WO2022146242A1

WO2022146242A1 - Composition of a nanoparticle charged bioadsorbent blood culture and its production method

Info

Publication number: WO2022146242A1
Application number: PCT/TR2020/051429
Authority: WO
Inventors: Kürşat Seyfi DEMİREZEN
Original assignee: Nanobi̇otech Arge İnovasyon Sağlik Ürünleri̇ Sanayi̇ Ve Ti̇caret Li̇mi̇ted Şi̇rketi̇
Priority date: 2020-12-29
Filing date: 2020-12-29
Publication date: 2022-07-07

Abstract

The invention relates to a blood culture composition containing fruit and seeds of carob bean tree, resin and shell of olibanum, iron nanoparticle and cellulose, and the production method of the said composition, for use in determining the status of antibiotic administration in the treatment of sepsis in the health sector.

Description

DESCRIPTION

COMPOSITION OF A NANOPARTICLE CHARGED BIOADSORBENT BLOOD CULTURE AND ITS PRODUCTION METHOD

Technical Field

The invention relates to a composition of a nanoparticle loaded bio adsorbent blood culture and the production method of the said composition for use in the health sector in defining the microbiological etiology in suspected infection cases.

Prior Art

Sepsis is a fatal infectious disease that involves many systems, especially causing hemodynamic changes, leading to shock, organ dysfunction, and organ failure.

About three out of 1000 people in the world experience sepsis, and 1.8 million people die due to sepsis every year. In addition to identifying microbial causes in suspected infection cases, blood cultures also play a role in guiding treatment. If it is not intervened early, the chance of the patient to survive decreases.

In the current applications, the survival rate of the patients who have bacteria in their blood and treated with antibiotics within the first 2 hours is 80 %, while in patients who started antibiotics after 12 hours, this rate drops by almost half. Following the technological developments, the pathogen detection rate in blood cultures has been increased with new methods. Examples of these are the development of new mediums, the addition of growth factors to the mediums, and the neutralization of growth inhibitors I metabolic products I antibiotic residues. However, the average positivity period of automated blood culture measurement systems and bottles is 5 - 7 days, and for other systems, the average is 15 hours. The value of conventional blood cultures is limited in slow or difficult breeds or in species that cannot be cultured and its sensitivity decreases in those who take antibiotic treatment and when the burden of microorganisms is low.

The false - positive blood cultures cause results to be interpreted with error, resulting in the incorrect use of antibiotics, additional laboratory tests, prolonged hospital stay and increased examination and treatment costs. Sepsis patients are given high doses of antibiotics. In the examination method, the blood taken from the patient is kept in a medium containing the nutrients of the pathogens that cause the infection, and pathogen growth is observed. In this context, the blood taken from the patient due to antibiotic treatment can suppress these pathogens for a period of time, and the amount of growth appears to be lower than it should be, or there are certain deviations. The BACTEC blood culture system (BD Diagnostics) produced for this purpose consists of an antibiotic binding resin on small glass beads, while the BacT / Alert blood culture system (BioMerieux Inc.) uses activated charcoal powder. In summary, in state of the art, it is aimed to retain the antibiotic with the material chosen as adsorbent.

In this context, new developments were needed to eliminate the effect of antibiotics on the pathogen by using nanoparticles that inhibit antibiotics and, accordingly, ensure accurate examination results.

In the patent and literature survey conducted for the known techniques, a patent application document numbered US4543328A is found. The said document describes a process and device for the detection of pathogens, such as bacteria, fungi, and viruses, in blood in the presence of an anticoagulant agent. In the invention described in the document, it is said that the polymer is selected from the group of polyacrylate, polymethacrylate, polyhydroxy ethyl methacrylate, an adsorber resin, synthetic crosslinked polystyrene, cellulose acetate, collodion, and nylon.

As a result, since many problems and drawbacks are experienced in the said technical area, as mentioned above, the existing applications are inadequate in solving these problems and drawbacks. This necessitates development and innovation in the technical field.

Brief Description of the Invention

The present invention relates to a composition of a nanoparticle loaded bio adsorbent blood culture and its production method that meets the above - mentioned requirements, eliminates all disadvantages, and brings some additional advantages.

