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1. (WO2019032057) METHOD FOR PREPARING NANO-PARTICLE OF TILMICOSIN AND PRODUCT THEREOF
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Title of the invention

METHOD FOR PREPARING NANO-P ARTICLE OF TILMICOSIN AND PRODUCT THEREOF

Field of the invention

The field of chemistry in relation to the synthesis of nanoparticles, especially preparation of tilmicosin nanoparticles.

Background of the invention

Tilmicosin is an antibiotic which belongs to the group of macrolide with broad effects in controlling and treating animal's respiratory diseases caused by Actinobacillus Microplasma Pasteurella Haemophilus wherein tilmiconsin will be absorbed and accumulated in cells of the respiratory tract and, thereby elicited bodily attack on the germs before being spread into lungs. Further, it is found that the accumulation of tilmicosin in the lung tissues, especially in the major white blood cells of the lungs, namely neutrophils and monocytes, can cause the destruction of bacteria in the area of infection quite effectively. Further, it is found that tilmicosin can work together with the major white blood cells to suppress Porcine reproductive and respiratory syndrome virus (PRRSV) as well.

Oral administration is the safe and most popular way, but it comes with certain limitations, such as Gastrointestinal epithelium, which creates a physical barrier for absorption of drugs into the blood circulation. Therefore, further investigation and development for better drug delivery system is needed in order to increase absorption capacity from digestive to blood circulation system.

Tilmicosin base is the raw material which has low solubility, but for ease of feeding to animals, the tilmicosin phosphate version is used to improve its water solubility by means of mixing tilmicosin base with phosphoric acid. However, the problem with tilmicosin phosphate has to do with its relatively low bioavailability. Even if one can increase efficacy or bioavailability by increasing dosage, there is the risk of rapid and acute cardiac toxicity as the level of toxicity is dose dependent. Therefore, it is necessary to develop a tilmicosin delivery system to improve tilmicosin efficacy and solve such problem as stated.

Vehicles that can be used to deliver drugs can be made of various materials that are biodegradable such as lipids wherein these vehicles shall be stable and toxic free in order to improve efficacy and safety of the drug being delivered. Presently, the lipid-based nanoparticles are very popular— from synthetic and natural sources— for utilizing as vehicles to improve bioavailability by means of controlled release. It is found that such lipid-based nanoparticles will improve absorption capacity through digestive tract tissues by increasing dissolution of drug, slowing gastric emptying rate and prolonging releasing rate. Further, lipids can boost circulation of lymph wherein the drug, encapsulated in these lipid nanoparticles, can be absorbed through lymph, together with lipids, which can also be called Trojan horse effect. Lipid nanoparticles can improve solubility and absorption of drug whereby these particles can encapsulate and protect drugs from being destroyed and can be mixed with water which acts as emulsifier in the digestive system. Further, the lipid nanoparticles can be absorbed through microfold or M cells in Peyer's patch and directly into the lymphatic system to prevent imperfect absorption and first-pass metabolism.

As for the prior inventions related to tilmicosin's compositions in the global patent databases, the following were found:

The invention "Tilmicosin liposome injection and preparation method thereof with publication number "CN 102327225" has disclosed tilmicosin production method wherein the particle size is in the range of 7-10 microns with the compositions of soy beam lecitin of 3-5 grams, tilmicosin of 5-10 grams, cholesterol of 1.5-2.5 grams, non-ionic surfactant of 2 grams, and water of 20 millilitres to produce liposome of tilmicosin. Nevertheless, such invention's liposome particles are difficult to scale up into manufacturing process.

Further, there are various other publications related to tilmicosin nano-emulsion as follows:

The invention "Preparation method of tilmicosin nano-emulsion antibacterial drug" with publication number "CN 101422432" discloses tilmicosin nano-emulsion wherein the particle size is in the range of 1-100 microns, comprising the following compositions: tilmicosin at 0.01-0.06 %, surfactant/co-surfactant of 20.0-40.0 %, oil of 2.2-10.0 %, and volume-adjusted with water. The preferable types of oil are isopropyl myristate (IPM) or ethyl acetate.

The invention "Compound tilmicosin nano-emulsion antibacterial agent and preparation method thereof with publication number "CN101983632" discloses tilmicosin nanoparticles with particle size of range 1-100 nanometers (nm) with the following ratio: tilmicosin of 0.01-15.2 %, florfenicol of 0.001-2.12 %, surfactant of 13.38-36 %, co-surfactant of 0-26.5% oil of 2.43-5.32 %, and volume-adjusted with water, wherein the preferably oil are chosen from isopropyl myristate (IPM) or ethyl acetate, tilmicosin solution of 14.89-84.17 % and florfenicol soluble of 0.004-2.12 %.

