Preparing Azithromycin Nanocrystals by Medium Grinding

Azithromycin nanocrystals were prepared by medium grinding method. The effects of machine speed, grinding time, size and dosage of zirconia beads, types and concentrations of surfactants, and concentration of azithromycin on particle size and polydispersion index were investigated. Scanning electron microscopy (SEM), particle size analyzer, X-ray powder diffraction (XRPD) and infrared spectrometer (FT-IR) were used to analyze and characterize the nanocrystals, and the dissolution properties of the nanocrystals and the bulk drug were studied. The experimental results showed that the average particle size of acimxol nanoparticles was 165 AM, and the crystal state of acimxol nanoparticles did not change significantly before and after grinding. Compared with bulk drug, the dissolution rate of nanocrystals has been significantly improved.

Azithromycin (AzM) is a new generation of macrolides antibiotics, which has a wide antibacterial spectrum. It not only has an effect on Gram-positive coccus, anaerobic bacteria, mycoplasma, chlamydia, but also has a good antibacterial effect on some gram-negative bacteria, including Gonorrhea, haemophilus influenzae, etc. The drug has high permeability and low solubility, which is a typical class ⅱ drug in the biopharmaceutical classification system, resulting in poor absorption in the human body and only 37 bioavailability, which reduces the clinical efficacy. The methods to solve this kind of problem are to make the drug into soluble salt, change the crystal shape to make amorphous drug, use the technology of micropowder and solid dispersion to increase the surface area of the drug, and make inclusion complex.

In the experiment, AZM ultrafine powder with average particle size of about 1.0 m was prepared by micropowder technology, which improved the solubility and dissolution of azithromycin to a certain extent, but there were also some problems such as high energy consumption, low product yield, easy pollution and so on. With the continuous progress of science and technology, nanocrystallization has been widely developed. Due to the rapid increase of specific surface area, nanocrystalline drugs can effectively overcome the problem of low bioavailability of insoluble drugs, and nano-drugs emerge with this trend.

Nanomeuticals refer to the combination of nanotechnology and pharmaceutical theory to make drugs into drug-carrying particles or nanocrystals with a particle size of 1 ~ 1 000 nm. Drug-carrying particles generally include nanoparticles, nanocapsules, nanoliposomes, etc., which are matrix skeleton or vesicle-type nanomeuticals. The preparation process is complex and the drug loading is usually low. The research on nanocrystals began in the 1990s, which is a kind of “pure” solid drug particles, containing a small amount of stabilizer colloidal dispersion system. At present, the preparation methods of nanocrystals mainly include medium grinding, high pressure homogenization, precipitation, emulsification and so on. The problem of organic solvent residue exists in the precipitation and emulsification methods. The high pressure homogenization method requires expensive equipment and high energy consumption. The medium grinding method only needs to put the liquid dispersed with drugs and the grinding medium together in the closed grinding chamber, relying on the operation of the machine to drive the impact between the drug particles and the drug particles and the grinding medium, the wall of the device, and reach the nanometer level. The medium grinding method is simple in operation, low in production cost and suitable for industrial production. Four nanocrystalline preparations have been successfully prepared and marketed by this method. In this paper, the author used crystalline AZM as raw material. AZM nanocrystalline suspension was prepared by medium grinding method. After freezing and drying, AZM nanocrystals with uniform particles, narrow particle size distribution and good water redispersion were obtained.

Ⅰ. Reagents and Instruments

1. Reagents

  • AZM API, Poloxam 188(P188), tocopherol succinic acid (TPGS, Hubei Hengshuo Chemical Co., LTD.);
  • Sodium dodecyl sulfate (SDS, Tianjin Damao Chemical Reagent Factory);
  • Tween 80(Tianjin Chemical Reagent No. 3 Factory);
  • Acetonitrile, methanol (chromatographic pure, Oceanp AK AlexativeChemical Compand, Ltd);
  • All other reagents were analytically pure, and the laboratory water was double distilled water.