The main purpose of the invention is to develop a new blood culture composition, especially for use in the examinations of sepsis patients. By means of the blood culture composition of the invention, the quality of the examination is improved, and the duration of the examination is shortened. Thus, early precautions can be taken by obtaining rapid results in the treatment of fatal sepsis patients.

Another purpose of the invention is to obtain a blood culture composition that gives faster and more accurate results than resin - containing applications in the current technique. The blood culture composition of the invention contains bioadsorbent material charged with iron nanoparticles obtained through herbal synthesis. For this purpose, a mixture comprising of the fruit and seeds of carob bean tree (Ceratonia siliqua), shell and resin of the olibanum (Boswellia carterii), as well as cellulose and flower - shaped iron hybrid nanostructure are used. In the process of obtaining the flower - shaped iron hybrid nano - structure, hydrosol obtained from the selected plant combinations as the reducing material is sufficient. It was determined that the composition containing the flower - shaped iron hybrid nanoparticle obtained using plant hydrosol not only retains the antibiotic in the environment but breaks it down, disrupting its structure.

Another object of the invention is to demonstrate that there is no need to use chemicals as reducers and stabilizers as in the current technique since the use of plant hydrosols is sufficient to obtain nanoparticles.

In the composition of the invention, all reducing, stabilizing agents are composed of plant - derived raw materials. Thus, it is possible to prevent the negative effects of nanoparticle formulations with the chemical synthesis on human health in the current technical field. Besides, due to the herbal synthesis nano iron solution, the antibiotic adhesion surface area, and the rate of degradation of the antibiotic are increased. The resin used in state of the art has a narrower surface area than nanoparticles, so it reaches saturation in a shorter time, and its performance decreases. The antibiotics, which cannot be retained by resin, give misleading results in diagnosis. Since the antibiotic in the environment is not only retained but also degraded employing the invention, the aforementioned problems can be avoided. In order to provide this solution, the invention comprises flower - shaped iron hybrid nanostructure and cellulose obtained using the fruit and seeds of carob bean tree and shell and resin of the Olibanum. The cellulose charged with flower - shaped iron hybrid nanoparticles synthesized by herbal methods in the scope of the invention can be easily applied to any environment in which human contact is made and is environmentally friendly. The innovative aspects of the product of the invention are listed below;

• since the antibiotic inhibition system is nanoparticle - based, the amount of antibiotic adsorbed with an extended surface area increases,

• thanks to the nanoparticle system, the antibiotic is adsorbed, and together with the deterioration of its structure, its effect is completely eliminated,

• by increasing the efficiency compared to the traditional methods used in blood culture bottles, reducing the costs of blood culture bottles by 40 %,

• reducing the incubation time,

• providing chance of rapid intervention to sepsis table,

• having an environmental production process with herbal synthesis,

• prevention of environmental damage by making the developed nanoparticles easily decomposable and recyclable.

The structural and characteristic features of the invention and all advantages thereof will be more clearly understood through the detailed description and figures. Therefore, the evaluation should be made considering the said detailed description and figures.

Brief Description of the Figures

Figure 1 shows The SEM image of plant synthesis flower - shaped iron hybrid nanostructure.

Figure 2 shows The TEM image of iron nanoparticles that form the center of the flower - shaped hybrid nanostructure.

Figure 3 shows the results of EDX analysis.

Figure 4 shows the results of FT - IR analysis.

Figure 5 shows the results of BET surface area analysis.

Detailed Description of the Invention In this detailed description, the preferred applications of the nanoparticle - loaded bio adsorbent blood culture composition, and production method of the invention are explained only for a better understanding of the subject and without any limiting effect.

The invention relates to the composition of a nanoparticle - charged bioadsorbent blood culture and the production method of the said composition for use in determining the state of antibiotic administration. The said blood culture composition comprises the fruit and seeds of carob bean tree and shell and resin of the Olibanum, iron, and cellulose.

Olibanum (Boswellia carterii) resin contains ester and acidic components and has high antimicrobial properties. In the composition of the invention, it is combined with carob bean tree hydrosols in the appropriate proportion to the composition, providing a flower - shaped organic - inorganic hybrid structure.

