Journal of Photochemistry and Photobiology B: Biology 149 (2015) 51–57
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Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Preparation and characterization of injectable Mitoxantrone poly (lactic acid)/fullerene implants for in vivo chemo-photodynamic therapy Zhi Li, Fei-long Zhang, Li-li Pan, Xia-li Zhu, Zhen-zhong Zhang ⇑ School of Pharmaceutical Sciences, Zhengzhou University, 100 Kexue Avenue, Zhengzhou 450001, PR China
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Article history: Received 5 January 2015 Received in revised form 21 May 2015 Accepted 21 May 2015 Available online 27 May 2015 Keywords: Fullerene (C60) Mitoxantrone Poly (lactic acid) Chemo-photodynamic therapy Implants Sustained release
a b s t r a c t Fullerene (C60) L-phenylalanine derivative attached with poly (lactic acid) (C60-phe-PLA) was developed to prepare injectable Mitoxantrone (MTX) multifunctional implants. C60-phe-PLA was self-assembled to form microspheres consisting of a hydrophilic antitumor drug (MTX) and a hydrophobic block (C60) by dispersion–solvent diffusion method. The self-assembled microspheres showed sustained release pattern almost 15 days in vitro release experiments. According to the tissue distribution of C57BL mice after intratumoral administration of the microspheres, the MTX mainly distributed in tumors, and rarely in heart, liver, spleen, lung, and kidney. Photodynamic antitumor efficacy of blank microsphere was realized. Microspheres afforded high antitumor efficacy without obvious toxic effects to normal organs, owing to its significantly increased MTX tumor retention time, low MTX levels in normal organs and strong photodynamic activity of PLA-phe-C60. These C60-phe-PLA microspheres may be promising for the efficacy with minimal side effects in future treatment of solid tumors. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Cancer is a major cause of death in human throughout the world. Current clinical strategies for treating cancer include surgery, radiation therapy, chemotherapy, and more recently, photodynamic therapy (PDT). PDT involves three components: a photosensitizer (PS), a special wavelength of drug-activating light and oxygen. A major advantage of PDT over conventional chemotherapy is that PS itself is minimally toxic in the absence of light. Furthermore, compared with radiotherapy, the activating light is non-ionizing and hence its effect on tissues without PS drug is not harmful [1–3]. Fullerene (C60), the third allotrope of carbon, are nano-scale carbon materials with unique photo-, electro-chemical, physical properties and low systemic toxicity [4–7]. In addition, C60 possess unique photochemical properties: under the irradiation of UV or visible light, C60 molecule is able to shift to the excitation triplet state and generate singlet and other active forms of oxygen [8,9]. Due to the enormous PDT potential of fullerene, there has been much interest in studying possible biological activities of fullerenes to use them as PS in medicine, for example, malonic acid derivatives of C60 have a obvious photosensitive effect and significant cytotoxicity in vitro experiments [10]. Recently, we reported the photocytotoxicity of fullerene aminoacid
⇑ Corresponding author. E-mail address: [email protected] (Z.-z. Zhang). http://dx.doi.org/10.1016/j.jphotobiol.2015.05.018 1011-1344/Ó 2015 Elsevier B.V. All rights reserved.
derivative, revealed that p38MAPK is activated by C60 nanoparticles through triggering reactive oxygens species generation, leading to cancer cell injuries in vitro [11]. In this work, PDT was conducted to obtain a synergistic effect on malignant tumor in C57 mice models by use of the C60 based drug delivery system. In most current clinical application, the PS is formulated in lipidic or organic excipients and given as an intravenously injection leading to unpredictable biodistribution [12,13]. While, biodegradable implants containing chemotherapeutic agents are clinically useful for both site-specific and systemic drug therapies in cancer. By intratumoral administration of an implant, it is possible to retain the drug in tumor at cytotoxicity levels for a long time and overcome the drawbacks, such as toxicity, metabolic deactivation and frequent, repeated intake of medicines [14,15]. Poly (lactic acid) (PLA), a biocompatible and biodegradable copolymer, has been mostly used for delivering drugs [16–18]. In this study, a fullerene L-phenylalanine derivative was synthesized, then fuctionalized by PLA, giving C60-phe-PLA with self-assembled ability, and then prepared microspheres consisting of Mitoxantrone (MTX) by dispersion–solvent diffusion method, in order to get a system for combination chemotherapy with PDT. MTX is an anthracycline anticancer drug. It has been effectively used in the treatment of ovarian and hepatic cancer, breast cancer, lymphoma and leukemia [19–21]. Although MTX has low toxicity, high doses of MTX or its frequent administration may cause lots
