DEVELOPMENT OF BIODEGRADABLE MICRO AND NANOSPHERE FOR MEDICAL APPLICATION

DEVELOPMENT OF BIODEGRADABLE MICRO AND

NANOSPHERE FOR MEDICAL APPLICATION

Sudaryanto1, Evi Yulianti1, Mujamilah1, Wahyudianingsih1, Ari Handayani1, and Abdul Mutholib2

1

Center for Technology of Nuclear Industry Material. BATAN 2

Center for Radioisotope and Radiopharmacy. BATAN Kawasan Puspiptek, Serpong, Tangerang 15314, Indonesia

e-mail: dryanto@batan.go.id

ABSTRACT

The development of biodegradable micro and nanosphere based on poly(lactic acid) for medical application has been done. The preparation was conducted using in water solvent evaporation method. Microsphere containing holmium nuclide (Ho2O3) having 20 – 50 μm in diameter were targeted in order to fulfill the application requirement. The existence of holmium inside the microsphere was determined by Neutron Activation Analysis (NAA) technique. The NAA results show that the encapsulation percentage of Ho2O3 was in the range of 87 to 100%. In other word, only a small number of holmium lost during the preparation of microsphere. Utilizing ultrasonic probe instead of stirrer as mixer in emulsification processes is yielding formation of nanosphere having several hundreds nanometer in size. Nanosphere containing Fe3O4 were also prepared successfully. The dimension as well as the dispersion of nanosphere having magnetic particles could be controlled through out the sonication time, the solvent composition and the polymer concentration as well. The existence of Fe3O4 encapsulated in the nanosphere could be confirmed by using NAA, X-Ray Difractometry (XRD) profiles, as well as Vibrating Sample Magnetometry (VSM) curvatures. The encapsulation percentage obtained in this research was very low (<25%) but tend to increase with the smaller size of nanosphere. The encapsulation of magnetic nanoparticle by polymer in form of nanosphere has made it stable in a colloidal system up to 21 days.

Keywords : Biodegradable, nanosphere, poly(lactic acid)

INTRODUCTION

Many attempts have been done to develop micro and nanosphere both in academicals and industrial scale [1]. Microspheres are generally defined as small spheres made of any material and sized from about 0.5 µm to 100 µm. Similar, but smaller spheres sized 10 to 500 nm are called nanospheres [2]. Depending on the material used, micro and nanopshere are found very wide applications such as agricultural, industrial, and medical field. Micro and nanosphere have many applications in medical field, with the main uses being for the drugs encapsulation.

236

unlike other drugs, is never released from the micro and nanosphere and acts from within over a radioisotope-typical distance.

Figure 1 shows application examples of micro and nanosphere containing  emitter radioactive particle for cancer and rheumatoid therapies. Figure 1A illustrated an example of cancer therapy using microsphere which known as radioembolization therapy. Figure 1B demonstrated the application of nanosphere for rheumatoid therapy, known as radiosynovectomy (RSV)[3].

(A) (B)

Figure 1: Example application of micro and nanosphere containing radioisotope for (A) cancer therapy and (B) rheumatoid therapy.

Biodistribution of the micro and nanosphere is highly dependent on the size and surface charge. Large sized radioactive microsphere (sized 10 to 30 μm), for example, are larger than capillaries and will be trapped in the first capillary bed that they encounter. Thus, microspheres are suitable for radioembolizations therapy in which they are injected into the artery that leads to the target. In other hand, the nanospheres having smaller size can circulated in the blood system for up to several days. The long-circulating nanosphere will then lead to higher concentration in tumor areas because of the leaky capillary system of the newly-growing tumor vasculature [4]. Such radioactive nanosphere could be used for imaging purposes. It was also reported that nanosphere based on DL-lactide/glycolide copolymer (PLGA) should be more suitable for delivery to inflamed synovial tissue than microsphere due to their ability to penetrate the synovium [5]. Therefore, the control of the shape and size as well as to make micro and nanosphere more homogenous is off important.

Several techniques such as solvent evaporation, phase separation, and spray drying are used to prepare micro and nanosphere. However, the solvent evaporation process is one of the most common methods. This process involves oil-in-water (o/w) emulsification prior to the solvent evaporation process. The final shape and size of micro and nanosphere are highly dependent on the emulsification process and solvent evaporation condition.