The carob bean tree (Ceratonia siliqua L.) plant contains a high percentage of polyphenols in its fruit. The polyphenol group consists of carbohydrates (48 - 56 %), condensate tannin (16 - 20 %). Polyphenols can reduce metals by forming complexes with metal ions. In the composition of the invention, it is combined with Olibanum hydrosols in the appropriate proportion to the composition, providing a flower - shaped organic - inorganic hybrid structure.

The production method of the composition of the invention, in its most basic form, comprises the following steps;

(i) Preparation of PBS (phosphate - buffered solution),

(ii) Preparation of iron solution with distilled water,

(iii) Production of plant hydrosol by adding Olibanum (Boswellia carterii), carob bean tree (Ceratonia siliqua) plants in the appropriate concentration of PBS (phosphate buffer solution),

(iv) Mixing the solution prepared in process step (iii) from the iron solution prepared in process step (ii),

(v) Obtaining cellulose from sunflower heart and stem pulp for adsorbent production, (vi) Combining substances prepared in the process steps (v) and (iv).

The structure of the nanoparticle loaded bio adsorbent blood culture composition of the invention comprises, by weight

• 2.5 - 4.2 % carob bean tree and Olibanum mixture hydrosol,

• 0.041 - 0.053 % flower - shaped iron hybrid nanoparticle,

• 95 - 98 % cellulose (adsorbent substance).

In one embodiment of the invention, blood culture composition preferably comprises, by weight

• 3.45 % hydrosol of carob bean tree and olibanum mixture,

• 0.047 % iron,

• 96.5 % cellulose (adsorbent substance).

In the production method of the composition of the nanoparticle loaded bio adsorbent blood culture of the invention, six different lidded glass bottles are used.

In glass bottle number 1 ; PBS (phosphate buffer solution), 35 - 45 gr NaCI is added to 100 ml distilled water and vortexed for 1 - 2 minutes. Then 0.5 - 1.5 g KCI is added to it and vortexed for 1 - 2 minutes. 6.8 - 7.5 g of Na2HPC .2H2O is slowly added to the resulting mixture and mixed for 2 - 3 minutes. Finally, 0.9 - 1 .5 gr of KH2PO4 is added and vortexed for 2 - 3 minutes, and after the volume is completed to 1000 ml with distilled water, it is mixed in a magnetic stirrer for 5 - 10 minutes, the pH value is adjusted to 6.7 - 8.3.

200 - 400 ml of PBS solution with pH = 6.7 - 8.3 is taken to bottle number 2, and 10 - 15 g of Olibanum plant and 20 - 30 g of carob bean tree plant are added, then mixed at 40 - 50 ‘C at 80 - 100 rpm while infusing for 30 - 40 minutes. At the end of these processes, the mixture is filtered through the filter paper and stored at + 4 ^<C.

In bottle number 3, 5 - 6 g FeCh.6H2O is added and dissolved in 100 ml distilled water.

200 - 400 g of sunflower heart and stem pulp is taken to bottle number 4, 800 - 1000 ml distilled water is added and heated to 80 - 90 °C at 50 - 70 rpm for 30 - 45 minutes. It is then crushed with a laboratory - type blender for 1 - 3 minutes, kept in the ultra sonication device for 10 - 15 minutes, and stored at + 4 ^<C.

After taking 150 - 200 ml of the mixture from bottle number 2 to bottle number 5, heated to 50 - 60 ^<C, then 0.041 - 0.053 % of the s olution in bottle number 3 in an ice bath is added slowly to the mixture, and vortexes for 3-5 minutes. At the end of the said process, it is kept at 0 - 5 <0 for 8 - 10 hours; it is centrifuged for 5 - 10 minutes at 4000 - 10000 rpm. In the next process step, this combination in bottle number 5 is taken into the oven, and its water is evaporated at 80 - 90 ^<C.

0.29 - 0.42 g of cellulose in bottle number 4 is added to bottle number 6, and 1 - 1.5 % citric acid is added and mixed for 20 - 25 minutes. Then, 0.9 - 1 .3 mg of flower - shaped iron hybrid nanomaterial obtained in process step 5 is added and mixed for 45 - 65 minutes at 300 - 350 rpm.