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potential toxic effects, one of the most serious adverse events associated with MTX treatment is cardiotoxicity [22,23]. Herein in this study, injectable C60-phe-PLA /MTX implants (Fig. 1) were developed and characterized by transmission electron microscopy (TEM), dynamic laser scattering (DLS), scanning electron microscopy (SEM) and its photodynamic efficacy, MTX release efficiency and its treatment effect in vivo were examined using melanoma tumor-bearing mice models. 2. Materials and methods 2.1. Materials Fullerene (C60, purity 95%) were purchased from Henan Fengyuan Chemicals Co. Ltd. Mitoxantrone HCl (MTX, 20120503, purity 98%) was gotten from Beijing Yi-He Biotech Co. Ltd. Poly (lactic acid) (PLA, average molecular weight 10,000– 18,000) was obtained from Sigma–Aldrich Co. LLC. Tin protochloride (SnCl2) was purchased from Aladdin chemistry Co. Ltd. Sulforhodamine B (SRB), DMEM cell culture medium, penicillin, streptomycin, fetal bovine serum (FBS) were bought from Gibco Invitrogen. L-phenylalanine (phe) and other reagents were acquired from China National Medicine Corporation Ltd. 2.2. Synthesis of C60-phe-PLA and characterization C60 Phenylalanine derivative (C60-phe) was synthesized according to the procedure of our previous study [11]. C60-phe was attached with PLA through ester bonds. C60-phe (100 mg) was suspended with dl-lactic acid (1 ml), 1% (w/w) SnCl2, PLA (1 g) was then added. The mixture was allowed to react at 110 °C for 1 h under N2 and then stirred at 140 °C for 2 h. After the conjugate was dissolved in dichloromethane, then the conjugate was precipitated using deionized water. The precipitated was purified by repeated rinsing with deionized water and filtrations to remove the unreacted reagents to obtain C60-phe-PLA complex. TEM (Tecnai G2 20, FEI) were used for morphological of C60-phe-PLA and C60-phe-PLA was dispersed in dichloromethane. FT-IR spectra were recorded in KBr pastilles on a Nicolet iS10 spectrometer (Thermo). Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Advance DPX300 (300 MHz) Fourier-transform NMR spectrometer (Bruker, Fallanden, Switzerland) from solutions in Dichloromethane-d2. The relative amount of PLA linked to C60-phe was tested using a thermal
gravimetric analysis (TGA, PerkinElmer) with the experimental conditions of scanning from 25 to 800 °C under nitrogen at a heating rate of 20 °C/min.
2.3. Microsphere formulation and characterization C60-phe-PLA (200 mg) was dissolved in 3 ml of dichloromethane. Then MTX saturated aqueous solution (1 ml) was added. The mixture solution was stirred at room temperature for 30 min. After that, the solution was dropped into 40 ml of glycerol by stirring at 1800 rpm for 10 min in an ice bath. The microsphere glycerol solution was dispersed in 5% (w/v) gelatin solution by stirring at 700 rpm for 2 h. The resulting microspheres were separated by filtration and then dried in vacuum at 50 °Cfor 24 h. Blank microspheres were prepared following the same procedure, but without adding MTX to C60-phe-PLA solution. Microspheres morphology was assessed with scanning electron microscopy (SEM). Dynamic lights scattering (DLS) measurements of particle size were carried out using a Zetasizer Nano-ZS90 light scattering instrument (Malvern Instruments, Enigma Business Park, UK). MTX content was determined by the following method. An accurately weighed amount of microspheres (about 10 mg) was dissolved in 3 ml of DMSO, the solution was centrifuged to remove precipitated polymer, and the drug concentration was analysed at 627 nm by UV spectrometer.
2.4. In vitro release studies Two groups were designed, one for 532 nm/laser (Diode laser, CW, Changchun laser research center, 100 mW/cm2, 1 min) everyday, the other without laser. The release experiments were conducted at 37 °C. 10 mg dried microspheres were accurately weighed and re-suspended in 20 ml release medium (PBS, pH 6.8), then was incubated at 37 °C with a shaking speed 100 rpm. At each specified intervals, 1 ml solution was drawn from the release medium, being replaced by the same volume of fresh PBS. The concentration of MTX released from microspheres into PBS solution was quantified using high performance liquid chromatography (HPLC, 1100 Agilent, USA) with the following conditions: an Eclipse XDB-C18 column (150 mm 4.6 mm, 5.0 lm); mobile phase acetate/methanol 64:36; column temperature 30 °C; detection wave length 627 nm; flow rate 1.0 ml/min.
Fig. 1. Scheme of fullerene-based multi-functional sustained-release microsphere and its bio-functions.
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Fig. 2. Characterization of C60-PHE-PLA. (A) FT-IR spectrum of C60-PHE (a), C60-phe-PLA (b); (B) 1H NMR of C60-phe-PLA; (C) 1H NMR of C60-phe; (D) and (E) TEM image of C60-phe and C60-phe-PLA; (F) TGA curves of C60- phe-PLA.
2.5. Evaluation of in vivo antitumor effects
2.6. Biodistribution studies
Xenograft tumor mouse model. All animal experiments were performed under a protocol approved by Henan laboratory animal center. Mice melanoma tumor models were generated by subcutaneous injection of 1 106 B16-F10 cells in 0.1 ml saline into the dorsal ventral of male C57 mice (18–22 g, Henan laboratory animal center). The mice were treated when the tumor volume reached 60–100 mm3 (7–10 days after tumor inoculation). In vivo, six therapeutic schedules (five mice per group) were designed as follows: Group 1 (blank microsphere); Group 2 (blank microsphere/532 nm laser); Group 3 (MTX loaded microsphere/532 nm laser, MTX dose: 2.5 mg/kg); Group 4 (saline); Group 5 (saline/532 nm laser). Group 6 (MTX/532 nm laser, MTX dose: 2.5 mg/kg). The mice were intratumoral injection every 6 days, the tumor regions were irradiated with 532 nm laser (100 mW/cm2, 1 min) every day. The tumor length and width were measured by a caliper every other day and calculated as the volume = (tumor length) (tumor width)2/2. Relative tumor growth rate (T/C, %) = (Treatment tumor volume at day 11 Treatment Tumor Volume at day 0)/(Control tumor volume at day 11 Control tumor Volume at day 0). After treatment for 11 days, the mice were killed to collect the tissues, and the collected tissues were soaked in 10% formalin solution, embedded with paraffin, then for H&E stain. Morphological changes were observed under a microscope.
