Synthesis of Calcium Phosphate Microspheres Using an Ultrasonic

Downloaded by UNIV OF FLORIDA on October 26, 2017 | http://pubs.acs.org Publication Date (Web): October 25, 2017 | doi: 10.1021/bk-2017-1253.ch006

Chapter 6

Synthesis of Calcium Phosphate Microspheres Using an Ultrasonic Spray–Pyrolysis Technique and Their Application as Novel Anti-Angiogenic Chemoembolization Agents for Cancer Treatment Mamoru Aizawa,*,1 Michiyo Honda,1 and Makoto Emoto2 1Department

of Applied Chemistry, School of Science and Technology, Meiji University, 1-1-1, Higashimita, Tama-ku, Kawasaki, Japan 2Department of Health and Welfare and Division of Preventive Medicine, Fukuoka Sanno Hospital, International University of Health and Welfare, 1-7-4, Momochihama, Sawara-ku, Fukuoka, Japan *E-mail: [email protected]

The purpose of the present study was to develop a novel process for chemoembolization to improve the therapeutic effectiveness and safety profile of cancer treatment. A chemoembolization approach was designed for treating human solid tumors using biodegradable calcium phosphate microspheres (hereafter, CPMs) prepared using an ultrasonic spray–pyrolysis technique combined with an anti-angiogenic agent (TNP-470; Takeda, Japan) that inhibits tumor vasculature formation in vivo. FU-MMT-3 human uterine sarcoma cells were used in this study because this type of tumor is aggressive and responds poorly to radiotherapy and currently used chemotherapy agents. In this chapter, preparation of biodegradable CPMs and their powder properties, including drug release characteristics, will be reviewed. We performed biological evaluations both in vitro and in vivo using CPMs loaded with TNP-470. The results of these tests indicated that microspheres loaded with TNP-470 inhibit i) the proliferation of FU-MMT-3 cells in a tumor model and ii) tumor enlargement in a model of nude mice injected with FU-MMT-3 cells. Biological evaluations demonstrated that

© 2017 American Chemical Society Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

CPMs loaded with TNP-470 exhibit excellent anti-tumorigenic effects. In addition to the above, this chapter will discuss i) the effects of particle size and CPM distribution on anti-angiogenic chemoembolization, ii) the creation of porous advanced carrier CPMs with nano-pores on the surface prepared via salt-assisted ultrasonic spray–pyrolysis, and iii) our future work.