The invention "Compound nanoemulsion of preventing and treating mgi (mycoplasma gallisepticum infection) and preparation method" with publication number "CN105640887" disclose tilmicosin nano-emulsion comprising 4-10 parts of tilmicosin, 10-25 parts of oil, 20-40 parts of surfactant, 8-16 parts of co-surfactant, 5-20 parts of spiramycin, and 0.1-5 parts of diprophyllin.

The inventions as described above are nano-emulsion particles of tilmicosin which are different from the tilmicosin nanoparticles of the present invention. The differences are related to the compositions of nano-emulsion which contains very high ratio of surfactant and thereby, causing the production to be more wasteful.

There are other inventions that are related to improved solubility of tilmicosin in the following examples:

The invention "Tilmicosin emulsion and preparation method thereof with the publication number "CN105640883" which discloses emulsion of tilmicosin wherein they are not formed into particles but merely process of dissolving tilmicosin comprising 2-20 % of lecithin, 2-20 % of tilmicosin, and 1-10 % of emulsifiers.

The invention "Suspension for injecting tilmicosin and preparation method thereof with publication number "CN103906039" discloses compositions that help to improve tilmicosin's solubility without forming into particles wherein said compositions comprises 5-30 % of tilmicosin, 0.1-5.0 % of surfactant, 0.01-1 % of antioxidant, and lipid emulsion.

Such inventions merely improve solubility but are not considered particle clustering; thus, the derived products exhibit different properties.

Further, there appears prior arts related to grouping of micro-emulsion of particles such as "Microemulsion for animals and preparation method thereof with publication number "CN101947202" or the invention that groups microsphere such as "Method for preparing veterinary tilmicosin microspheres" with publication number "CN101810582".

In fact, it is found that inventions related to the preparation of tilmicosin according to the prior arts have had the attempts to group together particles in order to improve solubility of tilmicosin by using various mixture ratio. Nevertheless, many particles derived are considered nano-emulsion, micro-emulsion, micro-encapsulation, liposome, or microsphere— which differ in their ratios or chemicals used, particle sizes, up to quality of the derived products; as well, the different types of tilmicosin used as raw materials have effects on the different types of end products. Namely, the prior inventions preferably used tilmicosin phosphate which has higher water solubility rate than tilmicosin base, but is highly toxic. Further research and development tasks are required to investigate the use of tilmicosin base, but are quite unpredictable and highly dependent upon someone with special skills in the related arts.

As for the tilmicosin nanoparticles according to this invention, the invention is different from any of the previously described inventions in both its size and property by developing superior absorption efficacy with tilmicosin through digestive tract of animals with tilmicosin base as tilmicosin nanoparticles.

Summary of the invention

Method for preparing nano-particle of tilmicosin and product thereof according to this invention is the method of synthesizing tilmicosin nanoparticles which is the antibiotic used for controlling and treating respiratory infections in animals. The compositions generally comprises tilmicosin base antibiotic : surfactant : oil : water in the following ratio: 0.3-0.6 : 3-6 : 0.5-5.5 : 88-96 % by weight.

Method for preparing nano-particle of tilmicosin and product thereof according to this invention has the main objective to develop absorption efficacy of tilmicosin drug through the animal digestive tract with high stability and non-toxic to the digestive system by means of synthesizing tilmicosin antibiotic base in the form of tilmicosin nanoparticles.

Brief description of the figures

Figure 1 illustrates the size of particle and surface charge of one example of the synthesized tilmicosin nanoparticle;

Figure 2 illustrates the morphology of one example of tilmicosin nanoparticle;

Figure 3 illustrates the morphology of one example of tilmicosin nanoparticle;

Figure 4 illustrates the stability of an example of tilmicosin nanoparticle;

Figure 5 illustrates the stability of an example of tilmicosin nanoparticle;

Figure 6 illustrates the stability of an example of tilmicosin nanoparticle;

Figure 7 illustrates the number of simulated cells inside the digestive system;

Figure 8 illustrates percentage of survival of simulated cells inside the digestive system;

Figure 9 illustrates simulated cells in the digestive system;

Figure 10 illustrates the percentage comparing the permeability through the simulated cells of the digestive system.