Heart, liver, spleen, lung, kidney, brain, and tumor tissue homogenate were collected from tumor C57 mice after treatment of MTX loaded microsphere (MTX dose: 2.5 mg/kg) for 1 d, 2 d, 3 d, 4 d, 5 d, then centrifuged, the supernatant (0.2 ml) was placed into 10 ml centrifuge tubes. Methanol–acetonitrile (9:1, 0.2 ml) was added to the above tubes and centrifuged after mixing by Vortex, followed by centrifugation (12,000 rpm, 15 min). The supernatant (20 lL) was determined by HPLC under the above chromatographic conditions. MTX tissue distribution = Dose of MTX/Organ weight (lg/g). 2.7. Statistical analysis All data are expressed as the mean ± standard deviation (SD). The statistical significance of differences was calculated by ANOVA analysis, p < 0.05 was regarded as a significant difference. 3. Results and discussions 3.1. Synthesis and characterization of C60-phe-PLA C60-phe was synthesized according to the procedure of our previous study [4]. Carboxyl (ACOOH) groups on the external surface of the C60-phe allowed these nanoparticles to be functionalized with a layer of PLA through ester bonds. The results of FT-IR
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Fig. 3. Characterization of microspheres based on C60-PHE-PLA (a): Photomicrographs of MTX loaded microsphere; (b) and (c): SEM image of MTX loaded microsphere with different magnification; (d): Dynamic light scattering analysis of MTX loaded microsphere; (e): In vitro release profiles of MTX from microsphere.
showed new peaks at AOAC@O (1092 cm 1) and ACHAOA (1188 cm 1) in the spectrum of C60-phe-PLA (Fig. 2A, b) compared with C60-phe (Fig. 2A, a), suggesting that the functionalization of C60-phe with PLA was successful. The typical peaks are observed at 1.55 and 5.3 ppm which are attributable to methyl and methyne protons in PLA chains [24]; AC6AH protons in the benzene ring block (d/ppm, 7.5–7.8) were clearly seen (Fig. 2B), the chemical shift in the signal of the AC6AH protons in C60-phe-PLA is different from that of C60-phe (Fig. 2C), these results also evidenced that the functionalized process of C60-phe-PLA was successful. C60-phe-PLA was not soluble in water but exhibited stability in dichloromethane. A clear coating layer can be observed on the TEM image of the C60-phe-PLA composite (Fig. 2E), while no coating layer can be observed on the surface of C60-phe (Fig. 2D). The relative amount of PLA grafted onto the surface of C60-phe was tested by TGA. PLA degraded completely at about 390 °C (Fig. 2F), C60-phe and C60-phe-PLA showed about 21.53% and 78.32% weight losses at 390 °C, respectively, thus the relative amount of PLA grafted onto C60-phe was 56.78%.
3.2. Microsphere formulation and characterisation We developed biodegradable microspheres based on C60-phe-PLA, and MTX was adsorbed on microspheres through a simple physical adsorption. As the photomicrographs show (Fig. 3a), the loaded microspheres were spherical shape, blue color and dispersible in water. These microspheres process a smooth surfaces and non-porous (Fig. 3b and c). Moreover, particle sizes distribution exhibits 1.0–10.0 lm as confirmed by DLS, and the average particle size is 5.37 lm (Fig. 3d). MTX content was 0.618 mg/10 mg of microspheres.
To investigate the release kinetics of the MTX from microspheres, we incubated the microspheres in PBS (pH 6.8), MTX was released from microsphere in a steady and gradual way (Fig. 3e). During this period, the cumulative release amount of MTX reached 87.56% of total loadings after 15 days. The burst release within the first day was about 10.38%. It is well known that PLA is a biodegradable biopolymer, when temperatures are at or above its glass transition temperature (55°–62 °C), PLA in the presence of water undergoes chemical hydrolysis, progressively reducing polymer molecular weight and ultimately releasing lactic acid [25,26]. In this work, the temperature of microspheres in PBS did not change with irradiating. As seen in Fig. 3e, irradiating would not affect the drug release in vitro, no obvious difference between these two groups was found (p > 0.05). 3.3. Evaluation of in vivo antitumor effects Treatments were done by intratumoral injection of blank microsphere, MTX loaded microsphere, MTX and saline (MTX dose: 2.5 mg/kg, once every 6 days) into tumor-bearing mice. PDT had been applied in the treatment of some malignant tumors [27,28], so in the in vivo antitumor study, we just irradiated tumor site, and that would reduce the side effects on normal tissues and organs. As shown in Fig. 4a, time-related changes of average fractional tumor volumes (V/V0) in tumor volume were observed in all groups. After 11 days treatment, saline group showed (V/V0) of 8.26 ± 1.03, saline/532 nm laser group and blank microsphere group showed (V/V0) of 7.37 ± 1.27 and 6.65 ± 0.63, respectively, suggesting that 532 nm laser alone and blank microsphere without irradiating would not affect the tumor growth. Recently, fullerene and fullerene derivatives have been widely explored in PDT, for example, doxorubicin-loaded PEI-derivatized fullerene have an
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Fig. 4. In vivo PDT treatments (a) Relative tumor volume (V/V0) of mice in different treatment groups within 11 days; 1–6: saline, saline/532 nm laser, blank microsphere, MTX/532 nm laser, blank microsphere/532 nm laser; MTX loaded microsphere/532 nm laser. Inset, a photo of representative tumors of the mice with different treatments (b) Changes of body weight of mice in different groups during treatment; (c) H&E stained tumor tissues harvested from the mice with different treatments, 1–6: saline, saline/ 532 nm laser, blank microsphere, blank microsphere/532 nm laser, MTX/532 nm laser, MTX loaded microsphere/532 nm laser. H&E stained heart tissue harvested from the mice with different treatments, 7–12: saline, saline/532 nm laser, blank microsphere, blank microsphere/532 nm laser, MTX/532 nm laser, MTX loaded microsphere/532 nm laser. Data was presented as mean standard deviation (n = 5).