2. Instruments

  • Qm-3sp04 Planetary ball mill (Nanjing University Instrument Factory);
  • Nano-$9O Laser particle size tester (Malvern Instrument Company, UK);
  • FTS — 135 Fourier Transform Infrared Spectrometer (Bole);
  • S-4800-i Cold Field Emission scanning electron microscope (HITACHI);
  • D/MAX a 2500 X-ray diffractometer (Rigaku, Japan);
  • 2010EV High Performance Liquid Chromatograph (Shimadzu Corporation, Japan);
  • Uv-2600 Ultraviolet Spectrophotometer (Shanghai Tianmei Scientific Instrument Co., LTD.);
  • Rz-8a Intelligent drug dissolution tester (Tianjin University Precision Instrument Factory)

Ⅱ.  Experimental Methods

1. Preparation of AZM nanocrystals

A certain amount of surfactant was accurately weighed and placed in a 100 mL grinding tank, then 15 mL of distilled water was added and ultrasound was performed for 2 rain. Take an appropriate amount of AZM API and place it in the above-mentioned grinding tank for ultrasound for several minutes. After the drug is fully dispersed, add a certain amount of zirconia beads. Install machine, adjust machine speed and run ball mill. Stop the machine for 15 min every 2 h, stop the machine after grinding for a certain period of time, stand for a period of time, freeze and dry the suspension for 8 h, and get AZM nanocrystals.

2. Characterization of Nanocrystals

The particle size and dispersion of AZM bulk drug and nanocrystalline were determined by particle size analyzer, and their morphology was confirmed by SEM. XRPD was used to analyze the crystal shape characteristics of nanocrystals. At the same time, ft-IR was used to scan at 400~4 000 cm-1 to compare the changes of drug chemical structure before and after grinding.

3. Dissolution Test

The dissolution rate of ZAM bulk drug and nanocrystals prepared under optimized conditions were determined in vitro by the dissolution rate determination method in Chinese Pharmacopoeia 2010 Edition (Part 2) (Appendix XC Method No. 2). After degassing the distilled water, 900 mL hydrochloric acid solution with pH= 1.0 was prepared as the dissolution medium. The stirring speed was set at 100 r/min, and the water bath temperature was adjusted to 37± 0.5 ℃. After the temperature was stabilized, the samples were put into each dissolution cup, and 5 mL samples were taken at 1, 2, 3, 5, 7, 10 O, 15, 20 O, 30 O, 45 and 60 min. The samples were filtered by 0.22 m microporous membrane, and 5 mL new media was added at the same time. The filtrate was diluted appropriately, and its peak area was determined by azithromycin content determination method (HPLC) according to Chinese Pharmacopoeia 2010 edition. The dissolution amount was calculated and the dissolution curve of the drug was drawn.

Ⅲ. Results and discussion

1. Investigation of Influencing Factors

A review was made of the main factors influencing grain size and polydispersity index (PI), such as machine speed, grinding time, zirconia bead size and dosage, surfactant type and concentration, and AZM concentration, to optimize the parameters and determine the optimal conditions for preparing AZM nanocrystalline.

(1) Machine Speed

Rotational speed is one of the important parameters of the grinding machine, which determines the kinetic energy of the whole system. Different surfactants with a mass fraction of 5.0 (relative main drug volume) were selected and the grinding time was controlled for 6 h. The effects of different rotational speeds on particle size and dispersion of the products were investigated, and the results were shown in Figure 1.

It can be seen from Figure 1 that at low rotational speed (450 r/rain), the particle sizes of the products prepared by using the four surfactants are all large, with PI values greater than 0.45, indicating poor uniformity of particles and wide particle size distribution of the products. With the increase of rotational speed, the particle size decreases continuously, and the dispersion of the particles also becomes better. Especially, when TPGS is used as the surfactant, the particle size of the product decreases nearly twice when the rotational speed is adjusted from 450 r/min to 500 r/min. When the rotational speed reaches 600 r/min, the particle size does not decrease with increasing rotational speed, and even tends to increase. Except for products using P188, the PI values of the other three particles basically do not change. In addition, under the condition of the same speed, the particle size of the products using TPGS and SDS as surfactants is smaller, and the particles are more evenly dispersed. Because in the grinding process, the drug particles by the high speed ball mill shear force and zirconia beads movement process formed extrusion pressure and constantly reduced: in the case of low speed, the system does not have enough kinetic energy, so that the drug particles, particles and zirconia beads between the collision effect is weak; When the rotation speed is high, the particle size of the product begins to increase again, which may be because the excessively high rotation speed causes the smaller particles to dissolve and then precipitate, leading to the larger particle size of the final product, or the change of system temperature before and after grinding, leading to this process. Considering the particle size and PI of the product, the optimal speed of the machine is 600 r/min.

