Preparation and distribution of 5-fluorouracil 125I sodium alginate-bovine serum albumin nanoparticles

Abstract

AIM: To prepare 5-FU sodium alginate ¹²⁵I bovine serum albumin nanoparticles (BSA NP), to de-termine the radioactive count in different organs of rats at different time points after oral adminis-tration of 5-FU ¹²⁵I sodium alginate-BSA NP and to calculate the kinetic parameters of its metabolism.

METHODS: Emulsion solidification method was used to prepare 5-FU ¹²⁵I sodium alginate-BSA NP, and to determine its diameter under trans-mission electronic microscope (TEM). Then the rate of NP and external drug releasing velocity were measured. Radioactive counting in different organs of rats wa s made after oral adminis-tration of the NP by GAMA Counter, and the ki-netic para meters of drug metabolism were calcu-lated by handling the data with the two-depart-ment model.

RESULTS: The average arithmatic diameter of the NP was 166 nm ± 34 nm, the rate of 5-FU was 32.8% and the cumulative external releasing ra-tio amounted to 84.0% within 72 hours. The NP was mainly distributed in the liver, spleen, lungs and kidneys after NP oral administration to rats. The mic ro-radioautographic experiment showed that NP was distributed in the Kupffers cells of liver, liver parenchymal cells and the phagocytes of spleen and lungs. The kinetic pa-rameters of matabolism were: T_1/2 = 9.42 h, C_max =2.45 × 10⁷ Bq, T_max = 2.18 h, AUC = 148 × 10⁹ Bq.

CONCLUSION: NP is difficult to pass through the blood-cerebral barrier,and ¹²⁵I sodium alginate-BSA NP enters the body circulation by gastroin testinal passage.

Key Words: 5-fluorouracil (5-FU), sodi-um alginate-al bumin, preparation of nanoparticle (NP), distribution

INTRODUCTION

Nanoparticle (NP) is an colloidal dispersion system, with diameters ranging from 10 nm to 1000 nm. The particles exist mainly in the organs rich in phago-cytes after absorption, such as liver, spleen and lymph system. Since its introduction in the 1980s, scientists have done a lot of researches on its preparation, stability and targeting[1,2]. Now most mate-rials used to prepare NP are synthetic substances, e.g. cyanoacrylate, methylacrylate and polylactic acid. However, we selected natural substances-sodi-um alginate and bovine serum albumin as carriers, and 5-fluorouracil (5-FU) as model drug to prepare NP by emulsion solidification, and studied its distri-bution after oral administration.

MATERIALS AND METHODS

Animal

The Kunming rats weighing 20 g ± 2 g, were provided by the Experimental Animal Centre of Tongji Medical University.

Reagent

Sodium alginate (chemical reagent), bovine serum albumin (Sigma), pentane dialdehyde (biochemical reagent, Merck), Na ¹²⁵ I (speific activity 7.78 TBq/L, Chinese Atomic Energy Institution), IV liquid nuclear emulsion (Physics Institute of Chinese Atomic Energy Research Institution).

Preparation of 125I bovine serum albumin (BSA)

The BAS (10 mg) was dissolved in 10 mL distilled water and marked with I¹²⁵ according to the chloro-amine-T method. After the reation was completed, the mixture was separated by column chromato-graphy (10 mm × 340 mm), using Sephadex G-50 as column material and Na₂S₂O₃ as eluant. Then the radio-chemical purity of eluate was determined by dichloroacetic acid method.

Preparation of 125I sodium alginate-BAS NP

According to the literature, 1 mL-above I¹²⁵-BAS (1 g/L, SpA 107 Bq) was added into sodium algi-nate solution to prepare sodium alginate- BSA NP.

Determination of the diameter of nanoparticles

The NP size in the suspension was detected on TEM.

Determination of the encapsulation efficiency

The standard curve of 5-FU was drawn first. One hundred and five mg of 5-FU was weighed precise-ly, dissolved in thin HCL solution (9-1000) and adjusted to given volume. This solution was diluted to its half concentration. After 3.0, 6.0, 9.0, 12.0 and 15.0 mL-diluted solution were taken into 100 mL-volumetric flasks and adjusted to given volume respectively, their absorbance (A) values were detected on ultraviolet-visible spectrophotometer at 265 nm. The following regression equation was obtained: C = 16.72A± 0.0416 ( r = 0.9998).