obvious photosensitive effect and significant cytotoxicity in vivo experiments [29]. In this work, PDT was also conducted in order to obtain a synergistic effect to malignant tumor in C57 mice models. Blank microsphere/532 nm laser resulted in V/V0 of 3.07 ± 1.07,
and had relative tumor growth rate (T/C) of 39.93%, showing that blank microspheres in PDT were achieved in vivo tumor treatment. Mice treated by MTX/532 nm laser showed (V/V0) of 5.04 ± 1.08; however, the treatment MTX loaded microsphere/532 nm laser
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Table 1 The biodistribution of MTX after intratumoral injection of MTX loaded microsphere based on C60-PHE-PLA in mice. Note: Data are presented as mean standard deviation (n = 5). p.s.: MTX tissue distribution = Dose of MTX/Organ weight (lg/g). Tissue
Tumor Heart Liver Spleen Lung Kidney
Time (days) 1
2
3
4
5
25.35 ± 1.17 (lg/g) 1.98 ± 0.43 (lg/g) – 6.23 ± 0.92 (lg/g) – –
16.02 ± 0.84 (lg/g) 1.85 ± 1.31 (lg/g) 3.50 ± 0.23 (lg/g) – – –
13.24 ± 0.22 (lg/g) – – – – –
10.66 ± 1.87 (lg/g) – – – – –
3.9 ± 1.12 (lg/g) – – – – –
resulted in V/V0 of 0.36 ± 0.16 and represented a T/C of 7.14%, suggesting that it is significantly more effective treatment efficacy than the other therapeutic groups (p < 0.05). The growth of tumor tissue was successfully suppressed by MTX loaded microsphere/532 nm laser. This high therapeutic efficacy originates from the high MTX accumulation and PDT from C60 in tumor tissue. Overall, these results not only demonstrated that C60-phe-PLA/MTX loaded microspheres are highly useful for chemotherapy of tumors but also revealed that C60-phe-PLA/MTX loaded microspheres were powerful for combination chemotherapy with photodynamic therapy of cancer in vivo. No weight loss was observed in all groups (Fig. 4b), implying that the toxicity of treatments was not obvious. The H&E staining tumor tissue in different treatment groups at day 11 post-treatment revealed that no damage was found in tumors of saline, saline/532 nm laser and blank microsphere alone (Fig. 4c). However, markedly increased necrotic tumor cells were found in all PDT treatment groups, and a large amount of cell death in tumor tissue was observed in the mice treated with MTX loaded microsphere/532 nm laser (Fig. 4c). The H&E staining heart tissue in MTX loaded microsphere/532 nm group showed vigorous growth, a tight arrangement and intact shape. While cell lysis and fragmentation to a certain extent was found in MTX/532 nm laser groups (Fig. 4c). These results indicated that microsphere was possible to maintain MTX stay in the tumor, overcome the cardiotoxicity of MTX, and also emerge photodynamic therapy in tumor.
3.4. Biodistribution MTX has cardiotoxicity, and is associated with decreased left ventricular ejection fraction and increased risk of congestive heart failure. To understand tumor treatment efficacy of MTX loaded microsphere, the biodistribution of MTX in tumor and various main organs was investigated. Significant differences of MTX tissue biodistributions were found at different days. Low MTX levels in the heart were found in 1–2 days after injection, whereas MTX was not detectable in next few days (Table 1). High MTX concentrations in the spleen were only found at 1 day after injection (Table 1). The distribution of MTX in the kidney and lung was not detectable at 1–5 day after injection (Table 1). MTX loaded microsphere showed noticeable MTX activity in tumor, the concentrations of MTX in tumor declined apparently slowly at 1–5 day after injection, whereas MTX levels in the other organs were much lower than in the tumor (p < 0.05), indicating that microsphere significantly increased MTX tumor retention time and reduced distribution in other tissues (Table 1). MTX releasing in vivo was much faster than in vitro, the complete microsphere could be observed at 15 day during in vitro release studies, while the complete microsphere could not be found at 5 day after injection, this might be related with PLA’s biodegradation in vivo.
4. Conclusion In summary, a fullerene-based multi-functional sustainedrelease microsphere was developed and applied this formulation to local cancer treatment in animals. The MTX loaded C60-phe-PLA microsphere showed negligible toxicity, and could serve not only as a powerful PDT but also as a sustained-release drug delivery carrier, indicating that there is a great potential of this microsphere for local cancer treatment.