Introduction Hydroxyapatite (Ca10(PO4)6(OH)2; HAp) and β-tricalcium phosphate (β-Ca3(PO4)2; β-TCP) are widely used as biomaterials substituting for human hard tissues (1). We previously synthesized HAp and other apatite-family compounds using an ultrasonic spray–pyrolysis (USSP) technique and examined the properties of the resulting powders (2–5). USSP techniques can be used to prepare powders via the liquid phase. USSP has the advantage of enabling the instantaneous stoichiometric preparation of homogeneous compounds by spraying solutions containing desired amounts of cations into the hot zone of an electric furnace (4, 5). Moreover, USSP is simple, provides a narrow particle size distribution, and can be used to prepare hollow spherical particles. Using USSP, we have thus far synthesized hollow biphasic calcium phosphate microspheres (hereafter, CPMs) consisting of β-TCP and calcium-deficient hydroxyapatite (Ca10-x(PO4)6-x(OH)2-x•nH2O; DAp) (6). In the present investigation, we explored the use of the resulting hollow biphasic microspheres as carriers for a drug delivery system (DDS) applicable to the medical treatment of cancers. Cancer is a serious worldwide problem. Since 1980, cancer has been the leading cause of mortality in Japan. Although uncommon, sarcomas (including carcinosarcomas) are the most aggressive neoplasms among uterine malignancies (7, 8). These tumors respond poorly to radiotherapy and any of the currently used chemotherapeutic agents with substantial toxic effects (7–10). The overall 5-year survival rate for all stages of uterine sarcomas is less than 40%, which is significantly lower than that for other uterine cancers (7). Except for the early stages, no standard treatment for these tumors has been developed; thus, new therapeutic strategies are urgently needed. Recent studies, including those of the authors, have shown that rapid growth and early metastasis of uterine sarcomas might be associated with high angiogenic potential in comparison with other uterine malignancies (10–12). The recent introduction of anti-angiogenesis therapies into clinical practice represented a turning point in cancer therapy (13). Emoto et al. reported that the agent TNP-470 (Takeda, Japan) is effective for anti-angiogenic therapy against highly aggressive human uterine carcinosarcomas both in vitro and in vivo (14–16). TNP-470 is a low-molecular-weight synthetic analogue of fumagillin, a natural compound secreted by Aspergillus fumigatus (17), and inhibits angiogenesis via endothelial cell cycle arrest in the late G1 phase (18). Methionine aminopeptidase2 has been identified as a molecular target of TNP-470 (19). TNP-470 exhibits antitumor activity against various human malignancies both in vitro and in vivo (20–24). 108 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Radiotherapy is currently one of the most effective means of treating cancers. For deep-seated cancers, however, external irradiation provides only small doses and often damages healthy tissues. Therefore, chemoembolization approaches that employ biomaterials loaded with anti-angiogenic agents may be effective for treating deep-seated solid tumors. β-TCP is a well-known biodegradable calcium phosphate ceramic material. Some studies have shown that TCP is an ideal biomaterial because it dissolves gradually without any cytotoxic or allergenic effects and elicits no immune response (25–27). Thus, we selected biodegradable, hollow CPMs as carriers for a DDS suitable for cancer treatment. Our goal is to develop novel chemoembolization processes to improve the therapeutic effectiveness and safety profile of cancer treatments. This chapter will discuss a chemoembolization approach designed for use in treating human solid tumors. The approach involves combining biodegradable, hollow CPMs prepared using a USSP technique and the anti-angiogenic agent TNP-470, which can inhibit tumor vasculature formation in vivo (28, 29). Figure 1 illustrates the process using a modification of the image reported by Kawashita et al. (30). In addition, we will discuss the effect of CPM particle size and distribution on anti-angiogenic chemoembolization (31) and the production of porous CPMs with nano-pores on the surface prepared via a salt-assisted USSP (SAUSP) approach for use as an advanced carrier (32–34), together with our future work.

Figure 1. Our novel chemoembolization approach involved combining biodegradable hollow CPMs prepared using an ultrasonic spray–pyrolysis technique with an anti-angiogenic agent (TNP-470) that inhibits tumor vasculature formation. 109 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Preparation of CPMs Containing an Anti-Angiogenic Agent and Their Application in Chemoembolization CPMs were prepared for anti-tumorigenesis experiments as previously reported (6, 28, 29). The starting solution, with a Ca/P ratio of 1.50, was prepared by mixing Ca(NO3)2, (NH4)2HPO4, and HNO3 to final concentrations of 0.60, 0.40, and 0.10 mol·dm−3, respectively. As shown in Figure 2(a), the lower-furnace temperature for drying the generated droplets was fixed at 300ºC, and the upper-furnace temperature for pyrolysis of the dried droplets was set at 850ºC. The solution was sprayed into the heating zone using an ultrasonic vibrator operated at a frequency of 2.4 MHz, and then the sprayed droplets were dried and pyrolyzed to form the CPMs, as shown in Figure 2(b). The resulting microspheres were washed with pure water and freeze dried to prepare the “washed powder” for analyses of anti-tumorigenic effects.

Figure 2. Synthesis of CPMs and their powder properties. (a) Overview of the USSP apparatus, (b) model diagram of CPM formation, (c) SEM image, and (d) TEM image.

