising cartilage.1 HA-protein interactions play crucial roles in cell adhesion, growth, and migration and HA acts as a signaling molecule in cell motility, inflammation, wound healing, and cancer metasis.1–5 HA is overexpressed at sites of tumor attachment to the mesentery and provides a matrix that facilitates invasion.6–9 Moreover, cellular HA receptors such as CD44 and
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RHAMM respond to HA as a signal.6,7,10 Especially, gliomas are existed and invaded to HA-rich environment, and express high levels of CD44 compared to normal brain tissue.11,12 Therefore, the targeting of anticancer agents to tumor cells and tumor metastases can be accomplished by the receptor-mediated uptake of bioconjugates between HA and anticancer agents.13 Since HA has immunoneutrality, it has been extensively used as a biocompatible and biodegradable biomaterial for tissue engineering and in drug delivery systems.13–16 Furthermore, hyaluronidase (HAse), a degrading enzyme of HA, is classified to HYAL1, HYAL2, HYAL3, HYAL4, PH20, and HYALP1 in human genome.15 Although the precise mechanism of HAse expression or secretion on invasive properties of glioma cells are not yet fully investigated, it was reported that the expression of HAse were positively correlated with invasion of tumor cells.12,17 Liu et al.18 reported that HAse was dominantly expressed at invasive malignant glioma cells while it was not expressed at normal tissues of brain. The expression of HAse are correlated with tumor malignancies and suggested to indicator of tumorigenesis. The secreted HAse to ECM inhibit cell adhesion to ECM and induce angiogenesis by releasing the growth factor from ECM.18 Therefore, bioconjugates or nanoparticles between HA and anticancer agent can be expected to deliver and selectively release the anticancer agents around the invasive tumor cells.13,16 Cisplatin is extensively used to treat various kinds of solid tumor.19 However, its clinical use is limited due to its severely toxic side effects, such as the emergence of intrinsic and acquired resistance, acute nephrotoxicity and chronic neurotoxicity.20,21 The development of a drug delivery system that can enhance cisplatin accumulation in the tumor, while reducing it in the kidney, would provide a promising approach to achieving enhanced antitumor activity as well as reduced nephrotoxicity in the clinical use of cisplatin. To improve the efficacy of cisplatin and control its release, several formulations utilizing delivery vehicles such as nanoparticles,22 liposomes,23 dextran conjugates,24 and polymeric micelles25 have been developed. Cisplatin-encapsulated nanoparticles of poly(DL-lactide-co-glycolide) (PLGA)/PEG block copolymers offer prolonged cisplatin residence in systemic blood circulation,22 however they have low drug loading efficiency and a high burst release effect. Nishiyama and Kataoka25 reported cisplatin-loaded block copolymer micelDOI 10.1002/jps
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les that were formed through the complexation between cisplatin and poly(ethylene glycol)-poly (aspartic acid) block copolymer PEG-P(Asp)] in an aqueous medium. The polymeric micelle drug carrier system is expected to afford long-term circulation in the bloodstream and eventually to accumulate in the solid tumor.26 The objectives of this study is to investigate possibility of anticancer agents-incorporated HA nanoparticles as a targeted drug carrier to a invasive and HAse-secreted glioma cells since secretion of HAse at gliomas are high level in the invasive areas.12 We prepared nanoparticles based on the formation of an ion complex between HA and cisplatin. The physicochemical properties of the cisplatin-incorporated nanoparticles of HA, as well as their cisplatin release behavior from the nanoparticles, were investigated.
MATERIALS AND METHODS Materials HA sodium salt (from Streptococcus equi sp.) was purchased from Fluka Chemie GmbH. HAse (999 units/mg solid) was purchased from Sigma (St. Louis, MO). Cisplatin was purchased from Wako Pure Chem. Ind. (Osaka, Japan). Dimethylsulfoxide (DMSO), dimethylformamide (DMF), and methanol were purchased from Aldrich Chemical Co. (Milwaukee, WI) as HPLC grade. Poly(ethylene glycol) (PEG) with Mn of 1000, 2000, 3400, 6000, 20000, 35000 g/mol, respectively, was purchased from Aldrich Chemical Co. Dialysis tubing [molecular weight cut-off (MWCO): 12000 g/mol] was purchased from Sigma and dialysis. The dialysis tubing with Spectra/Por dialysis membrane (MWCO 2000 g/mol) was purchased from Spectrum Laboratory (Gardena, CA). Dialysis membrane tubing used in this study was soaked in water for 3 h and subsequently rinsed it with deionized water before use. Degradation of HA Since HA has a large MW (1500000 g/mol), it was degraded to low MW HA using the procedure reported by Luo and Prestwich13 with a minor modification. Two grams of HA was dissolved in 500 mL PBS buffer (pH 6.5, 4 mg/mL) for 12 h and then HAse (10 units/mg of HA) was added. The degradation was carried out at room temperature stirring at 100 rpm for 5 min, followed by 958C for 15 min. The resulting solution was dialyzed JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 3, MARCH 2008
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against deionized water for 3 days using a dialysis tube (MWCO, 12000 g/mol), with the deionized water being exchanged every 3 h. The dialyzed solution filtered using a 0.2 mm cellulose acetate membrane and lyophilized. The resulting solid, viz. low MW HA (Yield: 60.0%, w/w), weighed 1.2 g. The degraded HA was used in all of the following experiments. The MW of degraded HA was evaluated by GPC system. The GPC system was composed of a Waters 515 HPLC pump, Waters 410 differential refractometer, Waters 486 tunable absorbance detector, and Waters Ultrahydrogel 250 and 2000 columns (7.8 mm i.d. 30 cm, flow rate: 5 mL/ min). The mobile phase was 150 mM phosphate buffer (pH 6.5)/methanol ¼ 80/20 (v/v) and the flow rate was 0.5 mL/min. PEG was used to determine MW of HA as a standard. Preparation of Cisplatin-Incorporated Nanoparticles of HA A measured amount of (100 mg) of HA and various amounts of cisplatin were dissolved in 10 mL of deionized water. The