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Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Preparation and characterization of injectable Mitoxantrone poly (lactic acid)/fullerene implants for in vivo chemo-photodynamic therapy Zhi Li, Fei-long Zhang, Li-li Pan, Xia-li Zhu, Zhen-zhong Zhang ⇑ School of Pharmaceutical Sciences, Zhengzhou University, 100 Kexue Avenue, Zhengzhou 450001, PR China
a r t i c l e
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Article history: Received 5 January 2015 Received in revised form 21 May 2015 Accepted 21 May 2015 Available online 27 May 2015 Keywords: Fullerene (C60) Mitoxantrone Poly (lactic acid) Chemo-photodynamic therapy Implants Sustained release
a b s t r a c t Fullerene (C60) L-phenylalanine derivative attached with poly (lactic acid) (C60-phe-PLA) was developed to prepare injectable Mitoxantrone (MTX) multifunctional implants. C60-phe-PLA was self-assembled to form microspheres consisting of a hydrophilic antitumor drug (MTX) and a hydrophobic block (C60) by dispersion–solvent diffusion method. The self-assembled microspheres showed sustained release pattern almost 15 days in vitro release experiments. According to the tissue distribution of C57BL mice after intratumoral administration of the microspheres, the MTX mainly distributed in tumors, and rarely in heart, liver, spleen, lung, and kidney. Photodynamic antitumor efficacy of blank microsphere was realized. Microspheres afforded high antitumor efficacy without obvious toxic effects to normal organs, owing to its significantly increased MTX tumor retention time, low MTX levels in normal organs and strong photodynamic activity of PLA-phe-C60. These C60-phe-PLA microspheres may be promising for the efficacy with minimal side effects in future treatment of solid tumors. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Cancer is a major cause of death in human throughout the world. Current clinical strategies for treating cancer include surgery, radiation therapy, chemotherapy, and more recently, photodynamic therapy (PDT). PDT involves three components: a photosensitizer (PS), a special wavelength of drug-activating light and oxygen. A major advantage of PDT over conventional chemotherapy is that PS itself is minimally toxic in the absence of light. Furthermore, compared with radiotherapy, the activating light is non-ionizing and hence its effect on tissues without PS drug is not harmful [1–3]. Fullerene (C60), the third allotrope of carbon, are nano-scale carbon materials with unique photo-, electro-chemical, physical properties and low systemic toxicity [4–7]. In addition, C60 possess unique photochemical properties: under the irradiation of UV or visible light, C60 molecule is able to shift to the excitation triplet state and generate singlet and other active forms of oxygen [8,9]. Due to the enormous PDT potential of fullerene, there has been much interest in studying possible biological activities of fullerenes to use them as PS in medicine, for example, malonic acid derivatives of C60 have a obvious photosensitive effect and significant cytotoxicity in vitro experiments [10]. Recently, we reported the photocytotoxicity of fullerene aminoacid
⇑ Corresponding author. E-mail address: [email protected] (Z.-z. Zhang). http://dx.doi.org/10.1016/j.jphotobiol.2015.05.018 1011-1344/Ó 2015 Elsevier B.V. All rights reserved.
derivative, revealed that p38MAPK is activated by C60 nanoparticles through triggering reactive oxygens species generation, leading to cancer cell injuries in vitro [11]. In this work, PDT was conducted to obtain a synergistic effect on malignant tumor in C57 mice models by use of the C60 based drug delivery system. In most current clinical application, the PS is formulated in lipidic or organic excipients and given as an intravenously injection leading to unpredictable biodistribution [12,13]. While, biodegradable implants containing chemotherapeutic agents are clinically useful for both site-specific and systemic drug therapies in cancer. By intratumoral administration of an implant, it is possible to retain the drug in tumor at cytotoxicity levels for a long time and overcome the drawbacks, such as toxicity, metabolic deactivation and frequent, repeated intake of medicines [14,15]. Poly (lactic acid) (PLA), a biocompatible and biodegradable copolymer, has been mostly used for delivering drugs [16–18]. In this study, a fullerene L-phenylalanine derivative was synthesized, then fuctionalized by PLA, giving C60-phe-PLA with self-assembled ability, and then prepared microspheres consisting of Mitoxantrone (MTX) by dispersion–solvent diffusion method, in order to get a system for combination chemotherapy with PDT. MTX is an anthracycline anticancer drug. It has been effectively used in the treatment of ovarian and hepatic cancer, breast cancer, lymphoma and leukemia [19–21]. Although MTX has low toxicity, high doses of MTX or its frequent administration may cause lots