(2) The Size and Dosage of Zirconia Beads

Zirconia beads are an important part of the preparation of nanocrystals by wet grinding. The large drug particles are constantly reduced by the extrusion and collision of zirconia beads. Therefore, it is very important to select the appropriate size and dosage of zirconia beads for the success of the experiment. In the experiment, ZrO2 beads with diameters of 0.5, 0.8, 1.0 and 3.0 mm were selected with the dosage of 20.o ~ 40.0 g and the grinding time was set to 6 h to investigate the effect of zirconia beads on the particle size and PI of the product. The results are shown in Figure 2. It can be seen that when the particle size of zirconia beads changes from 3.0mm to 0.5mm, the particle size also decreases. This is because the smaller the particle size of beads, in the same number of contact points and the number of more, and then increase the collision frequency between drugs and beads, improve the grinding effect. In addition, when the particle size of beads is small, the medium gap can play a filtering role, intercept and break the large particles, promote the reduction of particles, and improve the dispersion of particles.

It can also be seen from Figure 2 that it is very important to select the right amount of grinding media in order to obtain the smaller particle size. As shown in Figure 2, the particle sizes of the products prepared by grinding 30 g(diameter: 0.5, 0.8 mm) beads are 155,165 nm, and PI values are 0.175 and 0.181, respectively. The same number of zirconia beads with diameters of 1.0 and 3.0 mm were used to obtain larger particle sizes (~-220 nm). For each particle size of zirconia beads, the nanometer grain size decreases gradually with the increase of the amount of zirconia beads, and the particle size increases again when the amount exceeds a certain amount. This is because the number of contact points is reduced when the grinding tank contains less zirconia beads; However, when the amount increases, the impact strength of zirconia beads is weakened when the machine provides the same kinetic energy, so the grinding effect is not good. At the same time, it was found in the experiment that although small size zirconia beads (diameter 0.5 mm) can produce small size nanocrystals, but the product is difficult to separate from the zirconia beads and the loss of zirconia beads during the preparation process is more, so 30 g ZrO beads with a diameter of 0.8 mm are the best.

(3) Grinding Time

30 g ZrO zirconia beads were weighed, SDS was selected as surfactants, AZM mass concentration was 300 g/L, and the grinding time was controlled within 2.0-7.0 h. Table 1 reflects the variation of product particle size with the grinding time. It can be seen that with the extension of grinding time, the particle size and PI value of the product are significantly reduced, especially in the initial grinding, this trend of reduction is more obvious. When the grinding time is 6.0 h, the average particle size of the product is reduced to the minimum, and the PI value of the particle is also small. After that, when the grinding time was increased, the particle size tended to increase, and the PI value had the same phenomenon. This is because of the longer grinding time, the large drug particles can be fully shear, impact and broken into small particles, at the same time help surfactant adsorption on the drug surface to obtain a relatively stable system, and make the nanoparticles can be more evenly dispersed in the liquid phase. However, when the grinding time is too long, the particles may connect with each other and grow, and the temperature rise of the grinding system will lead to the aggregation of particles, which will affect the particle size and dispersion of the product. Therefore, the grinding time of 6.0 h is suitable.

(4) The Type and Concentration of Surfactant

When the particles are ground into nanocrystals, especially from several nanometers to tens of nanocrystals, the nanocrystals have a huge specific surface area and a sharp increase in surface energy. If there is no suitable stabilizer to wet the surface, the particles will slowly gather and form secondary particles, and then lose the unique physical and chemical properties of nanocrystals. Surfactants and polymers are commonly used as stabilizers, among which surfactants are often selected for their advantages of low viscosity of dispersed solution and multilayer adsorption on the surface of particles. In this experiment, the mass concentration of AZM was controlled at 300 g/L, and four surfactants, P188, SDS, TPGS and Tween 8O, were screened. The effects of their mass concentration (relative to main drug dosage) on the grain size and dispersion of AZM nanoparticles were investigated, and the results were shown in Figure 3.

