**4. Polyurethane microparticle as carrier of antituberculosis drug**

New polyurethane microcapsules incorporated with antituberculosis drug Is have been synthesized by interfacial polyaddition between toluene-2,4-diisocyanate (TDI) and various poly(ethylene glycol)s (PEG). Drug Is is hydrophilic water-soluble compound, and it is insoluble in toluene. Thus Is could be capsulated by interfacial polycondensation technique using water-in-toluene emulsion, which prevents transferring of Is to the external phase. And drug encapsulation is possible during the process of the polymer wall formation (Batyrbekov et al., 2009; Iskakov et al., 2004).

Isocyanate groups react with hydroxyl groups of PEG to form polyurethane chains according to the Scheme (Fig.1 a). TDI can also reacts with molecules of water at the border of reaction to form unstable NH-COOH group, which dissociates into amine and carbon dioxide (Fig.1 b). Chains with amine end-group reacts with the isocyanate groups of growing polymer with urea segments formation.

Polycondensation was carried out in a 1 L double-neck flask fitted with a stirrer. Polyethylene glycol with 4 various molecular weights 400, 600, 1000 and 1450 (Sigma, USA) - PEG 400, PEG 600, PEG 1000 and PEG 1450 respectively were used as diol monomers. TDI (Sigma, USA) was applied as a bifunctional monomer for the polycondensation. Three solutions were prepared separately. Solution A: 10 mg surfactant Tween 40 was dissolved in 100 ml of toluene; solution B: *x* mmol diol and Is were added in *y* ml of water; amount of Is was 10, 20, and 30 mol % from PEG; solution C: 2.5 *x* mmol TDI was dissolved in 10 ml of solution A. Water/oil ratio was 1:10 vol.%. Solution B was poured into the reaction flask, contaning 90 ml of solution A under the stirring at 1000 rpm during 15 minutes. After the formation of microemulsion, solution C was added dropwise. After 180 min the polymerization was stopped. Microparticles were filtered, carefully washed with distillated water and dried at ambient conditions. Yield of polymers was estimated from the total amount of introduced monomers compared to the weight of polycondensation products.

Polyurethane as Carriers of Antituberculosis Drugs 157

c

a

b

**Figure 2.** FT-IR-spectra of polyurethane microparticles containing isoniazid (a), polyurethane

Fig. 3 shows that composition of the water phase influences upon effectiveness of capsulation. Increase of PEG concentration results in decreasing effectiveness of capsulation and decrease of Is loading correspondingly. The high PEG concentration promotes miscibility of Is in the internal oil phase - toluene. Morphology of the surface of microparticles is very important factor, which affects release behavior of active agent. The wall structure depends on the conditions of interfacial polycondensation process, such as Mw and chemical structure of diol, the concentration of the monomers and other. The effect of water/PEG ratio in aqueous phase on morphology of microparticle was investigated. Microparticles prepared from PEG solutions of higher concentration have dense surface so that Is diffused much slower. At the high concentration of PEG reaction between PEG and TDI is significantly limited on the interface of the drops. Furthermore excessive PEG transfers to the external surface of microparticles and

Fig.4 shows SEM photos of interfacial polycondensation products prepared by reaction between TDI and PEG 400 at water/PEG ratio 11,8 : 88,2 vol.% in water phase. According to Figs. 4a and 4b two products of polycondensation with different structure were formed. PU microparticles have spherical shape and size about 5 - 10 μm (Fig. 4a). The secondary product has fibril structure with diameter less then 500 nm (Fig 4b). The effect of water/PEG ratio on morphology of microparticle walls is shown in Figs. 4c and 4d. PU microparticles prepared at water/PEG ratio 82,4 :17,6 (Fig. 4c) have rough surface. On the contrary the surface of PU microparticles prepared at water/PEG ratio 11,8 : 88,2 (Fig 4d) were dense and

microparticles (b) and isoniazid (c).

smooth.

reacts with TDI and less penetrable wall was formed.

**Figure 1.** Scheme of reaction between PEG and TDI with polyurethane formation.

The completion of polycondensation process was estimated by IR-spectra from decreasing of the absorption band at 2270-2320 cm-1, which correspond to -N=C=O isocyanate group. IR spectra were obtained by a Nicolet 5700 FT-IR (USA) infrared spectrophotometer in KBr.