The results of X-ray diffraction (XRD) analyses demonstrate that the resulting CPMs were biphasic, composed of β-TCP (~50 mass%) and CDAp (~50 mass%). Both β-TCP and DAp are well-known biodegradable ceramics. The Ca/P molar ratio, as determined by X-ray fluorescence spectrometry, was 1.49, in good agreement with the nominal composition of the starting solution. Although the powder before washing contained NO3− groups derived from Ca(NO3)2 in the starting material, the results of Fourier-transform infrared spectroscopy analyses indicated that the NO3− groups were removed by washing. The specific surface area (SSA) of the powder increased from ~10 m2·g−1 before washing to ~20 m2·g−1 after washing. Figure 2(c) and (d) show the particle morphology of the washed 110 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


powder as determined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), respectively. Figure 2(c) shows that the powder was composed of microspheres with a diameter of about 1 µm. When observed by TEM, the microspheres appeared transparent, suggesting that they were hollow. We then performed biological evaluations of the resulting hollow CPMs. The anti-angiogenic agent TNP-470 was dissolved in ethanol at 100, 500, and 1000 ppm (control, 0 ppm). TNP-470 was loaded by adding microspheres (0.05 g) to the ethanol solutions (1 cm3), followed by freeze drying. Microspheres loaded with TNP-470 were used as sample powders for in vitro and in vivo evaluations of anti-tumorigenic activity. In order to maintain the stability of TNP-470 in the medium used for cell culture, we added a medium-chain triglyceride (MCTG, ncaprylic acid) to some of the sample powders (35). The drug release profile of the TNP-470–loaded TCP microspheres was examined over a period of 25 h. The amount of TNP-470 in the supernatant was determined by high-performance liquid chromatography (HPLC). Seventy-five percent of the total TNP-470 was released relatively rapidly from the microspheres, within 30 min, and the remaining 25% was released more slowly up to 25 h following immersion, after which no TNP-470 was detected in the supernatant. Five samples of CPMs without TNP-470 and loaded with TNP-470 were evaluated, with a polystyrene cell culture plate serving as a control: i) microspheres (washed powder), ii) microspheres loaded with 1000 ppm TNP-470, iii) microspheres loaded with 500 ppm TNP-470/1 v/v% MCTG, iv) microspheres loaded with 100 ppm TNP-470, and v) microspheres loaded with 100 ppm TNP-470/1 v/v% MCTG. The anti-angiogenic activity of the microspheres was examined using FU-MMT-3 cells, which were established by Emoto et al. as a tumor model (36). A total of 5 × 105 cells were seeded in a 12-well plate and cultured for 1 day, and then the five different powder samples were set on Transwell® (Corning) membranes dipped in D-MEM/F12 (10% FBS) and cultured at 37°C in a 5% CO2 atmosphere for an additional 1 or 3 days, as illustrated in Figure 3(a). Cell proliferation was examined by counting cells using an erythrocytometer, and cell morphology was observed by phase-contrast microscopy. Powder compacts were prepared using microspheres loaded with various concentrations of TNP-470, and then the cell-culture test was performed. FU-MMT-3 cells were seeded in a 12-well plate and cultured for 1 d, and then the powder compacts were placed on the Transwell® membranes and cultured for up to 3 additional days. The results are shown in Figure 3(b). The cells proliferated in the control and powder compacts without TNP-470; however, cell proliferation was inhibited by the powder compacts of microspheres containing 100 ppm TNP-470 (Figure 3(b)). The number of dead cells increased with increasing TNP-470 concentration. Notably, the number of proliferating cells decreased drastically in the case of powder compacts of microspheres containing 500 and 1000 ppm TNP-470. The observed inhibition of cell growth is believed to have been due to the release of TNP-470 into the medium. Figure 3(c) and (d) show the morphologies of cells cultured with control and CPMs loaded with 1000 ppm TNP-470 after 3 days, respectively. In Figure 3(d), we can observe many the rounded-shaped cells. The excess TNP-470 may express the cytotoxicity. 111 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 3. In vitro evaluation of CPMs loaded with TNP-470. (a) Testing of cytotoxicity using Transwell® membranes, (b) cell proliferation, and (c, d) morphologies of cells after cell culture for 3 days; (c) Control, (d) CPMs loaded with 1000 ppm TNP-470.