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potential toxic effects, one of the most serious adverse events associated with MTX treatment is cardiotoxicity [22,23]. Herein in this study, injectable C60-phe-PLA /MTX implants (Fig. 1) were developed and characterized by transmission electron microscopy (TEM), dynamic laser scattering (DLS), scanning electron microscopy (SEM) and its photodynamic efficacy, MTX release efficiency and its treatment effect in vivo were examined using melanoma tumor-bearing mice models. 2. Materials and methods 2.1. Materials Fullerene (C60, purity 95%) were purchased from Henan Fengyuan Chemicals Co. Ltd. Mitoxantrone HCl (MTX, 20120503, purity 98%) was gotten from Beijing Yi-He Biotech Co. Ltd. Poly (lactic acid) (PLA, average molecular weight 10,000– 18,000) was obtained from Sigma–Aldrich Co. LLC. Tin protochloride (SnCl2) was purchased from Aladdin chemistry Co. Ltd. Sulforhodamine B (SRB), DMEM cell culture medium, penicillin, streptomycin, fetal bovine serum (FBS) were bought from Gibco Invitrogen. L-phenylalanine (phe) and other reagents were acquired from China National Medicine Corporation Ltd. 2.2. Synthesis of C60-phe-PLA and characterization C60 Phenylalanine derivative (C60-phe) was synthesized according to the procedure of our previous study [11]. C60-phe was attached with PLA through ester bonds. C60-phe (100 mg) was suspended with dl-lactic acid (1 ml), 1% (w/w) SnCl2, PLA (1 g) was then added. The mixture was allowed to react at 110 °C for 1 h under N2 and then stirred at 140 °C for 2 h. After the conjugate was dissolved in dichloromethane, then the conjugate was precipitated using deionized water. The precipitated was purified by repeated rinsing with deionized water and filtrations to remove the unreacted reagents to obtain C60-phe-PLA complex. TEM (Tecnai G2 20, FEI) were used for morphological of C60-phe-PLA and C60-phe-PLA was dispersed in dichloromethane. FT-IR spectra were recorded in KBr pastilles on a Nicolet iS10 spectrometer (Thermo). Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Advance DPX300 (300 MHz) Fourier-transform NMR spectrometer (Bruker, Fallanden, Switzerland) from solutions in Dichloromethane-d2. The relative amount of PLA linked to C60-phe was tested using a thermal
gravimetric analysis (TGA, PerkinElmer) with the experimental conditions of scanning from 25 to 800 °C under nitrogen at a heating rate of 20 °C/min.
2.3. Microsphere formulation and characterization C60-phe-PLA (200 mg) was dissolved in 3 ml of dichloromethane. Then MTX saturated aqueous solution (1 ml) was added. The mixture solution was stirred at room temperature for 30 min. After that, the solution was dropped into 40 ml of glycerol by stirring at 1800 rpm for 10 min in an ice bath. The microsphere glycerol solution was dispersed in 5% (w/v) gelatin solution by stirring at 700 rpm for 2 h. The resulting microspheres were separated by filtration and then dried in vacuum at 50 °Cfor 24 h. Blank microspheres were prepared following the same procedure, but without adding MTX to C60-phe-PLA solution. Microspheres morphology was assessed with scanning electron microscopy (SEM). Dynamic lights scattering (DLS) measurements of particle size were carried out using a Zetasizer Nano-ZS90 light scattering instrument (Malvern Instruments, Enigma Business Park, UK). MTX content was determined by the following method. An accurately weighed amount of microspheres (about 10 mg) was dissolved in 3 ml of DMSO, the solution was centrifuged to remove precipitated polymer, and the drug concentration was analysed at 627 nm by UV spectrometer.
2.4. In vitro release studies Two groups were designed, one for 532 nm/laser (Diode laser, CW, Changchun laser research center, 100 mW/cm2, 1 min) everyday, the other without laser. The release experiments were conducted at 37 °C. 10 mg dried microspheres were accurately weighed and re-suspended in 20 ml release medium (PBS, pH 6.8), then was incubated at 37 °C with a shaking speed 100 rpm. At each specified intervals, 1 ml solution was drawn from the release medium, being replaced by the same volume of fresh PBS. The concentration of MTX released from microspheres into PBS solution was quantified using high performance liquid chromatography (HPLC, 1100 Agilent, USA) with the following conditions: an Eclipse XDB-C18 column (150 mm 4.6 mm, 5.0 lm); mobile phase acetate/methanol 64:36; column temperature 30 °C; detection wave length 627 nm; flow rate 1.0 ml/min.
Fig. 1. Scheme of fullerene-based multi-functional sustained-release microsphere and its bio-functions.
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Fig. 2. Characterization of C60-PHE-PLA. (A) FT-IR spectrum of C60-PHE (a), C60-phe-PLA (b); (B) 1H NMR of C60-phe-PLA; (C) 1H NMR of C60-phe; (D) and (E) TEM image of C60-phe and C60-phe-PLA; (F) TGA curves of C60- phe-PLA.
2.5. Evaluation of in vivo antitumor effects
2.6. Biodistribution studies
Xenograft tumor mouse model. All animal experiments were performed under a protocol approved by Henan laboratory animal center. Mice melanoma tumor models were generated by subcutaneous injection of 1 106 B16-F10 cells in 0.1 ml saline into the dorsal ventral of male C57 mice (18–22 g, Henan laboratory animal center). The mice were treated when the tumor volume reached 60–100 mm3 (7–10 days after tumor inoculation). In vivo, six therapeutic schedules (five mice per group) were designed as follows: Group 1 (blank microsphere); Group 2 (blank microsphere/532 nm laser); Group 3 (MTX loaded microsphere/532 nm laser, MTX dose: 2.5 mg/kg); Group 4 (saline); Group 5 (saline/532 nm laser). Group 6 (MTX/532 nm laser, MTX dose: 2.5 mg/kg). The mice were intratumoral injection every 6 days, the tumor regions were irradiated with 532 nm laser (100 mW/cm2, 1 min) every day. The tumor length and width were measured by a caliper every other day and calculated as the volume = (tumor length) (tumor width)2/2. Relative tumor growth rate (T/C, %) = (Treatment tumor volume at day 11 Treatment Tumor Volume at day 0)/(Control tumor volume at day 11 Control tumor Volume at day 0). After treatment for 11 days, the mice were killed to collect the tissues, and the collected tissues were soaked in 10% formalin solution, embedded with paraffin, then for H&E stain. Morphological changes were observed under a microscope.