In the IR-spectra of microparticles the N-H stretching vibration were observed at 3450–3300 cm-1, absorption bands at 1740–1700 cm-1 for the C=O stretching of urethane and at 1690– 1650 cm-1 for urethane-urea formation were also present (Fig.2). Absorption bands are present at 1100 cm-1 for C-O-C ether group and at 2850 -2950 cm-1 for C-H. In FT-IR spectra of microparticles containing Is, the new stretching vibrations band appeared at 1350 cm-1, 1000 cm-1 and 690 cm-1, which were also present in FT-IR spectra of pure Is that indicates the physical mechanism of Is capsulation.

In the process of interfacial polycondensation, two PU products of reaction were detected: the main product - microparticles, and the secondary product - linear precipitated polyurethane. The increase of PEG content in water phase resulted in increased amount of the secondary product, and as the PEG content in water phase reached 60 vol.%, maximum of the secondary product was observed (about 40%).

Decreasing PEG concentration in water phase leads to increased yield of polyurethane microparticles. Maximum of yield was reached at PEG concentration 22 - 27 vol.% and in that condition whole olygomer reacted at surface of emulsion drops with microparticles formation. Reduction of microparticle yield after the maximum is due mainly to increasing contribution of the hydrolysis process of isocyanate groups.

Appearance of the secondary product and increase of its yield, probably, can be attributed to the increase of PEG concentration and results in PEG partially transfer from the water phase to the internal phase of toluene and the process of polycondensation between PEG and TDI takes place with linear polyurethane formation. At the end of reaction rate of PEG diffusion to surface, namely at the reaction region, seems to be a limit stage of the process. Reducing of PEG concentration causes to decrease of system viscosity. Effectiveness of Is capsulation in PU microparticles ranged from 3.4 to 41.7 % and significantly depended on water/PEG ratio in the water phase of emulsion.

m HO(CH2CH2O)nH + m

**b**

physical mechanism of Is capsulation.

of the secondary product was observed (about 40%).

water/PEG ratio in the water phase of emulsion.

contribution of the hydrolysis process of isocyanate groups.

**a**

CH3

[-O(CH2CH2O)n-C(O)-NH

N=C=O + H2O NH-COOH NH2 + CO2

The completion of polycondensation process was estimated by IR-spectra from decreasing of the absorption band at 2270-2320 cm-1, which correspond to -N=C=O isocyanate group. IR spectra were obtained by a Nicolet 5700 FT-IR (USA) infrared spectrophotometer in KBr.

In the IR-spectra of microparticles the N-H stretching vibration were observed at 3450–3300 cm-1, absorption bands at 1740–1700 cm-1 for the C=O stretching of urethane and at 1690– 1650 cm-1 for urethane-urea formation were also present (Fig.2). Absorption bands are present at 1100 cm-1 for C-O-C ether group and at 2850 -2950 cm-1 for C-H. In FT-IR spectra of microparticles containing Is, the new stretching vibrations band appeared at 1350 cm-1, 1000 cm-1 and 690 cm-1, which were also present in FT-IR spectra of pure Is that indicates the

In the process of interfacial polycondensation, two PU products of reaction were detected: the main product - microparticles, and the secondary product - linear precipitated polyurethane. The increase of PEG content in water phase resulted in increased amount of the secondary product, and as the PEG content in water phase reached 60 vol.%, maximum

Decreasing PEG concentration in water phase leads to increased yield of polyurethane microparticles. Maximum of yield was reached at PEG concentration 22 - 27 vol.% and in that condition whole olygomer reacted at surface of emulsion drops with microparticles formation. Reduction of microparticle yield after the maximum is due mainly to increasing

Appearance of the secondary product and increase of its yield, probably, can be attributed to the increase of PEG concentration and results in PEG partially transfer from the water phase to the internal phase of toluene and the process of polycondensation between PEG and TDI takes place with linear polyurethane formation. At the end of reaction rate of PEG diffusion to surface, namely at the reaction region, seems to be a limit stage of the process. Reducing of PEG concentration causes to decrease of system viscosity. Effectiveness of Is capsulation in PU microparticles ranged from 3.4 to 41.7 % and significantly depended on

CH3

NH-C(O)-]m

NCO OCN

**Figure 1.** Scheme of reaction between PEG and TDI with polyurethane formation.

**Figure 2.** FT-IR-spectra of polyurethane microparticles containing isoniazid (a), polyurethane microparticles (b) and isoniazid (c).