mixtures were stirred gently for 3 days under dark conditions. Subsequently, the resulting solution was dialyzed against deionized water for 6 h with stirring to remove unwanted trace elements. The dialyzed solution was lyophilized for 2 days or analyzed. To evaluate their drug content and loading efficiency, the lyophilized cisplatin-incorporated HA nanoparticles were redistributed in deionized water. To measure cisplatin content in the HA nanoparticles, liberation of whole cisplatin from HA nanoparticles was carried out previously reported method by Nishiyama and Kataoka.25 The whole entrapped cisplatin was released from the nanoparticles by keeping them in 0.3 M NaCl at 378C for 3 days. This solution was diluted with 1–10 with DMF. The content and loading efficiency of the cisplatin in the nanoparticles were determined by UV-spectrophotometer (Shimadzu UV-spectrophotometer, UV-1201) at 310 nm.27 The drug contents and loading efficiency were calculated as follows:
Drug content ¼ Loading efficiency ¼
Transmission Electron Microscopy (TEM) Observation To observe cisplatin-incorporated HA nanoparticles, TEM was used. A drop of polymeric micelle suspension was placed onto a carbon film coated on a copper grid for TEM observation. The observation was performed at 80 kV in a JEOL JEM2000 FX II. Particle Size and Zeta Potential Measurement The size of the nanoparticles was measured by dynamic laser scattering spectrophotometer (DLS-7000, Otsuka Electonics Co., Osaka, Japan). The zeta potential of the nanoparticles was measured using photon correlation spectroscopy (Zetasizer 3000, Malvern Instruments, Worcestershire, England) with an He–Ne laser beam at a wavelength of 633 nm at 258C (scattering angle of 908). A sample solution prepared by the dialysis method was used for the particle size measurement (concentration: 0.1 wt.%). X-Ray Powder Diffractometry (XRD) Measurement X-ray powder diffractograms were obtained with a Rigaku D/Max-1200 (Rigaku, Tokyo, Japan) using Ni filtered Cu Ka radiation (40 kV, 20 mA) to determine the crystallinity of drug. All experiments were performed at room temperature. The conditions of powder XRD measurement was as follows: Data Type ¼ Binary; Goniometer ¼ 1; Attachment ¼ 1; Scan mode ¼ Continuous. Mode 2 (R/T) ¼ Reflection; Scan axis ¼ 2Theta/Theta. Start angle ¼ 10000; Stop angle ¼ 80000; Scan speed ¼ 5000; Sampling interval ¼ 0.050; Theta angle ¼ 5000; 2 Theta angle ¼ 10000; Fixed time ¼ 0.01; Full scale ¼ 1000; Counting unit ¼ CPS; Target ¼ Cu. Wave length, Ka1 ¼ 1.540510; Wave length, Ka2 ¼ 1.544330; Wave length, Ka ¼ 1.541780; Wave length, Kb ¼ 1.392170. 40.0 kV; 20.0 mA.
Amount of liberated cisplatin from the nanoparticles 100 Weight of nanoparticles Lirated amount of cisplatin from the nanoparticles 100 Feeding amount of cisplatin
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Cisplatin, HA, and nanoparticles were used to measure powder XRD. For physical mixture of HA and cisplatin, 100 mg of HA was dissolved in 10 mL of deionized water. To this solution, 10 mg of cisplatin was added and instantly lyophilized to prevent ion complex formation between HA and cisplatin. Drug Release Study A measured amount (20 mg) of the nanoparticles was resuspended into 5 mL of phosphate-buffered saline (PBS, pH 7.4) and transferred to a dialysis tube (MWCO: 2000 g/mol). The dialysis tube was put into a 200 mL bottle with 95 mL of PBS. Cisplatin release was studied at 100 rpm (NB-205V, Shaking incubator, N-BIOTEC Co., Inc., Seoul, Korea) at 378C. At predetermined time intervals, 2 mL of the sample solution was taken and the solution was supplemented with 2 mL of fresh medium. The amount of the drug that was released was evaluated by UV-spectrophotometer at 310 nm.27 As a control, 2 mg of cisplatin itself was distributed in 5 mL PBS for 3 h at room temperature and introduced into dialysis (MWCO: 2000 g/mol) for release test. Release test of cisplatin itself was performed similar to HA nanoparticles. All experiments were triplicated and the data was expressed as mean SD. To examine the effect of HAse on the drug release, HAse (100 units) was added to the dialysis tube containing the nanoparticle solution. Hyaluronidase Extraction From Tumor Cell Lines For the extraction of HAse, U343MG and U87MG cell lines were cultured confluently in Dulbecco’s modified essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 378C in a 5% CO2 atmosphere. Following this, the culture medium was replaced with 2 mL of DMEM without FBS and cultured for 3 days in a 5% CO2 atmosphere. All the medium was harvested and dialyzed against deionized water using a dialysis tube (MWCO 2000 g/mol) for 2 days at 48C to remove the trace elements. The resulting solution was lyophilized for 3 days and then stored it at 208C until use. To study the effect of the extracts from the U343MG and U87MG cell lines, 1 mg of the solid extracts from the U343MG or U87MG cell line was added to the dialysis bag and a drug release DOI 10.1002/jps
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study was performed in a manner similar to that described above. Zymography of Hyaluronic Acid For the HA zymography, various glioma cell lines, viz. U343MG, 9L, C6, U373MG, U87MG, were cultured confluently in DMEM supplemented with 10% FBS at 378C in a 5% CO2 atmosphere. Following this, the culture medium was replaced with DMEM without FBS and cultured for 3 days in a 5% CO2 atmosphere. Subsequently, the culture medium were harvested and lyophilized for 3 days. The lyophilized solid was used for the HA zymography study. The existent of HAse was determined by zymography, as reported previously.28–30 Briefly, an electrophoresis run was performed in 8% SDS acrylamide gel (1-mm thickness) containing 170 mg/mL of HA (bulk HA, final concentration). After the electrophoretic run and 2.5% Triton X-100 incubation, the gels were incubated overnight at 378C in 0.1 M sodium formate, 0.15 M NaCl, pH 3.5 buffer, or in 50 mM citric acid– Na2HPO4, 0.15 M NaCl, pH 5.0 buffer. Finally, the gels were stained in 0.5% Alcian blue. The HAse activity was determined in the form of unstained bands corresponding to the migrated positions of the enzymes. HAse purchased from Sigma Chem. Co. was used as positive control.