The resulting iron nanoparticle loaded cellulose - based adsorbent material is added to existing blood culture bottles in the appropriate proportion instead of resin or activated carbon.

In order to solve the above - mentioned technical problems and realize all the advantages to be understood from the detailed description below, the invention is a blood culture composition containing carob bean tree and olibanum plant hydrosols, the iron, and cellulose.

In order to solve the above - mentioned technical problems and realize all the advantages to be understood from the detailed description below, the invention is a blood culture composition production method, characterized by comprising the following process steps of i. Preparation of the PBS solution ii. Obtaining of hydrosols of the fruit and seeds of carob bean tree, shell and resin of the olibanum in the PBS, iii. Thawing of iron, iv. Combining iron thawed in step (iii) and plant hydrosols treated with PBS in step (ii), v. Extraction of cellulose from sunflower heart and stem pulp, vi. Evaporation of the water of the mixture obtained in the process step (iii), vii. Charging the powdered iron nanoparticle obtained in the process step (vi) into the cellulose adsorbent obtained in the process step (iv).

Claims

9

CLAIMS A composition of a nanoparticle - charged adsorbent blood culture, characterized by comprising;

• fruit and seeds of carob bean tree,

• shell and resin of the olibanum hydrosol,

• flower - shaped iron hybrid nanoparticle and

• cellulose. The composition of a nanoparticle - charged adsorbent blood culture according to Claim 1 , characterized by comprising;

• 2.5 - 4.2 % of carob bean tree and olibanum mixture hydrosol by weight,

• 0.041 - 0.053 % of flower - shaped iron hybrid nanoparticle by weight,

• 95 - 98 % of cellulose by weight. The composition of a nanoparticle - charged adsorbent blood culture according to Claim 1 , characterized by comprising;

• 3.45 % of carob bean tree - olibanum plant hydrosol by weight,

• 0.047 % of flower - shaped iron hybrid nanostructure by weight and

• 96.5 % of cellulose by weight. A production method of the composition of a nanoparticle - charged adsorbent blood culture according to Claim 1 , characterized by comprising the following process steps of i. Preparation of the PBS solution ii. Obtaining of hydrosol of the fruit and seeds of carob bean tree, shell and resin of the olibanum in the PBS, iii. Thawing of iron, iv. Extraction of cellulose from sunflower heart and stem pulp, v. Combining iron thawed in step (iii) and plant hydrosols treated with PBS in step (ii), vi. Evaporation of the water of the composition obtained in step (iv), vii. Charging the composition, which is dehydrated at step (vi), into the cellulose obtained at step (v).

5. The production method of the composition of nanoparticle - charged adsorbent blood culture according to Claim 4, characterized in that in the process step (ii), mixing process is performed at 40 - 50 <C, at 80 - 100 rpm for 30 - 40 minutes.

6. The production method of the composition of nanoparticle - charged adsorbent blood culture according to Claim 4, characterized in that in process in step

(iv) is performed at 80 - 90 <C, at 50 - 70 rpm, an d for 30 - 45 minutes.

7. The production method of the composition of nanoparticle - charged adsorbent blood culture according to Claim 4, characterized in that in the process step

(iv), the resulting material is crushed with a blender for 1 - 3 minutes, and the ultrasonication process is performed for 10 - 15 minutes.

8. The production method of the composition of nanoparticle - charged adsorbent blood culture according to Claim 4, characterized in that in process in step

(v) it is rested for 8 - 10 hours at 0 - 5 <C, and then centrifuged for 5 - 10 minutes at 4000 - 10000 rpm to obtain the iron hybrid nanostructure.

9. The production method of the composition of nanoparticle - charged adsorbent blood culture according to Claim 4, characterized in that in process in step

(vi) the dehydration is performed at a temperature of 80 - 90 <C.

10. The production method of the composition of nanoparticle - charged adsorbent blood culture according to Claim 4, characterized in that in process in step

(vii) it is mixed at 300 - 350 rpm for 45 - 65 minutes to obtain iron hybrid nanostructure - charged cellulose adsorbent .