In order to prepare a radiopharmaceutical material for therapeutic application, we have carried out a series of study to synthesize and characterize beta-emitting micro and nanosphere. The development progress is reviewed in this paper.

EXPERIMENTAL METHOD

The micro and nanosphere were prepared by mean of in water solvent solution method which can be ilustrated as in Figure 2.

237 dried at 50 _˚C to remove the water completely. Detailed explanation of the preparation methods has been reported [6, 7].

Figure 2: Preparation of micro and nanosphere by solvent evaporation method.

The dimension of obtained micro/nanosphere was observed by scanning electron microscopy (SEM). The existent of particle inside micro/nanosphere was detected by X-Ray Difractometry (XRD) and the particle content was confirmed by neutron activation analysis (NAA) technique. The magnetic properties of nanosphere were characterized by Vibrating Sample Magnetometer (VSM).

RESULTS AND DISCUSSION

Micro and nanosphere were successfully prepared by in water solvent evaporation method. The dimension of the sphere could be controlled from the preparation condition such as stirring speed as well as the time [6], and polymer concentration [7]. Theoretical approach of the dimension control based on the vessel dimension was also successfully performed [9-11]. The main key to prepare a certain size of micro/nanosphere is the emulsification condition. Namely, the higher speed of the stirrer is used, the smaller sphere is obtained. Furthermore, utilizing ultrasonic probe instead of stirrer in emulsification process lead to smaller size of sphere ranged from micro to nanosized. Further condition to obtain a smaller nanosphere is controlling the sonication time as well as the polymer concentration and mixing composition.

Figure 3 show several examples of the micro and nanosphere. Figure 3(A) represents SEM picture of freshly prepared holmium contained microsphere with a loading of 17 % (w/w). The picture is demonstrating the spherical structure with diameter of 10-50 μm. Representative SEM picture of nanosized sphere prepared by using ultrasonic probe is shown in Figure 3(B). Figure 3(B) depicted a relative homogenous nanosphere with avarage diameter of about 800 nm[7]. Controlling the polymer concentration in the emulsion process leads to smaller size up to 100 nm as shown in Figure 3(C) [12].

(A) (B) (C)

Figure 3: Electron micrograph of (A) microsphere prepared by using stirrer, and (B), (C) nanospheres prepared by using ultrasonic probe.

238

encapsulated Fe3O4 nanoparticle were compared. Therefore, the existence of Fe3O4 inside the nanosphere could be confirmed based on the appearance of diffraction peak as shown by arrows. The diffraction peak at 23 is indicated the PLA [13].

Similar method using VSM was also applicable to confirm the particle existence as shown in Figure 5. The magnetic respond of pure Fe3O4 nanoparticle and Fe3O4 encapsulated in PLA nanosphere are shown as (A) and (B) curvatures respectively. The pure Fe3O4 shows saturated magnetization 72 emu/g-sample, while the PLA nanosphere shows 5 emu/g-sampel which can be normalized with the initial composition became 28 emu/g-Fe3O4. The magnetic responses of the nanosphere clearly show the existence of the Fe3O4 nanoparticle [14].

Figure 4: X-raydiffraction profile of (A) pure Fe3O4 nanoparticle and (B) Fe3O4 encapsulated in PLA nanosphere.

Figure 5: Magnetic hysteresis curve of (A) pure Fe3O4 nanoparticle and (B) Fe3O4 contained PLA nanosphere.

239 and Fe3O4 in PLA nanosphere determined by NAA, respectively. The calculation results show that the encapsulation percentage of Ho into PLA sphere was 87 to 100 %. In other word, only a small number of holmium lost during the preparation [15]. While the encapsulation percentage of Fe3O4 in PLA nanosphere was less than 25% but tends to increase with the decreasing of the nanosphere size.

Nanoscale sized and high particle contained was the target of the nanosphere preparation. Thus, more parameter exploration should be done to obtain the ideal one. However, the nonosphere containing Fe3O4 prepared in this study show a sufficient dispersion stability in water as shown in Figure 6. The PLA nanosphere has size average of 800 nm and can be dispersed in water stably for more than 21 days.