RESULTS AND DISCUSSION The formation of anionic polymer–metal complexes was previously reported by the Kataoka group25,26 and that of polymeric micelles through the development of ion complexes between poly(aspartic acid) and metal has been well studied. Since HA has an anionic character due to the carboxylic acid in the polysaccharide chain, cisplatin-incorporated nanoparticles were prepared through the anionic polymer–metal complexation between cisplatin and HA, as illustrated in Figure 1. The nanoparticles were spontaneously formed by the simple mixing of HA and cisplatin. Characterization of Degraded HA Bulk HA is a high molecular weight (1.5 MDa) polysaccharide and has high viscosity in aqueous JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 3, MARCH 2008
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Figure 1. Schematic illustrations of nanoparticle formation by ion complex between HA and cisplatin.
systems. Thus, this high molecular weight and viscous HA is not suitable for use as a drug delivery vehicle, due to its limited susceptibility to modification and poor solubility. Therefore, low molecular weight HA was produced by the degradation of bulk HA with HAse. As reported in a previous study,28 HAse degrades bulk HA to HA oligosaccharides with an N-acetylglucosamine moiety at the reducing terminus. The degradation of bulk HA in pH 6.5 PBS buffer with HAse produced partially degraded HA.11 HA oligosaccharides and buffer salts were removed by dialysis against deionized water. Subsequently, the degraded HA was harvested by lyophilization and analyzed by GPC, as shown in Figure 2: Mw ¼ 102500, Mn ¼ 92400, and polydispersity index (PDI) ¼ 1.11. This degraded low-MW HA was used in the following experiment. This degraded HA was more suitable to dissolve in water, that is, the degraded HA was dissolved in water at a concentration of more than 10 mg/mL.
Preparation of Cisplatin-Incorporated Nanoparticles
Figure 2. MW of degraded HA was performed by GPC system. PEG (Mn ¼ 3400 g/mol and MPEG (Mn ¼ 1000, 2000, and 5000) as a standard were used for evaluation of MW of HA.
Figure 3. Particle size distribution of cisplatinencapsulated HA nanoparticles. Particle size of cisplatin-encapsulated HA nanoparticles was measured by DLS at room temperature.
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As shown in Figure 3, narrowly distributed cisplatin-incorporated HA nanoparticles were formed and their particle size was distributed around 100–200 nm according to the HA/cisplatin weight ratio. Furthermore, according to the TEM
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indicating that at least 5 mmol of the disaccharides were required to induce 1 mmol of cisplatin to form a stable ion complex. The size of the nanoparticles increased slightly with increasing singular content of drug. The zeta potential of the nanoparticles was decreased with increasing singular content of drug. Figure 5 shows the XRD pattern of cisplatin, HA, the nanoparticles, and the physical mixture of cisplatin and HA. As shown in Figure 5a, cisplatin and HA have intrinsic crystal peaks. The specific peaks of cisplatin disappeared at cisplatin-incorporated HA nanoparticles as shown in Figure 5c and d, while the physical mixture showed both the peaks of HA and cisplatin. These results confirmed that cisplatin was complexed to the anionic domain of HA and then formed nanospherical aggregates. It is suggested that an ion complex formed between cisplatin and HA. Figure 4. Morphology of cisplatin-incorporated HA nanoparticles (100/10 of Tab. 1) observed by TEM.
Cisplatin Release From Nanoparticles observation, the cisplatin-incorporated HA nanoparticles have spherical shapes with a particle size of around 100–200 nm (100/10), as shown in Figure 4. The TEM observation confirmed that the nanoparticles were formed by the mixing of HA and cisplatin. During the complexation of HA and cisplatin, precipitation did not occur and the unloaded free cisplatin was removed by dialysis. As shown in Table 1, the drug contents increased, whereas the loading efficiency decreased, with increasing of feeding ratio of cisplatin. The drug contents were 3.9%, 7.0%, and 11.8% (w/w), for the feeding ratios of 100/5, 100/10, and 100/20, respectively. The loading efficiency was significantly decreased from 81.2% (w/w, 100/5) to 66.9% (w/w, 100/20), indicating that a lot of the drug was not complexed with HA and was removed during the dialysis procedure. The mol fraction of cisplatin versus HA varied from 5.6 to 17.6,
To test release characteristics of cisplatin from HA nanoparticles, dialysis method was used.25 Figure 6 shows the kinetics of the cisplatin release from the HA nanoparticles. As shown in Figure 6a, the kinetics of the cisplatin release from the nanoparticles showed two phases, that is, an initial burst release phase until 12 h followed by a continuous release phase until 4 days. These results might be due to that cisplatin complexed with HA around the surface of the nanoparticles predominantly released at initial stage of drug release and then controlled release according to the time course. The cisplatin release kinetics slightly decreased with increasing singular content of drug. Cisplatin itself liberated from dialysis membrane tubing was faster than HA nanoparticles. Since cisplatin is a hydrophobic compound, cisplatin as a solid compound was distributed into the PBS and introduced into
Table 1. Characterization of Cisplatin-Incorporated HA-Cisplatin Nanoparticles Particle Size (nm) Polymer/ Drug Ratio (mg/mg) 100/5 100/10 100/20
Drug Contents (%, w/w)
Loading Efficiency (%, w/w)
Mole Fraction of Disaccharide Unit/Cisplatina
Intensity Average
Weight Average
Number Average
Zeta Potential (mV)
3.9 7.0 11.8
81.2 75.3 66.9
17.1 10.0 5.6
102.0 56.3 125.0 41.6 183.9 49.3
90.8 48.9 123.2 61.3 171.6 44.3
81.3 38.6 119.6 56.7 167.4 43.0
16.2 10.6 9.2
a
Mole fraction of disaccharide/cisplatin ¼ (mol of cisplatin/mol of disaccharide of HA).
