Description:

FORM 2
THE PATENTS ACT, 1970
(39 OF 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10; rule 13)

1. Title of the invention – A PROCESS OF PREPARING STREPTOMYCIN COATED NANOMAGNETIC OXIDES
2. Applicant(s)
NAME: RK University
NATIONALITY: INDIAN
ADDRESS: RK University, School of Science, Bhavnagar Highway, Tramba, Rajkot-360020, Gujrat, India.

3. PREAMBLE TO THE DESCRIPTION

The following specification particularly describes the invention and the manner in which it is to be performed.

A PROCESS OF PREPARING STREPTOMYCIN COATED NANOMAGNETIC OXIDES
FIELD OF THE INVENTION
The present invention is relates to a process of preparing streptomycin coated nanomagnetic oxides. The present invention particularly relates to process for preparation of streptomycin coated nanomagnetic oxides which exhibits significant antibacterial effect for reducing or preventing growth of microorganisms.

BACKGROUND OF THE INVENTION
Microorganism resistance to antibiotic treatments has increased in recent decades and becoming a concern due to its harmful effects on human health. It happens when bacteria and fungi develop the ability to defeat the drugs which are designed to kill them. So, the germs are not killed and continue to grow. As a result, increasing occurrences of germs with higher resistance to antibiotics have been detected in humans and other animal species.

Nanotechnology has received wide attention globally due to the attractive modified surface properties of the nanoparticles. By using nanoparticles, a drug can be accurately delivered to the targeted region in the body. Metal oxide nanoparticles and particularly iron (Fe) based ferrites are having unique optical, electronic, magnetic and biomedical applications. Metal oxide nanoparticles are chemically stable and they are used in a variety of different applications such as adsorption, photocatalytic activities, antibacterial and antifungal activities.

Spinel nanomagnetic ferrites are fascinating nano materials that have ferromagnetic properties. The surface modification or functionalization of these ferrites creates a great impact on the biomedical field. There are several applications of drug-coated ferrite particles i.e. hyperthermia, brain imaging, targeted drug delivery and anti-microbial agent. These nanoparticles have several
properties like biocompatibility, biodegradability, possess high-temperature transition, and have high chemical stability.

The synthesis method plays a vital role in determining the properties of ferrite. There are several methods to synthesize ferrite nanoparticles like green synthesis, chemical co-precipitation, electrospinning, ultrasonic wave assisted ball milling, reverse micelle, hydrothermal and sol–gel auto combustion method. The sol-gel auto combustion method offers potential advantageous properties as compared to other methods such as good chemical homogeneity, fine particle size and narrow particle size distribution, high product purity and crystallinity and less complex process etc. Spinel nanomagnetic oxides are fascinating material for the guided drug delivery system.

There is a need to find new alternative techniques for the control of bacterial and fungal propagations in uncontrolled environments, several studies have been available using concepts regarding the interaction of nanostructured materials and microorganisms and studying the possible effects of this contact. There are various studies available on drug coated magnetic materials. Among the available drugs, streptomycin sulfate is a well-known for coating with good bacteriostatic effect.

Therefore the inventors of the present invention have developed a process for preparing nanomagnetic oxides by sol-gel combustion method and coated with streptomycin sulphate to check the effect against various bacteria. The prepared streptomycin coated nanomagnetic oxides exhibits notable antibacterial effect. So, far it has been exploited to achieve the following objective.

OBJECTIVE OF THE INVENTION
The main object of the present invention is to provide a process of preparing streptomycin coated nanomagnetic oxides.

Another object of the present invention is to provide a process of preparing streptomycin coated nanomagnetic oxides which is efficient process for synthesis and easier process.

Another objective of the present invention is to provide a process of preparing streptomycin coated nanomagnetic oxides which is having uniform particle size within the range of 10-300 nm.

Yet another object of the present invention is to provide a process of preparing streptomycin coated nanomagnetic oxides which is less complex and cost effective.

One other object of the present invention is to provide a process of preparing streptomycin coated nanomagnetic oxides which is having high potential for the targeted drug delivery.

SUMMARY OF THE INVENTION
The main aspect of the present invention is to provide a process of preparing streptomycin coated nanomagnetic oxides.