Figure 6: (A) Pure Fe3O4 and (B) Fe3O4 encapsulated in PLA nanosphere after 21 days in water.

Table 1: Encapsulation percentage of Ho2O3 in PLA based microsphere.

Sample No.

Ho content / sample

Encapsulation percentage (%)

Composition (mg/g) Measured by NAA (mg/g)

1 17,4 15,2 87,4

2 41,4 39,5 95,7

3 79,1 73,2 92,4

4 145,0 147,9 102,0

5 248,6 219,9 88,5

Table 2: Encapsulation percentage of Fe3O4 in PLA based nanosphere.

Sample No.

Diameter (nm)

Fe3O4 content/Sampel _{Encapsulation}

240

CONCLUSION

Micro and nanosphere based on PLA as particle container have been developed by in water solvent evaporation method. The microspheres containing holmium (Ho2O3) have been prepared successfully with 10-50μm in diameter. By using ultrasonic probe instead of stirrer to mix the oil and water in emulsification process, nanosphere containing Fe3O4 nanoparticle could be synthesized with several hundreds nanometer in size. By selecting the processing condition the desired size of micro and nanosphere could be produced The existence of particle either Ho2O3 or Fe3O4) inside the sphere could be determined both qualitatively and quantitatively. Based on the NAA results, it was found that the encapsulation percentage of Ho2O3 by PLA microsphere was in the range of 87 to 100%. While the encapsulation of Fe3O4 by PLA nanosphere was very low (<25%) but tend to increase with decreasing the size. The nonosphere containing Fe3O4 prepared in this study show a sufficient dispersion stability in water in which it can be dispersed stable in water for more than 21 days.

REFERENCES

[1]. Dagani R., Microsphere Play Role in Medical, Sensor, Energy, Space Technologies, C & EN, 19, (1994), 33-35

[2]. Hafeli U, Radioactive Microsphere for Medical Application, http://www.jradiology.org. accessed June 2001.

[3]. Mauricio S., James V. L.Jr., Adolfo L., Treatment of Hemophilia, 33, 2004; accessible from World Federation of Hemophilia’s web site www.wfh.org.

[4]. Ackerman. NB. Surgery 75: 589-596 (1974)

[5]. Horisawa E. , Kubota K., Izumi T., Sato K., Yamamoto H., Takeuchi H. And Kawashima Y.,Pharmaceutical Research, 19, 132 – 139, (2002).

[6]. Sudaryanto, Sudirman, Aloma Karo Karo, Indra Gunawan, Tri Darwinto, dan Wahyudianingsih, Prosiding Simposium Nasional Polimer IV, HPI, (2003), 181-187 [7]. Nono Ariyandi, Sudaryanto, Mersi Kurniati, Mujamilah and Ari H., Indonesian

Journal of Material Science, 8, 182-186, (2007).

[8]. Indra Gunawan, Sudaryanto and Tri Darwinto, Indonesian Journal of Material Science, 5, (2004), 44-47.

[9]. Indra Gunawan, Sudaryanto, Aloma K.K., Rochmadi and Nurul E.E., Indonesian Journal of Material Science, 6, (2005), 49-54.

[10]. Indra Gunawan, Aloma K.K., Rukihati and Sudaryanto, Indonesian Journal of Material Scienc, Special edition (2006), 113 – 117

[11]. Gunawan, I., Karo, A. K. and Sudirman, Indonesian Journal of Material Science, 8, (2007), 125-128.

[12]. Evi Yulianti, “Enkapsulasi Nanopartikel Magnetik Fe3O4 Menggunakan Polimer Poli Asam Laktat Dengan Ultrasonik Probe,” Thesis, Program Studi Magister Ilmu Kimia, Program Pascasarjana FMIPA UI, (2008)

[13]. Sudaryanto, Mujamilah, Wahyudianingsih, Ari Handayani, Ridwan, And Abdul Muthalib., Indonesian Journal of Material Science, 8, (2007), 134-138

[14]. Sonny Affandi, Mujamilah, Mersi Kurniati, And Sudaryanto, J Indonesian Journal of Material Science, Special edition (2007), 156 – 161