Detailed description of the invention

Method for preparing nano-particle of tilmicosin and product thereoft according to this invention comprising a tilmicosin base antibiotic, surfactanct, oil and water wherein the tilmicosin base antibiotic is prepared from tilmicosin solution of at least 95% by volume by which said tilmicosin base is a bioactive compound for controlling or treating respiratory infections in animals. The oil acts as a vehicle for transporting tilmicosin base so that the drug can be easily absorbed wherein the oil according to this invention is the synthesized oil or distilled oil from raw, natural material that is not toxic to animal. The oil according to this invention can be chosen from at least one of the following: a plant oil or an animal oil. The plant-based oil can be chosen from soy bean oil, corn oil, palm olein, palm oil, coconut oil, olive oil, sunflower oil, rice bran oil, camellia oleifera seed oil, or other types of plant-based oils as well as other oils that are by-products from industries. The animal-based oil can be chosen from fish oil, pork oil, or any other animal oils. Most preferably in one embodiment is the soy bean oil. The surfactant used in this invention is preferably without charge and is chosen from at least one of a poloxamer, polysorbate, or sorbitol ester, wherein the polysorbate is chosen from polyoxyethylene (20) sorbitan monooleate, polyoxyethylene (20) sorbitan monosterate, polyoxyethylene (20) sorbitan monopalmitate, polyoxyethylene (20) sorbitan monolaurate. The sorbitol ester is chosen from sorbitol monooleate, sorbitol monostearate,

sorbitol monopalmitate, or sorbitol monolaurate. Most preferred in one embodiment of this invention is polyoxyethylene (20) sorbitan monooleate or polyoxyethylene (20) sorbitan monolaurate, or are used together.

Tilmicosin nanoparticles in one embodiment according to this invention comprises the following composition:

- Tilmicosin antibiotic base 0.3-0.6 % by weight

- Surfactant 3-6 % by weight

- Oil 0.5-5.5 % by weight

- Water 88-96 % by weight

Optionally, an additive may be added wherein said addictive may improve characteristic or quality of the composition, preferably chosen from flavoring agent or coloring agent wherein flavoring agent is either natural or synthetic agent or their combination; coloring agent may also be chosen from natural or synthetic agent or their combination.

Without considering as limitations to any parts of the specification or patent claims, other embodiments of compositions of tilmicosin nanoparticles are shown subsequently wherein the embodiments do not limit the chemicals and other ratios which are known to the skilled person in the related arts that these chemicals have similar characteristics or produce similar result, and by which any improvements or modifications may be possible within the claims according to this invention.

Example 1: Composition for synthesizing tilmicosin nanoparticles comprises the following:

- Tilmicosin antibiotic base 0.5 % by weight

- Surfactant 5 % by weight

- Oil 1 % by weight

- Water 94 % by weight

Example 2: Composition for synthesizing tilmicosin nanoparticles comprises the following:

- Tilmicosin antibiotic base 0.5 % by weight

- Surfactant 5 % by weight

- Oil 1.5 % by weight

- Water 93 % by weight

Example 3: Composition for synthesizing tilmicosin nanoparticles comprises the following:

- Tilmicosin antibiotic base 0.5 % by weight

- Surfactant 5 % by weight

- Oil 2.5 % by weight

- Water 92 % by weight

Example 4: Composition for synthesizing tilmicosin nanoparticles comprises the following:

- Tilmicosin antibiotic base 0.5 % by weight

- Surfactant 5 % by weight

- Oil 4.5 % by weight

- Water 90 % by weight

In one embodiment of the preparation method of tilmicosin nanoparticles according to this invention, the method comprises the following steps:

1. Preparation:

- Mixing tilmicosin base at 0.3-0.6 % by weight and oil at 0.5-5.5 % by weight, then adding surfactant at 3-6 % by weight to completely form a homogeneous mixture;

- Adding water at 88-96 % by weight to fill up 100% volume within the given temperature wherein the temperature is preferably between 60-90 °C, most preferably between 70-80 °C.

2. Reduction of particle size:

- Reducing the particle size of the mixture from step 1 , preferably with a sonicator to reduce the size of the particles into nanometer scale, preferably with a sonicator probe at the energy level of 20-40 % amp, most preferably at 30% amp with the timeframe of 3-8 minutes, most preferably at 5 minutes. After the process of synthesizing tilmicosin nanoparticle mixture, the nano solution is settle at room temperature within some time to adjust the temperature and cool down to derive the tilmicosin nanoparticle solution with high absorption efficiency, stability, wherein the delivery of said solution to animal can be adjusted for controlling or treating respiratory infections in various kinds of animals in the portion which is suitable for each individual's weight or severity of the disease or the kind of animal being treated.