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Figure 5. XRD patterns of cisplatin-incorporated HA nanoparticles. (a) Cisplatin, (b) HA, (c) cisplatin-incorporated nanoparticles (100/5 in Tab. 1), (d) cisplatinincorporated nanoparticles (100/10), and (e) cisplatin/ HA physical mixture (1/10, w/w).
dialysis tube. It suggested that cisplatin powder was slowly dissolved and liberated out to the dialysis membrane due to its hydrophobicity. Figure 6b shows the effect of HAse on the cisplatin release from the nanoparticles. HA is overexpressed at the sites of tumor attachment to the mesentery and provides a matrix that facilitates invasion.6–9 Furthermore, HAse is known to be responsible for brain metastases.29 As shown in Figure 6b, the kinetics of the cisplatin release from the nanoparticles in the presence of HAse was faster than that in the absence of HAse. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 3, MARCH 2008
Figure 6. (a) The effect of drug contents and (b) presence of hyaluronidase (drug content: 7.0%, w/w) on the cisplatin release from HA nanoparticles. For comparison, cisplatin itself (2 mg/5 mL PBS) was introduced into dialysis tube and used for release experiment as a control. The experiment was triplicated and the data was expressed as mean SD.
These results indicated that the cisplatin-incorporated HA nanoparticles are responsive to HAse. Figure 7 shows the results of the HA zymography with various glioma cell lines. Among them, U343MG, C6, and 9L secreted HAse, while U373MG and U87MG did not show any HAse activity. Among these different cell lines, the lyophilized extracts from U343MG and U87MG were added to the release medium in order to study the effect of cellular-derived HAse on the release kinetics of cisplatin. As shown in Figure 8, the release of cisplatin was increased by the DOI 10.1002/jps
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Figure 7. HA zymography using glioma cell lines. As a control, hyaluronidase was loaded for HAse onto HA zymography and heat-inactivated (at 1008C for 5 min) hyaluronidase was loaded for HAse(þ).
addition of the extracts of U343MG, while the extracts of U87MG had no significant effect. These results indicated that the HA nanoparticles were not only responsive to the HAse activity, but also that they are potential vehicles for drug targeting. These results indicated that cisplatin-incorporated HA nanoparticles can be used as targetable drug carriers for invasive glioma via responsive to HAse secretion.
CONCLUSION Cisplatin-incorporated HA nanoparticles were prepared as a novel cisplatin drug delivery system. The morphological observation of the cisplatin-incorporated nanoparticles showed that they had spherical shapes with a particle size of around
Figure 8. The effect of conditioned media from U343MG and U87MG cell lines on the cisplatin release from HA nanoparticles. The experiment was triplicated and the data was expressed as mean SD. DOI 10.1002/jps
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100–200 nm. The loading efficiency of cisplatin in the nanoparticles was about 67–81% (w/w) and cisplatin was continuously released from the nanoparticles for 4 days. Especially, the cisplatin release rate from the nanoparticles increased when HAse was added to the release medium. In the results of the HA zymography, the U343MG cell line secreted HAse, while the U87MG cell line did not. When the extracts from U343MG were added to the release medium, the release rate of cisplatin was slightly increased, while the extracts from U87MG had no significant effect. Since the release of cisplatin from the nanoparticles was responsive to the secretion of HAse, the cisplatinincorporated HA nanoparticles are considered to be suitable vehicles for the antitumor drug targeting of HAse-related invasive gliomas.