Next, we examined the in vivo anti-tumorigenic activity of the microspheres using nude mice harboring tumors resulting from injection of FU-MMT-3 cells (37). To prepare the animal model, 6 × 104 FU-MMT-3 cells were injected subcutaneously into nude mice (weight ~20 g each), and the mice were then bred for 1 week. Washed powder and microspheres loaded with 1000 ppm TNP-470 (each 25 mg) were suspended in physiologic saline (1 cm3), and then 0.4 cm3 of the resulting suspension was injected around the tumor sites. The size of the tumors was measured using calipers over a period of 8 weeks and compared with control mice (no treatment) and mice injected with TNP-470 only. Based on the in vitro findings, we performed in vivo evaluations using microspheres prepared with 1000 ppm TNP-470. First, FU-MMT-3 cells were injected subcutaneously into nude mice (weight ~20 g) to induce the formation of tumors. After 1 week, microspheres loaded with TNP-470 were suspended in physiologic saline and then injected around the tumors. Figure 4(a) shows the change in tumor size over time. The control in Figure 4(a) refers to the case of no treatment after injection of FU-MMT-3 cells. The tumor size increased only minimally in the mice treated with microspheres loaded with TNP-470, in clear contrast to the cases of the control, injection of TNP-470 alone, and injection of empty microspheres alone. These results indicate that CPMs containing TNP-470 exhibit anti-tumorigenic effects. 112 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 4. In vivo evaluation of CPMs loaded with TNP-470. (a) Change in tumor volume over a period of 8 weeks. (b, c) Typical histologic images.

Histologic evaluations revealed that the CPMs were embolized in the feeding arteries of the xenografts in both treatment groups, with or without TNP-470. Fibrosis with granuloma formation and lymphocytic infiltration was observed in the lumen of the feeding arteries; no injury caused by microspheres was found in the vessel walls (Figure 4(b)). The TCP microspheres appeared as amorphous basophilic material upon hematoxylin and eosin staining and exhibited marked embolization in tumor microvessels in all mice of the CPM treatment groups (Figure 4(c)). Destruction of tumor microvessels and areas of coagulative necrosis were observed in tumors treated with TCP microspheres, with or without TNP-470. No significant loss of body weight was observed in any of the mice treated with TNP-470–loaded TCP microspheres compared with mice treated with TNP-470 alone. The mean body weight at 56 days in mice treated with TNP-470 alone, however, was 16.9 ± 2.55 g, which was significantly lower than that of mice treated with TNP- 470–loaded microspheres (25.17 ± 0.68 g) (p = 0.01). As indicated above, CPMs consisting of biphasic biodegradable β-TCP and DAp were used in the present study both as drug carriers and as an embolization material. In vitro, TNP-470 may be released in a sustainable two-step manner from the internal space and external surface of the hollow microspheres. Although the powder compacts of CPMs alone did not inhibit the proliferation of FU-MMT-3 cells in vitro (Figure 3), treatment with microspheres alone significantly inhibited the growth of the FU-MMT-3 xenografts in vivo, compared with the control (Figure 4). These results suggest that although CPMs alone do not inhibit sarcoma cell proliferation, a direct anti-tumor effect associated with this material occurs in vivo as a result of embolization. This is supported by histopathologic analyses, 113 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

which revealed marked embolization of microspheres in the feeding arteries of the xenografts, as well as in many tumor microvessels (Figure 4). This strong embolization effect exhibited by the CPMs alone is potentially useful for cancer treatment. In summary, a novel chemoembolization approach was developed for treating human solid tumors by combining biodegradable CPMs with an anti-angiogenic agent to inhibit tumor vasculature formation in vivo.


Effect of CPM Particle Size and Distribution on Anti-Angiogenic Chemoembolization In the previous section, we described the initial design of a novel biomaterial composed of resorbable hollow ceramic microspheres loaded with TNP-470 to target the tumor vasculature. We demonstrated the usefulness and safety of this approach using a highly aggressive human uterine sarcoma xenograft model. In the next step in the development of this new chemoembolization approach using CPMs, we designed a new type of anti-angiogenic microspheres as an advanced model to target (or measure) vascular heterogeneity in highly aggressive and highly angiogenic solid tumors. In this section, we describe the effect of CPM particle size and distribution on the anti-tumorigenic effect of CPMs loaded with TNP-470.