The following examples display results of the analysis or experimentation in various configurations including the comparison between the synthesized tilmicosin nanoparticles by processes according to the description with 4 examples and tilmicosin phosphate which is a commonly used product.

Table 1: Proportions of compositions used for synthesis of tilmicosin nanoparticles in the samples according to this invention for analysis and testing.


Particle size analysis (Hydrodynamic diameter) and surface charge (Zeta potential)

Figure 1 illustrates the particle size and zeta potential of a synthesized tilmicosin nanoparticles after measurement. It is found that the size of samples depended upon the proportion or volume of oil used.

Table 2: Particle size and surface charge of the samples of synthesized tilmicosin nanoparticles.

Particle size (nm) Surface charge (mV)

Sample 1 104.3 -23.7

Sample 2 151.6 -25.1

Sample 3 133.9 -23.5

Sample 4 127.0 -29.1

From the table 2, it is found that samples 1, 2, 3, and 4, which contained different concentrations of oil, namely 1, 1.5, 2.5, and 4.5 % by weight, respectively, had formed the particle sizes of 104.3, 151.6, 133.9, and 127.0 nm and surface charges of -23.7, -25.1, -23.5, and -29.1 millivolts (mV), respectively. The negative charge value of more than -20 mV is found. It can be concluded that tilmicosin nanoparticle synthesis according to this invention can produce tilmicosin nanoparticles at less than 200 nm with good stability.

Morphology evaluation with transmission electron microscope (TEM)

After analyzing the morphology of transmission electron microscope (TEM), it is found that the synthesized nanoparticles resemble a cluster of fatty acids at 100 nm wherein the derived size is similar to the Dynamic light scattering (DLC) technique. The particle size from TEM is slightly smaller as the measurement is on dried particles as shown in Figure 2 which displays morphological characteristics of tilmicosin nanoparticles through transmission electron microscope at magnifying power of 25,000x at scale bar of 500 nm Figure 3 shows another morphological characteristics of sampled tilmicosin nanoparticles from electron microscope at magnifying power of 200,000 at scale bar of 50 nm.

Particle stability analysis using hydrodynamic diameter and surface charge

Nanoparticle stability analysis can be divided into 2 stages: (1) Incubation of nanoparticles by acceleration (freeze thawing) at 6 rounds during 24 days period; (2) Incubation of nanoparticles by acceleration at different temperatures at 1 and 2 months with the following incubating temperatures:

(2.1) At 4 Degree Celsius; (2.2) At 25 Degree Celsius; (2.3) At 45 Degree Celsius

Table 3: Particle stability from size and surface charge at accelerated conditions

Particle size (nm) Surface charge (mV)

Before After Before After incubation incubation incubation incubation

Sample 1 104.3 142.7 -23.7 -40.9

Sample 2 151.6 130.7 -25.1 -36.6

Sample 3 133.9 129.2 -23.5 -35.7

Sample 4 127.0 120.6 -29.1 -32.0

Table 3 illustrates particle stability analysis at accelerated conditions wherein Sample 1 was found to exhibit clear changes in particle size from before incubation of 104.3 nm to 142.7 nm after incubation wherein before incubation provided surface charge of -23.7 mV to -40.9 mV after incubation. It is found that the particle size of sample 1 exhibited significant changes at accelerated conditions. Further, the particles did not create any precipitations or separations and the size and surface charge are also in acceptable range which can be used for real production or improvement.

At the same time, the particles of sample 2, sample 3, and sample 4 appeared to show little changes as depicted in Figure 4 by considering the size and surface charge at acceleration.

Sample 2 showed the change before incubation at 151.6 nm to after incubation at 130.7 nm with the surface charge of -25.1 mV to -35.7 mV, accordingly.

Sample 3 showed the change before incubation at 133.9 nm to after incubation at 129.2 nm with the surface charge of -23.5 mV to -35.7 mV, accordingly.

Sample 4 showed the change before incubation at 127.0 nm to after incubation at 120.6 nm with the surface charge of -29.1 mV to -32.0 mV, accordingly.