Heart, liver, spleen, lung, kidney, brain, and tumor tissue homogenate were collected from tumor C57 mice after treatment of MTX loaded microsphere (MTX dose: 2.5 mg/kg) for 1 d, 2 d, 3 d, 4 d, 5 d, then centrifuged, the supernatant (0.2 ml) was placed into 10 ml centrifuge tubes. Methanol–acetonitrile (9:1, 0.2 ml) was added to the above tubes and centrifuged after mixing by Vortex, followed by centrifugation (12,000 rpm, 15 min). The supernatant (20 lL) was determined by HPLC under the above chromatographic conditions. MTX tissue distribution = Dose of MTX/Organ weight (lg/g). 2.7. Statistical analysis All data are expressed as the mean ± standard deviation (SD). The statistical significance of differences was calculated by ANOVA analysis, p < 0.05 was regarded as a significant difference. 3. Results and discussions 3.1. Synthesis and characterization of C60-phe-PLA C60-phe was synthesized according to the procedure of our previous study [4]. Carboxyl (ACOOH) groups on the external surface of the C60-phe allowed these nanoparticles to be functionalized with a layer of PLA through ester bonds. The results of FT-IR
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Fig. 3. Characterization of microspheres based on C60-PHE-PLA (a): Photomicrographs of MTX loaded microsphere; (b) and (c): SEM image of MTX loaded microsphere with different magnification; (d): Dynamic light scattering analysis of MTX loaded microsphere; (e): In vitro release profiles of MTX from microsphere.
showed new peaks at AOAC@O (1092 cm 1) and ACHAOA (1188 cm 1) in the spectrum of C60-phe-PLA (Fig. 2A, b) compared with C60-phe (Fig. 2A, a), suggesting that the functionalization of C60-phe with PLA was successful. The typical peaks are observed at 1.55 and 5.3 ppm which are attributable to methyl and methyne protons in PLA chains [24]; AC6AH protons in the benzene ring block (d/ppm, 7.5–7.8) were clearly seen (Fig. 2B), the chemical shift in the signal of the AC6AH protons in C60-phe-PLA is different from that of C60-phe (Fig. 2C), these results also evidenced that the functionalized process of C60-phe-PLA was successful. C60-phe-PLA was not soluble in water but exhibited stability in dichloromethane. A clear coating layer can be observed on the TEM image of the C60-phe-PLA composite (Fig. 2E), while no coating layer can be observed on the surface of C60-phe (Fig. 2D). The relative amount of PLA grafted onto the surface of C60-phe was tested by TGA. PLA degraded completely at about 390 °C (Fig. 2F), C60-phe and C60-phe-PLA showed about 21.53% and 78.32% weight losses at 390 °C, respectively, thus the relative amount of PLA grafted onto C60-phe was 56.78%.
3.2. Microsphere formulation and characterisation We developed biodegradable microspheres based on C60-phe-PLA, and MTX was adsorbed on microspheres through a simple physical adsorption. As the photomicrographs show (Fig. 3a), the loaded microspheres were spherical shape, blue color and dispersible in water. These microspheres process a smooth surfaces and non-porous (Fig. 3b and c). Moreover, particle sizes distribution exhibits 1.0–10.0 lm as confirmed by DLS, and the average particle size is 5.37 lm (Fig. 3d). MTX content was 0.618 mg/10 mg of microspheres.
To investigate the release kinetics of the MTX from microspheres, we incubated the microspheres in PBS (pH 6.8), MTX was released from microsphere in a steady and gradual way (Fig. 3e). During this period, the cumulative release amount of MTX reached 87.56% of total loadings after 15 days. The burst release within the first day was about 10.38%. It is well known that PLA is a biodegradable biopolymer, when temperatures are at or above its glass transition temperature (55°–62 °C), PLA in the presence of water undergoes chemical hydrolysis, progressively reducing polymer molecular weight and ultimately releasing lactic acid [25,26]. In this work, the temperature of microspheres in PBS did not change with irradiating. As seen in Fig. 3e, irradiating would not affect the drug release in vitro, no obvious difference between these two groups was found (p > 0.05). 3.3. Evaluation of in vivo antitumor effects Treatments were done by intratumoral injection of blank microsphere, MTX loaded microsphere, MTX and saline (MTX dose: 2.5 mg/kg, once every 6 days) into tumor-bearing mice. PDT had been applied in the treatment of some malignant tumors [27,28], so in the in vivo antitumor study, we just irradiated tumor site, and that would reduce the side effects on normal tissues and organs. As shown in Fig. 4a, time-related changes of average fractional tumor volumes (V/V0) in tumor volume were observed in all groups. After 11 days treatment, saline group showed (V/V0) of 8.26 ± 1.03, saline/532 nm laser group and blank microsphere group showed (V/V0) of 7.37 ± 1.27 and 6.65 ± 0.63, respectively, suggesting that 532 nm laser alone and blank microsphere without irradiating would not affect the tumor growth. Recently, fullerene and fullerene derivatives have been widely explored in PDT, for example, doxorubicin-loaded PEI-derivatized fullerene have an
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Fig. 4. In vivo PDT treatments (a) Relative tumor volume (V/V0) of mice in different treatment groups within 11 days; 1–6: saline, saline/532 nm laser, blank microsphere, MTX/532 nm laser, blank microsphere/532 nm laser; MTX loaded microsphere/532 nm laser. Inset, a photo of representative tumors of the mice with different treatments (b) Changes of body weight of mice in different groups during treatment; (c) H&E stained tumor tissues harvested from the mice with different treatments, 1–6: saline, saline/ 532 nm laser, blank microsphere, blank microsphere/532 nm laser, MTX/532 nm laser, MTX loaded microsphere/532 nm laser. H&E stained heart tissue harvested from the mice with different treatments, 7–12: saline, saline/532 nm laser, blank microsphere, blank microsphere/532 nm laser, MTX/532 nm laser, MTX loaded microsphere/532 nm laser. Data was presented as mean standard deviation (n = 5).