Fig. 3 shows that composition of the water phase influences upon effectiveness of capsulation. Increase of PEG concentration results in decreasing effectiveness of capsulation and decrease of Is loading correspondingly. The high PEG concentration promotes miscibility of Is in the internal oil phase - toluene. Morphology of the surface of microparticles is very important factor, which affects release behavior of active agent. The wall structure depends on the conditions of interfacial polycondensation process, such as Mw and chemical structure of diol, the concentration of the monomers and other. The effect of water/PEG ratio in aqueous phase on morphology of microparticle was investigated. Microparticles prepared from PEG solutions of higher concentration have dense surface so that Is diffused much slower. At the high concentration of PEG reaction between PEG and TDI is significantly limited on the interface of the drops. Furthermore excessive PEG transfers to the external surface of microparticles and reacts with TDI and less penetrable wall was formed.

Fig.4 shows SEM photos of interfacial polycondensation products prepared by reaction between TDI and PEG 400 at water/PEG ratio 11,8 : 88,2 vol.% in water phase. According to Figs. 4a and 4b two products of polycondensation with different structure were formed. PU microparticles have spherical shape and size about 5 - 10 μm (Fig. 4a). The secondary product has fibril structure with diameter less then 500 nm (Fig 4b). The effect of water/PEG ratio on morphology of microparticle walls is shown in Figs. 4c and 4d. PU microparticles prepared at water/PEG ratio 82,4 :17,6 (Fig. 4c) have rough surface. On the contrary the surface of PU microparticles prepared at water/PEG ratio 11,8 : 88,2 (Fig 4d) were dense and smooth.

Polyurethane as Carriers of Antituberculosis Drugs 159

The release behavior of Is from PU microparticles was carried out and different conditions of synthesis such as water/PEG ratio, molecular weight of PEG and isoniazid concentration was investigated. The release behavior of microparticles loaded with isoniazid was studied with ultraviolet (UV) spectroscopy. For calibration, physiological solutions of isoniazid with concentration ranging from 0.004 to 0.05 mg/ml were prepared and their absorption was measured at 263.5 nm with Jasco UV/VIS 7850 spectrophotometer (Japan). 10 mg of isoniazid-loaded microparticles were dispersed in 10 ml of physiological solution under light stirring at constant temperature 37oC. After fixed time interval 2 ml of solution was taken out by the squirt equipped with the special filter. The efficiency of capsulation was calculated as ratio of introduced isoniazid to solution B compared with amount of delivered isoniazid into water during 3 weeks. Isoniazid loading was weight of isoniazid (mg)

Fig. 5 shows the release behavior of Is from PU microparticles, synthesized at different water/PEG ratio. The most part of the drug delivered during the first three hours, then slow release of the residual Is was observed during the next two weeks. Microparticles prepared with less concentration of PEG in the water phase demonstrated faster diffusion of Is through walls of microparticles. The increased PEG content in water phase of reaction, results in decreasing Is diffusion, due to formation of PU microparticles with denser polymer wall. Microparticles prepared with PEG concentration 17.6, 29.4 and 41.2 vol.% showed the release 58 - 66 % of Is during 3 h. However, due to denser wall of microparticles prepared with PEG 64.7 и 88.2 vol. % demonstrated the release no more 35% of the drug

**Figure 5.** Release of Is from PU microparticles synthesized at various water/PEG ratio: ● - 82.4:17.6, o -

02468

time (h)

The effect of molecular weight of PEG on release of Is from PU microparticles was investigated (Fig 6). Microparticles were prepared by using PEG with Mw 400, 600, 1000 and 1450. Increasing molecular weight of soft segments (PEG) results in the increase of diffusion

contained in 1 g of microparticles.

within the same time.

70.6:29, - 58.8:41.2, - 35.3:64.7 and - 11.8:88.2.

0

20

40

60

Release of isoniazid (%)

80

100

**Figure 3.** The effect of PEG content in water phase on effectiveness of encapsulation () and Is loading in PU microparticles ().