Through the analysis of changes in size and surface charge of tilmicosin nanoparticles, it is found that all samples were stable under accelerated conditions.

Table 4: Considerations of particle stability based on particle size and surface charge at accelerated conditions at different temperatures for 1 -month period.

Particle size (nm) Surface charge (mV)

Before Before

4 °C 25 °C 45 °C 4 °C 25 °C 45 °C incubation incubation

Sample 1 104.3 125.9 122.2 118.7 -23.7 -22.6 -22.6 -24.2

Sample 2 151.6 157.2 158.0 157.1 -25.1 -26.3 -22.2 -22.2

Sample 3 133.9 139.6 139.7 140.9 -23.5 -23.0 -22.3 -27.1

Sample 4 127.0 128.2 125.9 127.0 -29.1 -27.6 -27.6 -25.1

Table 4 illustrates the results of the particle stability by analyzing the particle sizes and surface charges at different accelerated conditions, namely 4, 25, and 45 °C for 1 month as illustrated in figure 5 wherein the stability of tilmicosin nanoparticles is determined by their particle sizes and surface charges at different accelerated conditions for 1 month. There were changes in particle sizes in the sample 1 from before incubation of 104.3 nm to 125.9, 122.2, and 118.7 nm at accelerated conditions of 4, 25, and 45 °C, respectively. There were also changes in surface charges from before incubation at -23.7 mV to -22.6, -22.6, and -24.2 mV at accelerated temperatures of 4, 25, and 45 °C, respectively.

There were changes in particle sizes in sample 2 from before incubation of 151.6 nm to 157.2, 158, and 157.1 nm at accelerated conditions of 4, 25, and 45 °C, respectively; as well, there were changes in surface charges before incubation of -25.1 mV to -26.3, -22.2, and -22.2 mV at accelerated conditions of 4, 25, and 45 °C, respectively.

There were changes in particle sizes in sample 3 from before incubation of 133.9 nm to 139.6, 139.7, and 140.9 nm at accelerated conditions of 4, 25, and 45 °C, respectively; as well, there were changes in surface charges before incubation of -23.5 mV to -23.0, -22.3, and -27.1 mV at accelerated conditions of 4, 25, and 45°C, respectively.

There were changes in particle sizes in sample 4 from before incubation of 127 nm to 128.2, 125.9, and 127.0 nm at accelerated conditions of 4, 25, and 45 °C, respectively; as well, there were changes in surface charges before incubation of -29.1 mV to -27.6, -27.6, and -25.1 mV at accelerated conditions of 4, 25, and 45 °C, respectively.

Table 5: Considerations of particle stability based on particle size and surface charge at different accelerated conditions for 2-months period.

Particle size (nm) Surface charge (mV)

Before Before

4 °C 25 °C 45 °C 4 °C 25 °C 45 °C incubation incubation

Sample 1 104.3 118.7 113.9 126.1 -23.7 -25.4 -24.0 -24.2

Sample 2 151.6 159.8 158.7 156.0 -25.1 -24.1 -23.6 -7.97

Sample 3 133.9 138.0 137.9 138.1 -23.5 -22.9 -23.4 -6.54

Sample 4 127.0 126.1 125.6 121.9 -29.1 -27.2 -27.4 -1 1.6

Table 5 displays particle stability analysis results based on particle sizes and surface charges at various accelerated temperatures, namely 4, 25, and 45 6C for the period of 2 months as shown in Figure 6 which shows the stability of tilmicosin nanoparticles based on particle size and surface charge at various accelerated conditions for 2-months period. There were changes in particle sizes in sample 1 from before incubation of 104.3 nm to 118.7, 113.9, and 126.1 nm at accelerated conditions of 4, 25, and 45 °C, respectively; as well, there were changes in surface charges from before incubation of -23.7 mV to -25.4, -24.0, and -24.2 mV at accelerated conditions of 4, 25, and 45 °C, respectively.

The changes in particle size as seen in sample 2 from before incubation of 151.6 nm to 159.8, 158.7, and 156.0 nm at accelerated temperatures, namely 4, 25, and 45 °C, respectively. The changes in the surface charges before incubation of -25.1 mV to -24.1, -23.6, and -7.97 mV at accelerated conditions of 4, 25, and 45 °C, respectively.

The changes in particle size as seen in sample 3 from before incubation of 133.9 nm to 138.0, 137.9, and 138.1 nm at accelerated temperatures, namely 4, 25, and 45 °C, respectively. The changes in the surface charges before incubation of -23.5 mV to -22.9, -23.4, and -6.54 mV at accelerated conditions of 4, 25, and 45 °C, respectively.