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Department of Neurosurgery, Chonnam National University Hwasun Hospital, Jeollanam-do 519-809, Republic of Korea
Received 13 June 2006; revised 30 March 2007; accepted 23 May 2007 Published online 2 August 2007 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21103
ABSTRACT: The aim of this study is to prepare cisplatin-incorporated nanoparticles based on ion complex formation between hyaluronic acid (HA) and cisplatin for antitumor drug delivery. To prepare nanoparticles using HA, bulk HA was degraded by hyaluronidases (HAses). Cisplatin-incorporated HA nanoparticles were prepared by mixing cisplatin with an aqueous solution of HA and then the nanoparticle solution was dialyzed to remove trace elements. Since glioma tumor cell lines are able to secrete HAse, extracts from U343MG and U87MG cell lines were used to test the release of cisplatin from the nanoparticles. The morphological observation of the cisplatin-incorporated nanoparticles showed that they had spherical shapes with a particle size around 100– 200 nm. The loading efficiency of cisplatin in the nanoparticles was about 67–81% (w/w) and cisplatin was continuously released from the nanoparticles for 4 days. Especially, the release rate of cisplatin from the nanoparticles increased when HAse was added to the release medium. In the results of the HA zymography, the U343MG cell line secreted HAse, while the U87MG cell line did not. When the extracts from U343MG were added to the release medium, the release rate of cisplatin was slightly increased, while the extracts from U87MG did not significantly affect the release rate of cisplatin. In conclusion, cisplatin-incorporated nanoparticles have sufficiently small particle sizes to use as a drug targeting system. The release of cisplatin from the nanoparticles was responsive to the secretion of HAse. These nanoparticles are suitable vehicles for an antitumor drug targeting system. ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:1268–1276, 2008
Keywords:
nanoparticles; cisplatin; hyaluronic acid; hyaluronidase; glioma cell
INTRODUCTION Hyaluronic acid (HA), a linear polysaccharide composed of alternating D-glucuronic acid and Nacetyl-D-glucosamine units, is one of the glycoCorrespondence to: Shin Jung (Telephone: þ82-61-3797666; Fax: þ82-61-379-7673; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 97, 1268–1276 (2008) ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association
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saminoglycan components of the extracellular matrix (ECM), the synovial fluid of joints, and the scaffolding comprising cartilage.1 HA-protein interactions play crucial roles in cell adhesion, growth, and migration and HA acts as a signaling molecule in cell motility, inflammation, wound healing, and cancer metasis.1–5 HA is overexpressed at sites of tumor attachment to the mesentery and provides a matrix that facilitates invasion.6–9 Moreover, cellular HA receptors such as CD44 and
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RHAMM respond to HA as a signal.6,7,10 Especially, gliomas are existed and invaded to HA-rich environment, and express high levels of CD44 compared to normal brain tissue.11,12 Therefore, the targeting of anticancer agents to tumor cells and tumor metastases can be accomplished by the receptor-mediated uptake of bioconjugates between HA and anticancer agents.13 Since HA has immunoneutrality, it has been extensively used as a biocompatible and biodegradable biomaterial for tissue engineering and in drug delivery systems.13–16 Furthermore, hyaluronidase (HAse), a degrading enzyme of HA, is classified to HYAL1, HYAL2, HYAL3, HYAL4, PH20, and HYALP1 in human genome.15 Although the precise mechanism of HAse expression or secretion on invasive properties of glioma cells are not yet fully investigated, it was reported that the expression of HAse were positively correlated with invasion of tumor cells.12,17 Liu et al.18 reported that HAse was dominantly expressed at invasive malignant glioma cells while it was not expressed at normal tissues of brain. The expression of HAse are correlated with tumor malignancies and suggested to indicator of tumorigenesis. The secreted HAse to ECM inhibit cell adhesion to ECM and induce angiogenesis by releasing the growth factor from ECM.18 Therefore, bioconjugates or nanoparticles between HA and anticancer agent can be expected to deliver and selectively release the anticancer agents around the invasive tumor cells.13,16 Cisplatin is extensively used to treat various kinds of solid tumor.19 However, its clinical use is limited due to its severely toxic side effects, such as the emergence of intrinsic and acquired resistance, acute nephrotoxicity and chronic neurotoxicity.20,21 The development of a drug delivery system that can enhance cisplatin accumulation in the tumor, while reducing it in the kidney, would provide a promising approach to achieving enhanced antitumor activity as well as reduced nephrotoxicity in the clinical use of cisplatin. To improve the efficacy of cisplatin and control its release, several formulations utilizing delivery vehicles such as nanoparticles,22 liposomes,23 dextran conjugates,24 and polymeric micelles25 have been developed. Cisplatin-encapsulated nanoparticles of poly(DL-lactide-co-glycolide) (PLGA)/PEG block copolymers offer prolonged cisplatin residence in systemic blood circulation,22 however they have low drug loading efficiency and a high burst release effect. Nishiyama and Kataoka25 reported cisplatin-loaded block copolymer micelDOI 10.1002/jps
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les that were formed through the complexation between cisplatin and poly(ethylene glycol)-poly (aspartic acid) block copolymer PEG-P(Asp)] in an aqueous medium. The polymeric micelle drug carrier system is expected to afford long-term circulation in the bloodstream and eventually to accumulate in the solid tumor.26 The objectives of this study is to investigate possibility of anticancer agents-incorporated HA nanoparticles as a targeted drug carrier to a invasive and HAse-secreted glioma cells since secretion of HAse at gliomas are high level in the invasive areas.12 We prepared nanoparticles based on the formation of an ion complex between HA and cisplatin. The physicochemical properties of the cisplatin-incorporated nanoparticles of HA, as well as their cisplatin release behavior from the nanoparticles, were investigated.