Another aspect of the present invention is to provide a process of preparing streptomycin coated nanomagnetic oxides comprises the steps of,
a) taking metal nitrates and dissolving in distilled water;
b) adding citric acid in step (a) metal nitrate solution;
c) stirring step (b) solution on a magnetic stirrer for 30 min;
d) adding ammonia solution dropwise in step (c) mixture till the pH found 9;
e) stirring step (d) mixture at 80°C constant temperature on a magnetic stirrer;
f) collecting metal ferrite nanomagnetic oxides from step (e) solution and sintering at 400°C for 3 h in a muffle furnace to remove impurities;
g) mixing metal ferrite nanomagnetic oxides from step (f) in distilled water;
h) dissolving streptomycin separately in distilled water;
i) mixing step (g) solution into step (h) solution and centrifuging for 30 min to get final streptomycin coated nanomagnetic functional oxides.

BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: Schematic diagram of the synthesis method of nanomagnetic oxides
Figure 2: Schematic diagram of streptomycin sulfate coating on Mg0.5Mn0.5 Fe2O4 nanoparticle
Figure 3: M–H hysteresis loop of (a) Mg0.5Mn0.5 Fe2O4, (b) MnFe2O4 nanoparticles
Figure 4: Thermogravimetric analysis of M2, M4, M6 and M8 specimens
Figure 5: TEM images of M1, M2 specimens and histogram using the ImageJ software of M1, M2 specimens
Figure 6: TEM images of M3, M4 specimens and histogram using the ImageJ software of M3, M4 specimens
Figure 7: TEM images of M5, M6 specimens and histogram using the ImageJ software of M5, M6 specimens
Figure 8: TEM images of M7, M8 specimens and histogram using the ImageJ software of M7, M8 specimens
Figure 9: SAED patterns of M1, M2, M3, M4, M5, M6, M7, M8 specimens
Figure 10: Particle size from DLS measurement of M1, M2, M3, M4, M5, M6, M7, M8 specimens

DESCRIPTION OF THE INVENTION
The main embodiment of the present invention is to provide a process of preparing streptomycin coated nanomagnetic oxides.

The detailed description set forth below is intended as a description of exemplary embodiments and is not intended to represent the only forms in which the exemplary embodiments may be constructed and/or utilized. The description sets forth the functions and the sequence of steps for constructing and/or operating the exemplary embodiments. However, it is to be understood that the same or equivalent functions and sequences which may be accomplished by different exemplary methods are also intended to be encompassed within the spirit and scope of the invention.

As defined herein, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs.

Although any process and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.

As stated in the present invention herein, the singular forms “a,” “an” and “the” specifically also encompass the plural forms of the terms to which they refer, unless the content clearly dictates otherwise. The term “about” is used herein to means approximately, in the region of, roughly, or around.

As stated herein, that it follows in a transitional phrase or in the body of a claim, the terms “comprise(s)” and “comprising” are to be interpreted as having an open ended meaning. That is, the terms are to be interpreted synonymously with the phrases “having at least” or “including at least”. When used in the context of a process, the term “comprising” means that the process includes at least the recited steps, but may include additional steps. When used in the context of a composition, the term “comprising” means that the composition includes at least the recited features or components, but may also include additional features or components.

As used herein “nanomagnetic oxides” are defined as magnetic iron oxide nanoparticles and having particles with diameter between 1 to 100 nm.

As used herein “metal ferrite nanomagnetic oxides” are defined as compounds composed of iron oxide nanoparticles combined chemically with one or more additional metallic elements.

As per main embodiment a process of preparing streptomycin coated nanomagnetic oxides comprises the steps of,
a) taking metal nitrates and dissolving in distilled water;
b) adding citric acid in step (a) metal nitrate solution;
c) stirring step (b) solution on a magnetic stirrer for 30 min;
d) adding ammonia solution dropwise in step (c) mixture till the pH found 9;
e) stirring step (d) mixture at 80°C constant temperature on a magnetic stirrer;
f) collecting metal ferrite nanomagnetic oxides from step (e) solution and sintering at 400°C for 3 h in a muffle furnace to remove impurities;
g) mixing metal ferrite nanomagnetic oxides from step (f) in distilled water;
h) dissolving streptomycin separately in distilled water;
i) mixing step (g) solution into step (h) solution and centrifuging for 30 min to get final streptomycin coated nanomagnetic functional oxides.