A total of 1.0 mL 5-FU NP suspension was added accurately to a test tube with 9 mL 6 mol/HCl. The mixture was hydrolyzed for 20 h at 100 °C water bath. The hydrolyzate was adjusted to given volume with distilled water to detect its A val-ue as standard curve item, the hydrolyzate of bland NP suspe nsion served as control. The result showed that the control group had no obvious absorption at 265 nm (A = 0.01). By putting the A value into the regression equation we worked out the concentration of 5-FU in the hydrolyzate and got the encapsu-lation efficiency consequently.

Determination of recovery rate

The same quantities of albumin, sodium alginate and pentane dialdehyde as “Prep aration of NP” items were mixed with 5-FU weighed precisely, the mixture was dissolved in distilled water. Its A value was detected according to the standard curve and the content of 5-FU was calculated.

Determination of drug release

Appropriate quantity of 5-FU NP suspension was taken precisely, washed for three times with dis-tilled water, and suspended in 200 mL-thin HCL solution (9-1000). The drug release state of the sus-pension was measured at 37 °C by paddle method (100 r/min). The samples were taken at the beginning, the 2nd, 4th, 8th, 12th, 24th, 48th and 72nd hour, and filtered through 0.2 μm microporous membrane. The filter liquor was detected at 265 nm to get its A value and cumulative rate of drug release.

The distribution of 125I sodium alginate-BSA in rats

Fourty-five Kunming rats were divided into 15 groups (3 rats per group). After starvation for 16 h, the rats were given the 5-FU NP orally 148 × 10⁸ Bq per rat. Right after its blood was taken from the eye at different time points (heparin was added to prevent coagulation), the rats were sacrificed by breaking its spine. The relevant tissues and organs were taken. When the water had been absorbed with filter paper after washing, they were weighed. The amounts of the NP in unit weight in different tissues were measured by GAMA Counter. In accor-dance with the counting result, we calculated the tissue blood ratio at different time points and used two-department model to process the data in order to get pertinent kinetic parameters.

Radioautography

While carrying out the histological experiment, we chose the tissues of liver, spleen, lungs, and kidneys at some time points (the 4th, 8th and 12th hour), and frozen sections and paraffin wax sections about 6 μm-thick were made. IV liquid emulsion was used for microradioautography.

RESULTS

Diameter of NP

On the 5-time enlarged photograph (diagram 1) of TEM, the average arithmetic mean diameter of 300 detected NPs was 166 nm ± 33 nm, 8% < 130 nm, 36% 130 nm-150 nm, 42% 150 nm-170 nm and 14% > 180nm.

Encapsulation efficiency

After two batches of 5-FU NPs were hydrolyzed in acid medium, their encapsulation efficiency was calculated (Table 1).

Table 1 Encapsulation efficiency of 5-FU in NP.

Weight of drug added (g)	A value	Weight of drug encapsulated (g)	Encapsulation efficiency (%)
0.6162	0.679	0.208	33.8
0.6113	0.625	0.193	31.6
0.5917	0.676	0.195	32.9

Rate of recovery

The rate of recovery of three batches of samples is shown in Table 2.

Table 2 Rate of recovery of 5-FU.

Weight of 5-FU added (g)	Weight of 5-FU determined (g)	Rate of recovery (%)
0.1348	0.1326	98.4
0.1463	0.1449	99.1
0.1429	0.1395	97.6

Varitation of drug releasing ratio of 5-FU NP before and after storage

The cumulative ratio of drug release within 72 h decreased from 84.8% before storage to 80.6%(Figure 1) after storage under 40 °C ± 1 °C for three months.

Figure 1 Releasing of 5-FU from NP.

The pharmacokinetics of ¹²⁵I sodium alginate-BAS NP in rats

Thirty min after the rats administrated ¹²⁵I sodium alginate-BSA NP orally, obvious radioactivity was shown in its blood. Eight hours after the administration, the radioactivity of their liver, spleen and lungs was 2.08 times, 2.32 times and 1.60 times, as much as that of blood, while 24 h after the ad-ministration, they decreased to 1.18, 1.22 and 0.87 times respectively (Table 3).

Table 3 Distribution of ¹²⁵I sodium alginate-BSA NP in rats (n = 3, dosage 148 × 10⁸ Bq per rat, po.).