MATERIALS AND METHODS Materials HA sodium salt (from Streptococcus equi sp.) was purchased from Fluka Chemie GmbH. HAse (999 units/mg solid) was purchased from Sigma (St. Louis, MO). Cisplatin was purchased from Wako Pure Chem. Ind. (Osaka, Japan). Dimethylsulfoxide (DMSO), dimethylformamide (DMF), and methanol were purchased from Aldrich Chemical Co. (Milwaukee, WI) as HPLC grade. Poly(ethylene glycol) (PEG) with Mn of 1000, 2000, 3400, 6000, 20000, 35000 g/mol, respectively, was purchased from Aldrich Chemical Co. Dialysis tubing [molecular weight cut-off (MWCO): 12000 g/mol] was purchased from Sigma and dialysis. The dialysis tubing with Spectra/Por dialysis membrane (MWCO 2000 g/mol) was purchased from Spectrum Laboratory (Gardena, CA). Dialysis membrane tubing used in this study was soaked in water for 3 h and subsequently rinsed it with deionized water before use. Degradation of HA Since HA has a large MW (1500000 g/mol), it was degraded to low MW HA using the procedure reported by Luo and Prestwich13 with a minor modification. Two grams of HA was dissolved in 500 mL PBS buffer (pH 6.5, 4 mg/mL) for 12 h and then HAse (10 units/mg of HA) was added. The degradation was carried out at room temperature stirring at 100 rpm for 5 min, followed by 958C for 15 min. The resulting solution was dialyzed JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 3, MARCH 2008
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against deionized water for 3 days using a dialysis tube (MWCO, 12000 g/mol), with the deionized water being exchanged every 3 h. The dialyzed solution filtered using a 0.2 mm cellulose acetate membrane and lyophilized. The resulting solid, viz. low MW HA (Yield: 60.0%, w/w), weighed 1.2 g. The degraded HA was used in all of the following experiments. The MW of degraded HA was evaluated by GPC system. The GPC system was composed of a Waters 515 HPLC pump, Waters 410 differential refractometer, Waters 486 tunable absorbance detector, and Waters Ultrahydrogel 250 and 2000 columns (7.8 mm i.d. 30 cm, flow rate: 5 mL/ min). The mobile phase was 150 mM phosphate buffer (pH 6.5)/methanol ¼ 80/20 (v/v) and the flow rate was 0.5 mL/min. PEG was used to determine MW of HA as a standard. Preparation of Cisplatin-Incorporated Nanoparticles of HA A measured amount of (100 mg) of HA and various amounts of cisplatin were dissolved in 10 mL of deionized water. The mixtures were stirred gently for 3 days under dark conditions. Subsequently, the resulting solution was dialyzed against deionized water for 6 h with stirring to remove unwanted trace elements. The dialyzed solution was lyophilized for 2 days or analyzed. To evaluate their drug content and loading efficiency, the lyophilized cisplatin-incorporated HA nanoparticles were redistributed in deionized water. To measure cisplatin content in the HA nanoparticles, liberation of whole cisplatin from HA nanoparticles was carried out previously reported method by Nishiyama and Kataoka.25 The whole entrapped cisplatin was released from the nanoparticles by keeping them in 0.3 M NaCl at 378C for 3 days. This solution was diluted with 1–10 with DMF. The content and loading efficiency of the cisplatin in the nanoparticles were determined by UV-spectrophotometer (Shimadzu UV-spectrophotometer, UV-1201) at 310 nm.27 The drug contents and loading efficiency were calculated as follows:
Drug content ¼ Loading efficiency ¼
Transmission Electron Microscopy (TEM) Observation To observe cisplatin-incorporated HA nanoparticles, TEM was used. A drop of polymeric micelle suspension was placed onto a carbon film coated on a copper grid for TEM observation. The observation was performed at 80 kV in a JEOL JEM2000 FX II. Particle Size and Zeta Potential Measurement The size of the nanoparticles was measured by dynamic laser scattering spectrophotometer (DLS-7000, Otsuka Electonics Co., Osaka, Japan). The zeta potential of the nanoparticles was measured using photon correlation spectroscopy (Zetasizer 3000, Malvern Instruments, Worcestershire, England) with an He–Ne laser beam at a wavelength of 633 nm at 258C (scattering angle of 908). A sample solution prepared by the dialysis method was used for the particle size measurement (concentration: 0.1 wt.%). X-Ray Powder Diffractometry (XRD) Measurement X-ray powder diffractograms were obtained with a Rigaku D/Max-1200 (Rigaku, Tokyo, Japan) using Ni filtered Cu Ka radiation (40 kV, 20 mA) to determine the crystallinity of drug. All experiments were performed at room temperature. The conditions of powder XRD measurement was as follows: Data Type ¼ Binary; Goniometer ¼ 1; Attachment ¼ 1; Scan mode ¼ Continuous. Mode 2 (R/T) ¼ Reflection; Scan axis ¼ 2Theta/Theta. Start angle ¼ 10000; Stop angle ¼ 80000; Scan speed ¼ 5000; Sampling interval ¼ 0.050; Theta angle ¼ 5000; 2 Theta angle ¼ 10000; Fixed time ¼ 0.01; Full scale ¼ 1000; Counting unit ¼ CPS; Target ¼ Cu. Wave length, Ka1 ¼ 1.540510; Wave length, Ka2 ¼ 1.544330; Wave length, Ka ¼ 1.541780; Wave length, Kb ¼ 1.392170. 40.0 kV; 20.0 mA.