As per one embodiment of the present invention, metal ferrite nanomagnetic oxides such as Mg0.5Mn0.5Fe2O4, MnFe2O4, and MgFe2O4 were synthesized via sol–gel auto combustion method.

As per one embodiment, magnesium manganese–mixed ferrite nano oxides of Mg0.5Mn0.5Fe2O4 (M1) is synthesized by using metal nitrates of Mg (II), Mn (II), Fe (III) and it was coated by streptomycin sulfate (SS) to get final SS coated Mg0.5Mn0.5Fe2O4 (M2).

As per one embodiment, magnesium ferrite nano oxides of MgFe2O4 (M3) is synthesized by using metal nitrates of Mg (II), Fe (III) and it was coated by streptomycin sulfate (SS) to get final SS coated MgFe2O4 (M4).
As per one embodiment, manganese ferrite nano oxides of MnFe2O4 (M5) is synthesized by using metal nitrates of Mn (II), Fe (III) it was coated by streptomycin sulfate (SS) to get final SS coated MnFe2O4 (M6).

As per one embodiment, Fe3O4 (M7) is procured from Sigma-Aldrich Ltd with purity =99% and it was coated by streptomycin sulfate (SS) to get final SS coated Fe3O4 (M8).

As per one embodiment, the present invention includes streptomycin sulfate (SS) coated nanomagnetic oxides which are having very high potential for the targeted delivery of therapeutic agents. The large surface area of the modified nano particles increase the fast absorption of the drug molecules due to high vascularization and the elimination of the first-pass effect.

As per one embodiment, the streptomycin coated nanomagnetic oxides are having particle size in the range from 1-500 nm, more preferably 5-400 nm and most preferably 10-300 nm.

As per one embodiment, after the coating of streptomycin sulfate (SS) on metal ferrite nano oxides, the M1 to M8 samples and pure streptomycin sulfate (SS) were tested against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus on Mueller Hinton agar plates using disc diffusion assay.

The invention is further illustrated by the following examples which are provided to be exemplary of the invention and do not limit the scope of the invention. While the present invention has been described in terms of its specific embodiments, certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the present invention.

EXAMPLE 1: PROCESS OF PREPARING STREPTOMYCIN COATED NANOMAGNETIC OXIDES
A process of preparing streptomycin coated nanomagnetic oxides comprises the steps of,
a) taking metal nitrates and dissolving in distilled water;
b) adding citric acid in step (a) metal nitrate solution;
c) stirring step (b) solution on a magnetic stirrer for 30 min;
d) adding ammonia solution dropwise in step (c) mixture till the pH found 9;
e) stirring step (d) mixture at 80°C constant temperature on a magnetic stirrer;
f) collecting metal ferrite nanomagnetic oxides from step (e) solution and sintering at 400°C for 3 h in a muffle furnace to remove impurities;
g) mixing metal ferrite nanomagnetic oxides from step (f) in distilled water;
h) dissolving streptomycin separately in distilled water;
i) mixing step (g) solution into step (h) solution and centrifuging for 30 min to get final streptomycin coated nanomagnetic functional oxides.

EXAMPLE 2: MAGNETIZATION STUDY
Figure 3 (a) and (b) shows the M-H hysteresis loops of the M1 and M3 nanoparticle respectively, measured at 300 K with a maximum applied field of 10 kOe. Various magnetic parameters of M1 and M3 nanoparticles like coercive field (Hc), saturation magnetization (Ms), remanent magnetization (Mr), the squareness of hysteresis loop (Q), and experimental magnetic moment per molecule (µ) were calculated from hysteresis loop as shown in Table 1. The experimental magnetic moment per molecule in Bohr magnetons was calculated from M-H hysteresis curves data using the relation µ= MwMs/5585, where Mw is the molecular weight in grams. The coercivity results indicated the super paramagnetic nature of present nanoparticles.