Table 1. Preparation and powder properties of CPMs of different sizes loaded with TNP-470.a Sample name 1510(-) 1510(+) 6040(-) 6040(+) 9060(-) 9060(+)

CPM (Ca: 0.15, PO4: 0.10)

(Ca: 0.60, PO4: 0.40)

(Ca: 0.90, PO4: 0.60)

Mix(-)

TNP-470 Non-loaded Loaded Non-loaded Loaded Non-loaded Loaded Non-loaded

Mix(+)

(Ca: 0.15, PO4: 0.10) (Ca: 0.60, PO4: 0.40) (Ca: 0.90, PO4: 0.60)

Loaded

Crystalline phases

Particle sizec

46% CDApb + 54% β-TCP

1.8 µm


2.6 µm


3.0 µm

Mixed powder prepared from the above three CPMs (1:1:1 [w/w/w])

1.8 µm + 2.6 µm + 3.0 µm

(Ca:0.60, PO4:0.40) → Ca2+: 0.60 mol/dm3, PO43-: 0.40 mol/dm3. deficient hydroxyapatite. c Particle size: median size.

a

b

CDAp: Calcium-

CPMs with different particle sizes (i.e., 1510[−], 6040[−], and 9060[−]), were prepared by varying the concentration of Ca2+ (0.15, 0.60, 0.90 mol·dm−3) and PO43− (0.10, 0.40, 0.60 mol·dm−3) ions in the starting solution, as previously reported (6, 28, 29). The resulting microspheres were washed with pure water and 114 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


then freeze dried. Next, TNP-470 was dissolved in ethanol to a concentration of 2000 ppm then loaded by adding the microspheres (0.25 g) to the above-mentioned ethanol solution (1 cm3), followed by freeze drying. Microspheres loaded with TNP-470 (i.e., 1510[+], 6040[+], and 9060[+]) were used as sample powders for in vivo evaluations of anti-tumorigenic effects. Table 1 summarizes the preparation of the CPMs with and without TNP-470, together with their powder properties: crystalline phases and median diameter. Figure 5 shows the particle morphology of the CPMs, together with the release profile of TNP-470 from the CPMs over a period of 120 h. The SEM micrographs show that the resulting powders were composed of spherical particles. The diameter of the particles decreased with decreasing concentration of the starting solution. The spherical particles were formed via the following process: (i) removal of the solvent from the droplet surface, (ii) formation of microcrystalline calcium phosphates, and (iii) growth of calcium phosphate crystals. The diameter of the resulting spherical particles is dependent upon the starting solution droplet size. The droplet size can be decreased by decreasing the concentration of the starting solution, which in turn reduces the diameter of the spherical particles. The release profile data showed that 90% of the total TNP-470 was released from the CPMs slowly over a period of 20 h. The amount of TNP-470 released from the 1510(+) powder was the highest among the examined sample powders.

Figure 5. Profile of drug release from CPMs loaded with TNP-470, together with typical CPMs composed of various particle sizes. 115 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 6 shows the weekly change in mean tumor volume during CPM injection using the FU-MMT-3 xenograft model. Figure 6(a) illustrates the effect of CPM particle size on the anti-tumorigenic effect, with and without TNP-470. Microspheres with a smaller particle size inhibited tumor growth more effectively. Figure 6(b) illustrates the effect of particle size distribution on the anti-tumorigenic effect of CPMs with and without TNP-470. The particle morphology of Mix(+) is inserted into Figure 6(b). Both the Mix(+) and 6040(+) treatments significantly inhibited the growth of FU-MMT-3 xenografts in comparison with the controls (p < 0.05). In addition, the Mix(+) treatment significantly suppressed tumor growth in comparison with the 6040(+) and 9060(+) treatments (p < 0.05). No significant loss of body weight was observed in any mouse treated with any size of TNP-470–loaded CPMs. Histopathologic analyses indicated marked embolization of the CPMs in the feeding arteries in the peripheral areas of the xenografts in vivo, as well as in many of the tumor microvessels.