Amount of liberated cisplatin from the nanoparticles 100 Weight of nanoparticles Lirated amount of cisplatin from the nanoparticles 100 Feeding amount of cisplatin
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Cisplatin, HA, and nanoparticles were used to measure powder XRD. For physical mixture of HA and cisplatin, 100 mg of HA was dissolved in 10 mL of deionized water. To this solution, 10 mg of cisplatin was added and instantly lyophilized to prevent ion complex formation between HA and cisplatin. Drug Release Study A measured amount (20 mg) of the nanoparticles was resuspended into 5 mL of phosphate-buffered saline (PBS, pH 7.4) and transferred to a dialysis tube (MWCO: 2000 g/mol). The dialysis tube was put into a 200 mL bottle with 95 mL of PBS. Cisplatin release was studied at 100 rpm (NB-205V, Shaking incubator, N-BIOTEC Co., Inc., Seoul, Korea) at 378C. At predetermined time intervals, 2 mL of the sample solution was taken and the solution was supplemented with 2 mL of fresh medium. The amount of the drug that was released was evaluated by UV-spectrophotometer at 310 nm.27 As a control, 2 mg of cisplatin itself was distributed in 5 mL PBS for 3 h at room temperature and introduced into dialysis (MWCO: 2000 g/mol) for release test. Release test of cisplatin itself was performed similar to HA nanoparticles. All experiments were triplicated and the data was expressed as mean SD. To examine the effect of HAse on the drug release, HAse (100 units) was added to the dialysis tube containing the nanoparticle solution. Hyaluronidase Extraction From Tumor Cell Lines For the extraction of HAse, U343MG and U87MG cell lines were cultured confluently in Dulbecco’s modified essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 378C in a 5% CO2 atmosphere. Following this, the culture medium was replaced with 2 mL of DMEM without FBS and cultured for 3 days in a 5% CO2 atmosphere. All the medium was harvested and dialyzed against deionized water using a dialysis tube (MWCO 2000 g/mol) for 2 days at 48C to remove the trace elements. The resulting solution was lyophilized for 3 days and then stored it at 208C until use. To study the effect of the extracts from the U343MG and U87MG cell lines, 1 mg of the solid extracts from the U343MG or U87MG cell line was added to the dialysis bag and a drug release DOI 10.1002/jps
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study was performed in a manner similar to that described above. Zymography of Hyaluronic Acid For the HA zymography, various glioma cell lines, viz. U343MG, 9L, C6, U373MG, U87MG, were cultured confluently in DMEM supplemented with 10% FBS at 378C in a 5% CO2 atmosphere. Following this, the culture medium was replaced with DMEM without FBS and cultured for 3 days in a 5% CO2 atmosphere. Subsequently, the culture medium were harvested and lyophilized for 3 days. The lyophilized solid was used for the HA zymography study. The existent of HAse was determined by zymography, as reported previously.28–30 Briefly, an electrophoresis run was performed in 8% SDS acrylamide gel (1-mm thickness) containing 170 mg/mL of HA (bulk HA, final concentration). After the electrophoretic run and 2.5% Triton X-100 incubation, the gels were incubated overnight at 378C in 0.1 M sodium formate, 0.15 M NaCl, pH 3.5 buffer, or in 50 mM citric acid– Na2HPO4, 0.15 M NaCl, pH 5.0 buffer. Finally, the gels were stained in 0.5% Alcian blue. The HAse activity was determined in the form of unstained bands corresponding to the migrated positions of the enzymes. HAse purchased from Sigma Chem. Co. was used as positive control.
RESULTS AND DISCUSSION The formation of anionic polymer–metal complexes was previously reported by the Kataoka group25,26 and that of polymeric micelles through the development of ion complexes between poly(aspartic acid) and metal has been well studied. Since HA has an anionic character due to the carboxylic acid in the polysaccharide chain, cisplatin-incorporated nanoparticles were prepared through the anionic polymer–metal complexation between cisplatin and HA, as illustrated in Figure 1. The nanoparticles were spontaneously formed by the simple mixing of HA and cisplatin. Characterization of Degraded HA Bulk HA is a high molecular weight (1.5 MDa) polysaccharide and has high viscosity in aqueous JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 3, MARCH 2008
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Figure 1. Schematic illustrations of nanoparticle formation by ion complex between HA and cisplatin.
systems. Thus, this high molecular weight and viscous HA is not suitable for use as a drug delivery vehicle, due to its limited susceptibility to modification and poor solubility. Therefore, low molecular weight HA was produced by the degradation of bulk HA with HAse. As reported in a previous study,28 HAse degrades bulk HA to HA oligosaccharides with an N-acetylglucosamine moiety at the reducing terminus. The degradation of bulk HA in pH 6.5 PBS buffer with HAse produced partially degraded HA.11 HA oligosaccharides and buffer salts were removed by dialysis against deionized water. Subsequently, the degraded HA was harvested by lyophilization and analyzed by GPC, as shown in Figure 2: Mw ¼ 102500, Mn ¼ 92400, and polydispersity index (PDI) ¼ 1.11. This degraded low-MW HA was used in the following experiment. This degraded HA was more suitable to dissolve in water, that is, the degraded HA was dissolved in water at a concentration of more than 10 mg/mL.
Preparation of Cisplatin-Incorporated Nanoparticles
Figure 2. MW of degraded HA was performed by GPC system. PEG (Mn ¼ 3400 g/mol and MPEG (Mn ¼ 1000, 2000, and 5000) as a standard were used for evaluation of MW of HA.
Figure 3. Particle size distribution of cisplatinencapsulated HA nanoparticles. Particle size of cisplatin-encapsulated HA nanoparticles was measured by DLS at room temperature.
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As shown in Figure 3, narrowly distributed cisplatin-incorporated HA nanoparticles were formed and their particle size was distributed around 100–200 nm according to the HA/cisplatin weight ratio. Furthermore, according to the TEM
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indicating that at least 5 mmol of the disaccharides were required to induce 1 mmol of cisplatin to form a stable ion complex. The size of the nanoparticles increased slightly with increasing singular content of drug. The zeta potential of the nanoparticles was decreased with increasing singular content of drug. Figure 5 shows the XRD pattern of cisplatin, HA, the nanoparticles, and the physical mixture of cisplatin and HA. As shown in Figure 5a, cisplatin and HA have intrinsic crystal peaks. The specific peaks of cisplatin disappeared at cisplatin-incorporated HA nanoparticles as shown in Figure 5c and d, while the physical mixture showed both the peaks of HA and cisplatin. These results confirmed that cisplatin was complexed to the anionic domain of HA and then formed nanospherical aggregates. It is suggested that an ion complex formed between cisplatin and HA. Figure 4. Morphology of cisplatin-incorporated HA nanoparticles (100/10 of Tab. 1) observed by TEM.