The results are shown below:

Sample code Hc (Oe) Ms
(emu/gm) Mr
(emu/gm) Q µ
(experimental)
M1 96.43 53.31 7.47 0.14 1.82
M3 258.44 41.32 21.8 0.53 1.71

EXAMPLE 3: THERMOGRAVIMETRIC ANALYSIS (TGA)
Thermogravimetric analysis (TGA) is known to provide quantitative information of chemically synthesized samples. Here, for the present work, four drug-coated nanomagnetic samples M2, M4, M6, and M8 were considered for confirming the presence of SS on them. As per Fig. 4, the weight loss of the SS-coated magnetic particles was observed in the range of 28–700 °C. Below 250 °C, weight loss was due to the removal of absorbed water and surface hydroxyl groups in the SS-coated samples, while SS begun to decompose at about 300 °C and the final decomposition temperature was at 700 °C. The total weight losses due to SS decomposition were 15.77%, 5.39%, 5.97%, and 3.6% for the M2, M4, M6, and M8 respectively. The highest weight loss was observed for the SS-coated Mg0.5Mn0.5Fe2O4 (M2) nanomagnetic specimen. These results confirm the loading of SS on the prepared nanomagnetic ferrites.

EXAMPLE 4: TRANSMISSION ELECTRON MICROSCOPY (TEM) ANALYSIS
TEM images of M1 and M2 are shown in Fig. 5 (a) and 5 (b) respectively. The coating of streptomycin sulfate on nano ferrites has been observed in Fig. 5 (b). The histograms are plotted from all the TEM images and shown as Figs. 5(c), 5 (d) for M1 and M2 specimen, respectively. Similarly, TEM images of M3, M4, M5, M6, M7, and M8 specimens are shown in Figs. 6(a), 6(b), 7(a), 7(b), 8(a), and 8(b) respectively. The coating of streptomycin sulfate on nano ferrites has been observed in the figures. The average particle sizes were calculated using the ImageJ software and listed in Table 1. The histograms are plotted from all the TEM images and shown as Figs. 6(c), 6 (d), 7(c), 7(d), 8(c), and 8(d) for M3, M4, M5, M6, M7, and M8 specimens, respectively. The average particle size for M7 and M8 is seen 102 nm and 224 nm from the TEM images. The agglomeration of nanoparticles and film structure is observed in Fig. 8(a) and 8(b), which can be reflected and affected antimicrobial activity of the present study.

Name of the nanoparticles Sample code Particle size (nm)-
TEM measurement
Mg0.5Mn0.5Fe2O4 M1 5
SS coated Mg0.5Mn0.5Fe2O4 M2 17
MnFe2O4 M3 35
SS coated MnFe2O4 M4 58
MgFe2O4 M5 13
SS coated MgFe2O4 M6 23
Fe3O4 M7 102
SS coated Fe3O4 M8 224
Streptomycin sulfate SS --
Table: 1 Coding and particle sizes (TEM measured) of coated and uncoated specimens

The selected area electron diffraction (SAED) pattern provides structural information of nanoparticles. The SAED pattern of all the coated and uncoated specimens is presented in Fig. 9 (a)–(g). The SAED pattern is showing the cubic spinel structure of the M1, M3, M5, and M7 nanoparticles belonging to the Fd3m space group as per Fig. 9 (a), (c), (e), and (g). Figure 9 (b), (d), (f), and (h) displays distortion in SEAD patterns which confirmed the coating of SS on the nanoparticles.

EXAMPLE 5: DYNAMIC LIGHT SCATTERING MEASUREMENT
Dynamic light scattering (DLS) measurement is a vital tool to give hydrodynamic diameter of nanoparticles present in the solution. In this, the laser beam is incident on the specimen and the variation of the scattered light is detected at a known scattering angle ? by a fast photon detector to determine the mean particle size in the nm regime. The hydrodynamic diameter of the nanoparticle is an important parameter for bacteriostatic application. Figure 10 (a)–(h) shows the hydrodynamic size of all the nanoparticles (M1 to M8) which are identical to the particle size calculated using the ImageJ software from the TEM images. The average particle sizes were calculated listed in Table 2.