Figure 6. Change in tumor volume over a period of 8 weeks after injection of CPMs of different sizes loaded with TNP-470; the effect of (a) CPM particle size and (b) particle size distribution on the anti-tumorigenic effect with and without TNP-470 agent, together with particle morphology of “Mix(-)” powder. In order to adapt our biomaterial for chemoembolization, in the present study, the biodegradable hollow CPMs were further refined to target morphologic heterogeneity in the vasculature. The in vitro results showed that the amount of TNP-470 released from the 1510(+) powder was the highest among the examined sample powders. The 1510(−) powder consists of microspheres of the smallest diameter; therefore, this powder has the largest specific surface area (20 m2/g) in comparison with the 6040(−) (14.4 m2/g) and 9060(−) (16.3 m2/g) powders. Thus, the 1510(−) powder can accommodate the greatest amount of TNP-470. As the 1510 powder consists of microspheres of the smallest diameter, the 1510(+) solution would have the largest microsphere surface area; thus, this solution could 116 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


incorporate a greater amount of TNP-470 in comparison with the 6040(+) and 9040(+) powders. As TNP-470 is released from the internal space and external surface of the hollow CPMs in a two-step sustainable fashion in vitro, the smaller microspheres would theoretically be the most useful drug carriers. However, the in vivo results of the present study showed that the Mix(+) treatment tended to achieve a better suppression of FU-MMT-3 tumor growth compared with the 1510(+) treatment. This result suggests that as the feeding arteries and tumor microvessels vary in diameter (they exhibit vascular morphologic heterogeneity), these vessels might have been more effectively embolized by the different sizes of CPMs or their aggregates in the Mix(+) treatment. Additionally, the diameter of the CPMs was approximately 0.5-3 μm, and there was no evidence of blood vessel–related injury, such as a marked hemorrhaging or hematoma formation, in any of the treated mice. Thus, this new DDS using a mixture of CPMs of different sizes might be a useful approach for chemoembolization in the treatment of many solid tumors.

Porous CPMs with Nano-Pores on the Surface Prepared via SAUSP as Advanced Carriers, and Our Future Work In a previous study, we proposed that CPMs loaded with TNP-470 would be effective DDS carriers for novel chemoembolization treatment of cancers, based on the results of in vivo experiments (28, 29, 31). However, in vitro, the microspheres used in those studies released 80% of the total TNP-470 within 1 h. In order to achieve controlled release of TNP-470, we prepared novel microspheres using a SAUSP approach with NaCl as the salt and attempted to form nano-pores on the surface of the microspheres (33, 38). As the SAUSP technique was originally developed to prepare nano-dispersive powders (39), we clarified the optimal concentration of salt at which nano-pores form on the surface of the microspheres. Unfortunately, the resulting microspheres exhibited several problems: (i) decreased solubility, and (ii) cytotoxicity due to the elution of chloride ions. In order to overcome these problems, in the present study, we prepared novel microspheres using a SAUSP approach with potassium nitrate (KNO3) serving as the salt instead of NaCl. In addition, we examined the efficacy of the resulting hollow microspheres as DDS carriers for the treatment of cancers using the anti-angiogenic agent TNP-470 as a model drug (37). We then compared the drug release profile of the novel microspheres with nano-sized pores on the surface with that of traditional microspheres lacking nano-sized pores. Starting solution with a Ca/P ratio of 1.50 was prepared by mixing Ca(NO3)2·4H2O, (NH4)2HPO4 and HNO3 to final concentrations of 0.60, 0.40, and 0.10 mol·dm−3. The concentration of KNO3 in the starting solution was in the range 0.20 to 1.20 mol·dm−3. The upper- and lower-furnace temperatures were fixed at 850 and 300°C, respectively. These solutions were sprayed into the heating zone using an ultrasonic vibrator operated at a frequency of 2.4 MHz, and then the sprayed droplets were dried and pyrolyzed to form CPMs incorporating a KNO3 phase. The resulting microspheres were washed with pure water and freeze dried to prepare the washed powders. 117 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


