DEVELOPMENT OF BIODEGRADABLE MICRO AND NANOSPHERE FOR MEDICAL APPLICATION

  MATERIALS SCIENCE and TECHNOLOGY

  Edited by Evvy Kartini et.al.

  

DEVELOPMENT OF BIODEGRADABLE MICRO AND

NANOSPHERE FOR MEDICAL APPLICATION

  1

  1

  1

  1

  1 Sudaryanto , Evi Yulianti , Mujamilah , Wahyudianingsih , Ari Handayani , and

  2 Abdul Mutholib

  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 (Ho

  2 O 3 ) 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 Ho

  2 O 3 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 Fe

  3 O 4 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 Fe

  

3 O

4 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.

  Micro and nanosphere containing radionuclide are used in medical applications both for diagnostic and therapeutic purposes. The radioactive micro and nanosphere are generally used in a similar fashion to non-radioactive ones. However, the radioactivity,

  Materials Science and Technology

  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.

  Firstly, water containing polyvinyl alcohol (PVA) was mixed with chloroform containing polymer poly(lactic acid) and particle such as Holmium (III) oxide (Ho

  2 O 3 ) or

  magnetic nanoparticle Fe

  3 O 4 using stirrer or ultrasonic probe to prepare emulsion. Secondly,

  the obtained emulsion was suspended into water. Finally, the suspension was cleaned and

  Development of Biodegradable Micro and Nanosphere……

  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.

  The existence of particle encapsulated in the micro/nanosphere could be confirmed by several methods such as XRD, VSM, and NAA. Figure 4 shows an example of determination of magnetic particle inside the nanosphere using XRD. X-ray diffraction profile of pure and

  Materials Science and Technology

  encapsulated Fe

  3 O 4 nanoparticle were compared. Therefore, the existence of Fe

  3 O 4 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 Fe

  3 O 4 nanoparticle and Fe

  3 O 4 encapsulated

  in PLA nanosphere are shown as (A) and (B) curvatures respectively. The pure Fe

  3 O 4 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-Fe O . The magnetic

  3

  4

  responses of the nanosphere clearly show the existence of the Fe

  3 O 4 nanoparticle [14].

  O nanoparticle and (B) Fe O encapsulated in PLA Figure 4: X-ray diffraction profile of (A) pure Fe ³

⁴

^{3 4}

nanosphere.

  

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

_{3 4 3 4} After irradiated by thermal neutron inside nuclear reactor, Fe as well as Ho will

  emmite gamma rays with a specific energies. Detecting the radioactivity will give information the existance of the particle encapsulated in the spherical PLA quantitavely. Table 1 and Table 2 show the encapsulation percentage of holmium inside PLA microsphere

  Development of Biodegradable Micro and Nanosphere……

  and Fe

  3 O 4 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 Fe

3 O 4 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 Fe O prepared in this study show a sufficient

  3

  4

  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 Fe ^{3 O 4 and (B) Fe 3 O 4 encapsulated in PLA nanosphere after 21 days in water.}

  Table 1: Encapsulation percentage of Ho O in PLA based microsphere. _{2 3} Ho content / sample Encapsulation percentage Sample No.

  (%) 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 Fe O in PLA based nanosphere. _{3 4} Fe ^{3 O 4 content/Sampel} Encapsulation

  Sample Diameter percentage

  

Composition Measured by NAA

No. (nm) (%) (mg/g) (mg/g)

  1 721 166,7 10,69 6,41 2 543 166,7 18,86 13,43 3 419 166,7 29,74 17,84 4 382 166,7 41,58 24,94 5 182 166,7 33,28 19,96 6 150 166,7 22,39 16,74

  Materials Science and Technology CONCLUSION

  REFERENCES

  Material Science, Special edition (2007), 156 – 161

  [14]. Sonny Affandi, Mujamilah, Mersi Kurniati, And Sudaryanto, J Indonesian Journal of

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

  Asam Laktat Dengan Ultrasonik Probe,” Thesis, Program Studi Magister Ilmu Kimia, Program Pascasarjana FMIPA UI, (2008)

  4 Menggunakan Polimer Poli

  3 O

  [11]. Gunawan, I., Karo, A. K. and Sudirman, Indonesian Journal of Material Science, 8, (2007), 125-128. [12]. Evi Yulianti, “Enkapsulasi Nanopartikel Magnetik Fe

  Material Scienc, Special edition (2006), 113 – 117

  [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

  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).

  [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

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

  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.

  Micro and nanosphere based on PLA as particle container have been developed by in water solvent evaporation method. The microspheres containing holmium (Ho

  4

  3 O

  The nonosphere containing Fe

  3 O 4 by PLA nanosphere was very low (<25%) but tend to increase with decreasing the size.

  Fe

  2 O 3 by PLA microsphere was in the range of 87 to 100%. While the encapsulation of

  quantitatively. Based on the NAA results, it was found that the encapsulation percentage of Ho

  3 O 4 ) inside the sphere could be determined both qualitatively and

  2 O 3 or Fe

  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 Ho

  3 O 4 nanoparticle

  prepared successfully with 10-50 μm in diameter. By using ultrasonic probe instead of stirrer to mix the oil and water in emulsification process, nanosphere containing Fe

  2 O 3 ) have been

  [15]. Sudaryanto, Wahyudianingsih, Aloma Karo Karo, Ari Handayani, Sutisna Dan Abdul Muthalib, Indonesian Journal of Material Science, 8, 65-68, (2006).