Cisplatin Release From Nanoparticles observation, the cisplatin-incorporated HA nanoparticles have spherical shapes with a particle size of around 100–200 nm (100/10), as shown in Figure 4. The TEM observation confirmed that the nanoparticles were formed by the mixing of HA and cisplatin. During the complexation of HA and cisplatin, precipitation did not occur and the unloaded free cisplatin was removed by dialysis. As shown in Table 1, the drug contents increased, whereas the loading efficiency decreased, with increasing of feeding ratio of cisplatin. The drug contents were 3.9%, 7.0%, and 11.8% (w/w), for the feeding ratios of 100/5, 100/10, and 100/20, respectively. The loading efficiency was significantly decreased from 81.2% (w/w, 100/5) to 66.9% (w/w, 100/20), indicating that a lot of the drug was not complexed with HA and was removed during the dialysis procedure. The mol fraction of cisplatin versus HA varied from 5.6 to 17.6,
To test release characteristics of cisplatin from HA nanoparticles, dialysis method was used.25 Figure 6 shows the kinetics of the cisplatin release from the HA nanoparticles. As shown in Figure 6a, the kinetics of the cisplatin release from the nanoparticles showed two phases, that is, an initial burst release phase until 12 h followed by a continuous release phase until 4 days. These results might be due to that cisplatin complexed with HA around the surface of the nanoparticles predominantly released at initial stage of drug release and then controlled release according to the time course. The cisplatin release kinetics slightly decreased with increasing singular content of drug. Cisplatin itself liberated from dialysis membrane tubing was faster than HA nanoparticles. Since cisplatin is a hydrophobic compound, cisplatin as a solid compound was distributed into the PBS and introduced into
Table 1. Characterization of Cisplatin-Incorporated HA-Cisplatin Nanoparticles Particle Size (nm) Polymer/ Drug Ratio (mg/mg) 100/5 100/10 100/20
Drug Contents (%, w/w)
Loading Efficiency (%, w/w)
Mole Fraction of Disaccharide Unit/Cisplatina
Intensity Average
Weight Average
Number Average
Zeta Potential (mV)
3.9 7.0 11.8
81.2 75.3 66.9
17.1 10.0 5.6
102.0 56.3 125.0 41.6 183.9 49.3
90.8 48.9 123.2 61.3 171.6 44.3
81.3 38.6 119.6 56.7 167.4 43.0
16.2 10.6 9.2
a
Mole fraction of disaccharide/cisplatin ¼ (mol of cisplatin/mol of disaccharide of HA).
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Figure 5. XRD patterns of cisplatin-incorporated HA nanoparticles. (a) Cisplatin, (b) HA, (c) cisplatin-incorporated nanoparticles (100/5 in Tab. 1), (d) cisplatinincorporated nanoparticles (100/10), and (e) cisplatin/ HA physical mixture (1/10, w/w).
dialysis tube. It suggested that cisplatin powder was slowly dissolved and liberated out to the dialysis membrane due to its hydrophobicity. Figure 6b shows the effect of HAse on the cisplatin release from the nanoparticles. HA is overexpressed at the sites of tumor attachment to the mesentery and provides a matrix that facilitates invasion.6–9 Furthermore, HAse is known to be responsible for brain metastases.29 As shown in Figure 6b, the kinetics of the cisplatin release from the nanoparticles in the presence of HAse was faster than that in the absence of HAse. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 3, MARCH 2008
Figure 6. (a) The effect of drug contents and (b) presence of hyaluronidase (drug content: 7.0%, w/w) on the cisplatin release from HA nanoparticles. For comparison, cisplatin itself (2 mg/5 mL PBS) was introduced into dialysis tube and used for release experiment as a control. The experiment was triplicated and the data was expressed as mean SD.
These results indicated that the cisplatin-incorporated HA nanoparticles are responsive to HAse. Figure 7 shows the results of the HA zymography with various glioma cell lines. Among them, U343MG, C6, and 9L secreted HAse, while U373MG and U87MG did not show any HAse activity. Among these different cell lines, the lyophilized extracts from U343MG and U87MG were added to the release medium in order to study the effect of cellular-derived HAse on the release kinetics of cisplatin. As shown in Figure 8, the release of cisplatin was increased by the DOI 10.1002/jps
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Figure 7. HA zymography using glioma cell lines. As a control, hyaluronidase was loaded for HAse onto HA zymography and heat-inactivated (at 1008C for 5 min) hyaluronidase was loaded for HAse(þ).
addition of the extracts of U343MG, while the extracts of U87MG had no significant effect. These results indicated that the HA nanoparticles were not only responsive to the HAse activity, but also that they are potential vehicles for drug targeting. These results indicated that cisplatin-incorporated HA nanoparticles can be used as targetable drug carriers for invasive glioma via responsive to HAse secretion.
CONCLUSION Cisplatin-incorporated HA nanoparticles were prepared as a novel cisplatin drug delivery system. The morphological observation of the cisplatin-incorporated nanoparticles showed that they had spherical shapes with a particle size of around
Figure 8. The effect of conditioned media from U343MG and U87MG cell lines on the cisplatin release from HA nanoparticles. The experiment was triplicated and the data was expressed as mean SD. DOI 10.1002/jps
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100–200 nm. The loading efficiency of cisplatin in the nanoparticles was about 67–81% (w/w) and cisplatin was continuously released from the nanoparticles for 4 days. Especially, the cisplatin release rate from the nanoparticles increased when HAse was added to the release medium. In the results of the HA zymography, the U343MG cell line secreted HAse, while the U87MG cell line did not. When the extracts from U343MG were added to the release medium, the release rate of cisplatin was slightly increased, while the extracts from U87MG had no significant effect. Since the release of cisplatin from the nanoparticles was responsive to the secretion of HAse, the cisplatinincorporated HA nanoparticles are considered to be suitable vehicles for the antitumor drug targeting of HAse-related invasive gliomas.
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