**Figure 4.** SEM photographs of products of interfacial polycondensation . between TDI and PEG 400 at 60oC. PU microparticles (a) and PU secondary product (b)synthesized at water/PEG ratio 11.8 : 88.2 in water phase. Surface of PU microparticles prepared at water/PEG ratio 82.4 :17.6 (c) and 11.8 : 88.2 (d) in water phase.

The release behavior of Is from PU microparticles was carried out and different conditions of synthesis such as water/PEG ratio, molecular weight of PEG and isoniazid concentration was investigated. The release behavior of microparticles loaded with isoniazid was studied with ultraviolet (UV) spectroscopy. For calibration, physiological solutions of isoniazid with concentration ranging from 0.004 to 0.05 mg/ml were prepared and their absorption was measured at 263.5 nm with Jasco UV/VIS 7850 spectrophotometer (Japan). 10 mg of isoniazid-loaded microparticles were dispersed in 10 ml of physiological solution under light stirring at constant temperature 37oC. After fixed time interval 2 ml of solution was taken out by the squirt equipped with the special filter. The efficiency of capsulation was calculated as ratio of introduced isoniazid to solution B compared with amount of delivered isoniazid into water during 3 weeks. Isoniazid loading was weight of isoniazid (mg) contained in 1 g of microparticles.

158 Polyurethane

in PU microparticles ().

in water phase.

**Figure 3.** The effect of PEG content in water phase on effectiveness of encapsulation () and Is loading

**Figure 4.** SEM photographs of products of interfacial polycondensation . between TDI and PEG 400 at 60oC. PU microparticles (a) and PU secondary product (b)synthesized at water/PEG ratio 11.8 : 88.2 in water phase. Surface of PU microparticles prepared at water/PEG ratio 82.4 :17.6 (c) and 11.8 : 88.2 (d)

Fig. 5 shows the release behavior of Is from PU microparticles, synthesized at different water/PEG ratio. The most part of the drug delivered during the first three hours, then slow release of the residual Is was observed during the next two weeks. Microparticles prepared with less concentration of PEG in the water phase demonstrated faster diffusion of Is through walls of microparticles. The increased PEG content in water phase of reaction, results in decreasing Is diffusion, due to formation of PU microparticles with denser polymer wall. Microparticles prepared with PEG concentration 17.6, 29.4 and 41.2 vol.% showed the release 58 - 66 % of Is during 3 h. However, due to denser wall of microparticles prepared with PEG 64.7 и 88.2 vol. % demonstrated the release no more 35% of the drug within the same time.

**Figure 5.** Release of Is from PU microparticles synthesized at various water/PEG ratio: ● - 82.4:17.6, o - 70.6:29, - 58.8:41.2, - 35.3:64.7 and - 11.8:88.2.

The effect of molecular weight of PEG on release of Is from PU microparticles was investigated (Fig 6). Microparticles were prepared by using PEG with Mw 400, 600, 1000 and 1450. Increasing molecular weight of soft segments (PEG) results in the increase of diffusion rate of Is into solution. This phenomenon can be attributed to increasing Mw of PEG which leads to accelerating diffusion of water-soluble Is through hydrophilic PEG chains.

Polyurethane as Carriers of Antituberculosis Drugs 161

PU microparticles were administrated subcutaneously to mice BL/6. Histologic analyses of the underskin tissue was carried out at a different period of microparticles administration in the mice by using electron microscope LEO F360, equipped with X-ray analyzer EDS Oxford

Fig 8 shows histologic analyses of tissue under skin. Within 5 days of the microparticles deposition, the thickening of the surrounding tissue due to primary macrophage reaction and fibrillar tissue formation were detected as shown in Fig. 8b. On day 21, some enzymatic lysis of polyurethane – С(О)–NH – group probably took place (Fig. 8c) and partial biodegradation of PU microparticles was observed. For all experimental animals no casting-

**Figure 8.** Histological slices of tissue under mice skin 1 (а), 5 (b), and 21 (c) days after deposition of iscontaining PU microparticles to BL/6 mice provided by transparent electron microscope with 400x