The changes in particle size as seen in sample 4 from before incubation of 127 nm to 126.1, 125.6, and 121.9 nm at accelerated temperatures, namely 4, 25, and 45 °C, respectively. The changes in surface charges before incubation of -29.1 mV to -27.2, -27.4, and -11.6 mV at accelerated conditions of 4, 25, and 45 °C, respectively.

Through the analysis of sizes and surface charges, together with the physical characteristics of accelerated conditions at various temperatures, namely at 4, 25, and 45 °C for the periods of 1 and 2 months, the stability of particles at 2 months are equal to 1 year through Arrhenius's equation, which was used for controlling the stability of tilmicosin nanoparticles before use. The test results showed the size of particles and surface charge did not show any significant changes and did not show any precipitation or separation of layers of tilmicosin nanoparticles. It can be concluded that tilmicosin showed good stability according to the prepared samples and can be used for production or further development.

Toxicity testing of tilmicosin nanoparticies and tilmicosin phosphate standard medication

The toxicity of tilmicosin nanoparticies that derived from the synthesis according to this invention can be carried out by creating a set of simulated cells of animal's digestive system and by comparing between tilmicosin phosphate medication and 4 tilmicosin nanoparticle samples with concentrations in the range of 25-200 ppm for 48 hours after simulated cells of animal's digestive system were incubated with 4 tilmicosin nanoparticies and tilmicosin phosphate medication. CellTiter-Glo® Luminescent Cell Viability Assay was used to measure survival rate of cells wherein cells shall be stained with LIVE/DEAD stain kit, Invitrogen, Ltd. as illustrated in Figure 7 which displays number of simulated cells in digestive system after incubated with tilmicosin nanoparticies and tilmicosin phosphates at 200 ppm. The result of the analysis shall be illustrated with fluorescent light by which living cells will be illustrated in green and dead cells in red according to Figure 8 wherein the survival percentage of cells of the digestive system after incubating with 4 tilmicosin nanoparticle sampels and tilmicosin phosphate medication at various concentrations. It is found that the concentrations used for testing of 4 tilmicosin nanoparticies and tilmicosin phosphate medication were not toxic to living organisms.

Comparison of absorption capacity of tilmicosin nanoparticies and tilmicosin phosphate standard medication

The absorption capacity of synthesized tilmicosin nanoparticies according to this invention can be carried out by creating set of simulated cells of animal's digestive system and by comparing tilmicosin phosphate standard medication and 4 samples of tilmicosin nanoparticies wherein the permeability testing of Caco-2 monolayer— which is a simulated absorption testing of the digestive tract tissue using Caco-2 incubated for 21 -23 days by testing for transfer from apical (AP) side to basolateral (BL) side of monolayer in the opposite side (BL-AP). Figure 9 illustrates simulated cells in the digestive system for absorption capacity testing of tilmicosin nanoparticies and tilmicosin phosphate standard medication wherein the sample solutions were collected from receiver at various times during 30-120 minute period and the amount of tilmicosin was analysed in the 4 samples with MaxSignal Tilmicosin ELISA Test Kit, Bioo Scientific. It was found that Caco-2 monolayer permeability of 4 tilmicosin nanoparticle samples occurred faster than tilmicosin phosphate standard medication as shown

in Figure 10 which displays the percentage permeability of 4 tilmicosin nanoparticles and tilmicosin phosphate standard medication through digestive system cell simulation at various timing.

Products that are derived from the tilmicosin nanoparticle preparation according to this invention will result in tilmicosin nanoparticles with less than 200 nm or within 100-160 nm range and with surface charges of less than -20 mV with stability. The particle size and surface charge did not show any significant changes. The products were not toxic to living organisms, and the absorption capacity was high and better than tilmicosin phosphate standard medication.

The finished tilmicosin nanoparticles derived from the preparation method according to this invention can be further developed or incorporated with other methods to derive any appropriate forms— such as in liquid, solid, or powdered forms or any other forms of choices with no limitations.

Although this invention has been disclosed in the context of certain embodiment and examples, it will be understood by those skilled in the art that the present invention extends beyond the specifically disclosed embodiment to other alternative embodiments and/or uses of the invention and obvious modifications and equivalents thereof.

Best mode of the invention

In accordance with the detailed description of the invention.