Figure 3 shows that when TPGS and SDS are used, the particle size of the product is below 473 nm, and the particle size can be reduced to 165 nm when the optimal dosage is used. The effect of P188 as stabilizer is worse than that of the former two, while when Tween 80 is used as stabilizer, the minimum particle size of the product is the change of table L particle size and dispersion with the grinding time is 602 nm. The PI value of nanocrystals is also large. Analysis of the reasons shows that P188 and Tween 80 are non-ionic surfactants, and their hydrophobic groups combine with drugs to form steric hindrances, making the distance between particles larger to prevent particle agglomeration. However, the experiment found that during the high speed running of the ball mill, Tween 😯 was easily foamed by violent impact. This is also the main reason why Tween 80 has stronger hydrophobic effect than P188, but the particle size and PI value of the prepared product are larger. TPGS are also non-ionic surfactants with a smaller hydrophilic and oleophilic equilibrium value, so the more TPGS adsorbed on the drug surface, the more hydrophobic it will be, thus forming a stable and dispersed system. At the same time, the viscosity of TPGS solution is smaller, so compared with P188, the nano grain size prepared by TPGS is greatly reduced. After grinding with TPGS of high concentration, the system becomes paste, which makes it difficult to separate the product and zirconia beads and greatly weakens the grinding effect. Therefore, TPGS with mass fraction of 5.0, 8.0 and 12.0 in Figure 3 did not get corresponding products. However, when the TPGS mass fraction is reduced to 0.1, the AZM particle size becomes larger. SDS is a kind of anionic surfactant, its stability of the system mainly depends on the electrostatic repulsion generated by electric charge. When SDS is adsorbed on the drug surface, the drug can be properly wettable and dynamic stability can be achieved under the action of electrostatic repulsion. The results in Figure 3 also show that SDS can be used as a stabilizer to prepare nanocrystals with smaller particle sizes.

It can also be seen from Figure 3 that the concentration of surfactant also has certain limits. Except TPGS, when the dosage of the other three stabilizers increased, the particle size of the nanocrystals did not decrease, and the particle size of the products prepared by SDS and P188 even increased, while the dispersion also began to deteriorate. This is because the high surface energy of nanocrystals increases the frequency of collisions between particles to reduce the energy of the system and achieve a stable state. Excessive surfactant can provide a bridging effect for particles to collide with each other, resulting in Ostwald phenomenon and speeding up particle agglomeration. The experimental results showed that SDS 5.0 was the best stabilizer for wetting and dispersing drugs.

2. Study on in Vitro Dissolution Performance

The Noyes-Whitney equation is often used to describe the dissolution rate of drugs. When the particle size decreases to the nanometer level, the specific surface area of drugs increases, thus speeding up the dissolution of drugs and improving the dissolution rate. The in vitro dissolution properties of nanocrystals (surfactants are SDS, TPGS, P188 and Tween 80) prepared by AZM and the optimal technological conditions were studied experimentally. The dissolution curves are shown in Figure 8. It can be seen that the dissolution rate of AZM nanocrystals increases significantly. When 10 rain is cast, the cumulative dissolution rate of AZM nanocrystals using SDS as surfactants can reach 75, which is about 7.5 times of the dissolution amount of bulk drug at the same time. At 45 min, the nanocrystals could be dissolved completely, while the total amount of dissolution was only about 21 at the same condition. In addition, FIG. 8 also shows the difference in dissolution performance of products prepared by different surfactants. The total dissolution of products prepared by SDS and TPGS exceeds 90 at 60 rain. P188 and Tween 80 products were 72.5 and 55.1, respectively, and still showed an increasing trend, which fully indicated that decreasing particle size can improve the dissolution performance of AZM.

Ⅲ. Conclusion

AZM nanometer suspension was prepared by medium grinding method, and nanocrystalline was obtained by freezing and drying. The effects of machine speed, grinding time, size and dosage of zirconia beads, type and concentration of surfactants, concentration of AZM and other factors on the size and dispersion of nano-grains were investigated. The optimum process conditions were determined as follows: SDS with mass fraction of 5.0 / G as stabilizer, AZM mass concentration of 300 G/L, distilled water volume of 15 mL, 30 G (diameter of 0.8 mm) as zirconia bead, rotation speed of 600 r/min, grinding time of 6.0 h. The nanocrystals prepared have regular oblong shape with an average particle size of 165 nm and the particle size distribution ranges from 100 nm to 300 nm. XRPD and FT-IR analysis showed that the product was still crystalline AZM with no change in chemical structure. The dissolution rate and total amount of AZM nanocrystals were improved obviously compared with that of bulk drug. This method has the advantages of simple preparation, low cost, small particle size, good dispersibility and good application prospect.

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