Thus, the data obtained in the present work have demonstrated the possibility of using PU microparticles as a carrier for the controlled delivery of antituberculosis drug Is. PU microparticles were prepared by interfacial reaction between PEG and TDI in water in toluene emulsion. The effect of water/PEG ratio on the morphology of microparticles and release behavior was shown. The low PEG content in aqueous phase results in the formation of microparticles with rough surface, which demonstrate faster diffusion of Is in comparison to PU microparticles produced from more concentrated PEG solutions, they have smooth surface and less penetrable walls for Is. Increased Mw of PEG and Is loading leads to increased diffusion rate of isoniazid from polyurethane microparticles. For PU microparticles administered in mice BL/6 subcutaneously, biodegradation was observed due to enzymatic lysis of polyurethane group. Preliminary data indicate that PU microparticles could be perspective carriers for controlled delivery and respirable administration of

The use of soft PU foams as carriers of antituberculosis drugs is of considerable interest. In such systems pharmaceutical agents are dispersed or dissolved in the PU carrier and the

**5. Polyurethane foams as carriers of antituberculosis drugs** 

ISI 300.

magnification.

antituberculosis drug Is.

off or necrosis of tissue were observed.

a b c

**Figure 6.** Release of Is from PU microparticles synthesized at various Mw of PEG. MW = 400 (o), 600 (●), 1000 () and 1450 ().

Microparticles with different Is loading, namely 18.4, 35.3 and 65.6 mg/g were produced. In Fig 7 release behavior of Is is shown.

**Figure 7.** Release of Is from PU microparticles with different Is loading: 18.4 (), 35.3 () and 65.6 mg/g (●).

Microparticles with higher Is loading demonstrate faster release rate of the drug due to increased gradient of concentrations between the external solution and core of microparticles.

PU microparticles were administrated subcutaneously to mice BL/6. Histologic analyses of the underskin tissue was carried out at a different period of microparticles administration in the mice by using electron microscope LEO F360, equipped with X-ray analyzer EDS Oxford ISI 300.

160 Polyurethane

1000 () and 1450 ().

mg/g (●).

microparticles.

Fig 7 release behavior of Is is shown.

0

20

40

60

Release of isoniazid (%)

80

100

0

20

40

Release of isoniasid

 (%)

60

80

100

rate of Is into solution. This phenomenon can be attributed to increasing Mw of PEG which

**Figure 6.** Release of Is from PU microparticles synthesized at various Mw of PEG. MW = 400 (o), 600 (●),

02468

time (h)

Microparticles with different Is loading, namely 18.4, 35.3 and 65.6 mg/g were produced. In

**Figure 7.** Release of Is from PU microparticles with different Is loading: 18.4 (), 35.3 () and 65.6

Microparticles with higher Is loading demonstrate faster release rate of the drug due to increased gradient of concentrations between the external solution and core of

02468

time (h)

leads to accelerating diffusion of water-soluble Is through hydrophilic PEG chains.

Fig 8 shows histologic analyses of tissue under skin. Within 5 days of the microparticles deposition, the thickening of the surrounding tissue due to primary macrophage reaction and fibrillar tissue formation were detected as shown in Fig. 8b. On day 21, some enzymatic lysis of polyurethane – С(О)–NH – group probably took place (Fig. 8c) and partial biodegradation of PU microparticles was observed. For all experimental animals no castingoff or necrosis of tissue were observed.

**Figure 8.** Histological slices of tissue under mice skin 1 (а), 5 (b), and 21 (c) days after deposition of iscontaining PU microparticles to BL/6 mice provided by transparent electron microscope with 400x magnification.

Thus, the data obtained in the present work have demonstrated the possibility of using PU microparticles as a carrier for the controlled delivery of antituberculosis drug Is. PU microparticles were prepared by interfacial reaction between PEG and TDI in water in toluene emulsion. The effect of water/PEG ratio on the morphology of microparticles and release behavior was shown. The low PEG content in aqueous phase results in the formation of microparticles with rough surface, which demonstrate faster diffusion of Is in comparison to PU microparticles produced from more concentrated PEG solutions, they have smooth surface and less penetrable walls for Is. Increased Mw of PEG and Is loading leads to increased diffusion rate of isoniazid from polyurethane microparticles. For PU microparticles administered in mice BL/6 subcutaneously, biodegradation was observed due to enzymatic lysis of polyurethane group. Preliminary data indicate that PU microparticles could be perspective carriers for controlled delivery and respirable administration of antituberculosis drug Is.