XRD analyses indicated that the unwashed powders with KNO3 at a concentration of 0.20 to 1.0 mol·dm−3 were composed of a mixture of KNO3 and HAp with low crystallinity, whereas the washed powders consisted only of an HAp phase with low crystallinity. This result shows that KNO3 used in SAUSP technique can be removed by the washing process. In the case of powder prepared with KNO3 at a concentration of 1.20 mol·dm−3, the XRD patterns indicated that the unwashed powder consisted of HAp with low crystallinity, KNO3, and KCaPO4. The washed powder, by contrast, was composed of HAp with low crystallinity and KCaPO4. Figure 7 shows the typical particle morphology of the washed powders, together with a schematic illustration of the formation of porous CPMs with nano-pores on the surface prepared using the SAUSP approach. In addition, SEM micrographs indicated that the washed powders were composed of microspheres with a diameter of 0.5-3.0 µm. Upper part in Figure 7 shows a high-magnification image of the particle morphology of powders prepared with KNO3 at a concentration of 1.00 mol·dm−3. Small pores with a sizes of ~50 nm were present on the surface of the microspheres. However, in the case of powders prepared without KNO3 addition, these small pores were not present on the surface of the microspheres, as shown in lower part in Figure 7.

Figure 7. Particle morphologies of CPMs with nano-sized pores on the surface (upper part), together with a model illustrating the formation process (lower one); 1) Formation of droplet, 2) decrease of droplet volume by evaporating the solve, 3) Formation of highly-viscus layer on the droplet surface, 4) Formations of calcium phosphate and added salt particles, 5) Formations of hollow spherical agglomerates (secondary particle), and 6) Formation of fragment particles by bursting the hollow particle. 118 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


TNP-470 was dissolved in ethanol to a concentration of 2000 µg/cm3 and loaded by adding microspheres (0.06 g) to the ethanol solution (12 cm3), followed by freeze drying for 24 h. The TNP-470 release profile was determined using HPLC. Figure 8 shows the drug release profile for microspheres loaded with TNP470. In the case of powders prepared without KNO3, 90% of the total amount of TNP-470 was released from the microspheres within 1 h, and total amount of TNP470 released from the microspheres was 20 µg·mg−1. For powders prepared with KNO3 at a concentration of 1.00 mol·dm−3 (Figure 7), 70% of the total TNP-470 was released from the microspheres within 1 h. The remaining 30% was slowly released up to 24 h following immersion, and the total amount of TNP-470 released from these microspheres was 42 µg·mg−1. The amount of drug loaded depended on the SSA; of the sample powders examined, the washed powders prepared using KNO3 at a concentration of 1.00 mol·dm−3 released the greatest amount of drug. Notably, microspheres with nano-sized pores on the surface that were prepared using KNO3 at a concentration of 1.00 mol·dm−3 exhibited a two-step release profile, perhaps due to release of the drug from the surface and interior of the microspheres, as illustrated in Figure 1.

Figure 8. Two-step release profiles of drug from TNP-470–loaded CPMs with nano-sized pores on the surface, together with typical particle morphologies. We evaluated the effect of CPMs loaded with TNP-470 described above upon human uterine sarcoma cells (FU-MMT-3 (36)) transplanted into nude mice. Although the detailed data will be reported elsewhere as a future work, we report here that compared with control mice, the size of the tumor increased only minimally in mice treated with the CPMs loaded with TNP-470. In addition, the lifespan of mice treated with TNP-470–loaded CPMs prepared with KNO3 increased relative to the control mice. Here, “control” refers to no treatment 119 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

after injection of FU-MMT-3 cells. In addition, histologic analyses indicated that many of the CPMs remained in the tumor blood vessels. Collectively, these results show that CPMs with nano-sized pores loaded with TNP-470 may be effective for advanced chemoembolization.


Conclusion Our purpose was to develop a novel process for chemoembolization to improve the therapeutic effectiveness and safety profile of cancer treatment. This study showed that calcium phosphate ceramic microspheres loaded with TNP-470 inhibit the growth of human uterine sarcoma in vivo via a physiochemical mechanism. This new chemoembolization method incorporating an anti-angiogenic agent may contribute to the development of more effective treatments for locally advanced or recurrent solid tumors.

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