obvious photosensitive effect and significant cytotoxicity in vivo experiments [29]. In this work, PDT was also conducted in order to obtain a synergistic effect to malignant tumor in C57 mice models. Blank microsphere/532 nm laser resulted in V/V0 of 3.07 ± 1.07,
and had relative tumor growth rate (T/C) of 39.93%, showing that blank microspheres in PDT were achieved in vivo tumor treatment. Mice treated by MTX/532 nm laser showed (V/V0) of 5.04 ± 1.08; however, the treatment MTX loaded microsphere/532 nm laser
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Table 1 The biodistribution of MTX after intratumoral injection of MTX loaded microsphere based on C60-PHE-PLA in mice. Note: Data are presented as mean standard deviation (n = 5). p.s.: MTX tissue distribution = Dose of MTX/Organ weight (lg/g). Tissue
Tumor Heart Liver Spleen Lung Kidney
Time (days) 1
2
3
4
5
25.35 ± 1.17 (lg/g) 1.98 ± 0.43 (lg/g) – 6.23 ± 0.92 (lg/g) – –
16.02 ± 0.84 (lg/g) 1.85 ± 1.31 (lg/g) 3.50 ± 0.23 (lg/g) – – –
13.24 ± 0.22 (lg/g) – – – – –
10.66 ± 1.87 (lg/g) – – – – –
3.9 ± 1.12 (lg/g) – – – – –
resulted in V/V0 of 0.36 ± 0.16 and represented a T/C of 7.14%, suggesting that it is significantly more effective treatment efficacy than the other therapeutic groups (p < 0.05). The growth of tumor tissue was successfully suppressed by MTX loaded microsphere/532 nm laser. This high therapeutic efficacy originates from the high MTX accumulation and PDT from C60 in tumor tissue. Overall, these results not only demonstrated that C60-phe-PLA/MTX loaded microspheres are highly useful for chemotherapy of tumors but also revealed that C60-phe-PLA/MTX loaded microspheres were powerful for combination chemotherapy with photodynamic therapy of cancer in vivo. No weight loss was observed in all groups (Fig. 4b), implying that the toxicity of treatments was not obvious. The H&E staining tumor tissue in different treatment groups at day 11 post-treatment revealed that no damage was found in tumors of saline, saline/532 nm laser and blank microsphere alone (Fig. 4c). However, markedly increased necrotic tumor cells were found in all PDT treatment groups, and a large amount of cell death in tumor tissue was observed in the mice treated with MTX loaded microsphere/532 nm laser (Fig. 4c). The H&E staining heart tissue in MTX loaded microsphere/532 nm group showed vigorous growth, a tight arrangement and intact shape. While cell lysis and fragmentation to a certain extent was found in MTX/532 nm laser groups (Fig. 4c). These results indicated that microsphere was possible to maintain MTX stay in the tumor, overcome the cardiotoxicity of MTX, and also emerge photodynamic therapy in tumor.
3.4. Biodistribution MTX has cardiotoxicity, and is associated with decreased left ventricular ejection fraction and increased risk of congestive heart failure. To understand tumor treatment efficacy of MTX loaded microsphere, the biodistribution of MTX in tumor and various main organs was investigated. Significant differences of MTX tissue biodistributions were found at different days. Low MTX levels in the heart were found in 1–2 days after injection, whereas MTX was not detectable in next few days (Table 1). High MTX concentrations in the spleen were only found at 1 day after injection (Table 1). The distribution of MTX in the kidney and lung was not detectable at 1–5 day after injection (Table 1). MTX loaded microsphere showed noticeable MTX activity in tumor, the concentrations of MTX in tumor declined apparently slowly at 1–5 day after injection, whereas MTX levels in the other organs were much lower than in the tumor (p < 0.05), indicating that microsphere significantly increased MTX tumor retention time and reduced distribution in other tissues (Table 1). MTX releasing in vivo was much faster than in vitro, the complete microsphere could be observed at 15 day during in vitro release studies, while the complete microsphere could not be found at 5 day after injection, this might be related with PLA’s biodegradation in vivo.
4. Conclusion In summary, a fullerene-based multi-functional sustainedrelease microsphere was developed and applied this formulation to local cancer treatment in animals. The MTX loaded C60-phe-PLA microsphere showed negligible toxicity, and could serve not only as a powerful PDT but also as a sustained-release drug delivery carrier, indicating that there is a great potential of this microsphere for local cancer treatment.
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