Organ	Value of radioactive counting of 100mg tissue in 30s at different time points
	1 h	4 h	8 h	16 h	24 h	36 h
Blood	502 ± 100	601 ± 135	373 ± 22	298 ± 19	191 ± 17	129 ± 23
Heart	436 ± 110(0.86)	801 ± 66(0.33)	265 ± 23(0.71)	230 ± 23(0.77)	190 ± 10(0.99)	87 ± 35(0.67)
Liver	436 ± 110(0.86)	908 ± 100(1.51)	777 ± 69(2.08)	405 ± 97(1.36)	226 ± 16(1.18)	153 ± 21(1.19)
Spleen	663 ± 140(1.32)	1029 ± 168(1.71)	834 ± 272(2.32)	436 ± 195(1.46)	233 ± 25(1.22)	169 ± 27(0.95)
Lungs	717 ± 314(1.42)	818 ± 172(1.36)	597 ± 121(1.60)	443 ± 31(1.49)	166 ± 4(0.87)	119 ± 27(0.92)
Kidneys	526 ± 139(1.05)	185 ± 39(0.14)	401 ± 71(1.08)	387 ± 105(1.30)	133 ± 13(0.69)	123 ± 14(0.95)
Brain	105 ± 31(0.21)	112 ± 49(0.19)	111 ± 47(0.30)	96 ± 27(0.32)	50 ± 5(0.26)	39 ± 11(0.30)
Stomach	89654 ± 7143	28674 ± 3647	8362 ± 1902	4641 ± 1400	698 ± 34	527 ± 114
Intestine	765 ± 137	5573 ± 891	30 ± 599	403 ± 118	115 ± 64	204 ± 46
Bowel	1256 ± 664	639 ± 1023	30 ± 599	3221 ± 863	125 ± 60	256 ± 102

The data in the parentheses show the ratio of radioactivity of the relative organ to that of blood. The data in Table 3 were put into computer, and processed according to the two-department model to calculate the relevant pharmacokinetical parameters. The results are shown in Table 4.

Table 4 Pharmacolinetical parameters of ¹²⁵I sodium alginate-BSA NP in rats.

Parameter	Unit	Value
A	Bq	3022 × 102
α	1/h	0.07
B	Bq	3.03 × 103
β	1/h	-0.11
Ka	1/h	1.46
V/F	Bq	1428 × 103
T1/2α	h	9.42
T1/2β	h	-6.07
T1/2Ka	h	0.47
K21	1/h	-0.11
K10	1/h	0.07
K12	1/h	-0.00035
AUC	Bq × h	3.9 × 109
CL(s)	Bq × h/Bq	105 × 103
T(peak)	h	2.18
Cmax	Bq	2.45 × 107

Radioautography

The microradioautographical experiment indicated that the ¹²⁵I sodium alginate-BSA NP were mainly distributed in the Kupffer cells of liver and liver parenchymal cells (Figure 2) after oral administration to rats. And there were Ag particles in the phagocytes of spleen (Figure 3) and also in the pulmonary cells (Figure 4).

Figure 2 NP in liver. × 1000

Figure 3 NP in spleen. × 1000

Figure 4 NP in lungs. × 1000

DISCUSSION

Since the introduction of NP in the early 80s, scientists have done a lot of researches in its preparation, stability, distribution and targeting. It is suggested that the stability of NP is better than that of lipo-some and it can carry more drug than liposome.

At present, the materials, which are reported to prepare NP in literature abroad, are all man-made synthetic materials. While NP is mostly ad-ministrated in travenously, we chose natural polysac-charide and protein to compose the carrier, and observed its distribution and metabolism after oral administration to rats.

Thirty minutes after the rats administrated ¹²⁵I sodium alginate-BSA NP orally, the radioactive substances in their blood were detected.

The results indicate that NP is mainly distributed in the liver, spleen and lungs, quantitatively in heart and slightly in brain. This shows NP is difficult in passing through the blook-cerebral barrier. The experiments also display that the ¹²⁵I sodium al-ginate-BSA NP enters the body-circulation by gas-trointestinal passage and is chiefly distributed in the tissues rich in phagocytes, such as in the liver and spleen.

In the study of its distribution, we discovered that there is still a large amount of radioactivity in the gastrointestinal passage 12 h after the ad-ministration. This illustrates that the absorption of NP is not complete because NP exists in the suspension as cloudy polymer but not mono-dispersion, and the large diameter of the polymer makes it difficult for the NP to be absorbed by the gastrointestinal passage.