Name of the nanoparticles Sample code Particle size (nm)-
DLS measurement
Mg0.5Mn0.5Fe2O4 M1 7
SS coated Mg0.5Mn0.5Fe2O4 M2 21
MnFe2O4 M3 40
SS coated MnFe2O4 M4 50
MgFe2O4 M5 17
SS coated MgFe2O4 M6 25
Fe3O4 M7 108
SS coated Fe3O4 M8 234
Streptomycin sulfate SS --
Table: 2 Coding and particle sizes (DLS measured) of coated and uncoated specimens

EXAMPLE 6: ANTIMICROBIAL ACTIVITY
The antibacterial activity of streptomycin coated and uncoated nanoparticles and streptomycin sulfate (SS) was investigated against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus. As-prepared nanoparticles (M1, M3, M5, and M7) showed no antimicrobial activity against the tested pathogens while activity was significantly increased in SS-coated nanoparticles as listed in Table 3. SS-coated nanoparticles (M2, M4, M6, and M8) showed the highest antimicrobial activity against E. coli, while only SS showed comparatively lower activity against all tested pathogens. To study the antimicrobial activity for SS drugs, percentage loading was considered evaluated from total weight losses in the TGA measurements which are tabulated as SS (Mg, Mn), SS (Mn), SS (Mg), SS (Fe) respectively in Table 3. MIC determination revealed that among all other bacteria, P. aeruginosa was the most sensitive to all antibiotic conjugated nanoparticles as indicated by very low MIC equivalent to 9, 9, 12, and 7 µg/mL for M2, M4, M6, and M8, respectively as seen in Table 4. Unconjugated nanoparticles failed to show any inhibition and hence represented as ND (not determined). MIC value for free streptomycin sulfate was in a range from 1 to 4 µg/mL for all test pathogens. Higher MIC values for the conjugated samples may be attributed to slow and sustained release of drugs which is significantly (*P< 0.05) different to free drug. This strategy of sustained release of drugs can help for controlling microbial resistance. Table 5 shows the minimum bactericidal concentration (MBC) for the nanoconjugates which revealed that E. coli and S. aureus are killed at identical concentration (64 µg/ mL) of nanoconjugate M2. Likewise, M6 also kills E. coli at the same concentration. Other streptomycin functionalized nanoconjugates exhibited MBC value in a significantly variable range (*P<0.05).

Bacteria Zone diameter (mm)
M1 M2
* SS
(Mg
Mn)* M3 M4
* SS
(Mn)
* M5 M6* SS
(Mg)
* M7 M8
* SS
(Fe)
*
Escherichia coli NIL 21 18 NIL 20 19 NIL 24 18 NIL 22 19
Pseudomonas aeruginosa NIL 9 7 NIL 9 6 NIL 12 6 NIL 7 6
Bacillus subtilis NIL 16 13 NIL 16 15 NIL 21 14 NIL 15 14
Staphylococcus aureus NIL 16 14 NIL 13 11 NIL 20 14 NIL 14 13
Table: 3 Antimicrobial activity of streptomycin sulfate (SS) coated and uncoated nanomagnetic specimens

Note: All the experiments were performed in triplicate, and standard deviations were negligible. *P<0.05; the mean difference in the zone diameter is significant among the bacteria at the 0.05 level by two factor ANOVA. Nil = no zone of inhibition.

Bacteria Minimum inhibitory concentration (MIC)* in µg/mL
M1 M2 M3 M4 M5 M6 M7 M8 SS
Escherichia coli ND 64 ND 32 ND 32 ND 32 1
Pseudomonas aeruginosa ND 256 ND 128 ND 256 ND 256 2
Bacillus subtilis ND 128 ND 128 ND 128 ND 64 2
Staphylococcus aureus ND 64 ND 128 ND 128 ND 128 4
Table: 4 Minimum inhibitory concentration (MIC) of streptomycin sulfate (SS) coated and uncoated nanomagnetic specimens against bacterial pathogens.

Bacteria Minimum bactericidal concentration (MBC)* in µg/mL
M1 M2 M3 M4 M5 M6 M7 M8 SS
Escherichia coli ND 128 ND 128 ND 64 ND 128 4
Pseudomonas aeruginosa ND >512 ND 512 ND >512 ND 512 64
Bacillus subtilis ND >512 ND >512 ND >512 ND >512 32
Staphylococcus aureus ND 256 ND 256 ND 256 ND 256 8
Table: 5 Minimum bactericidal concentration (MBC) of streptomycin sulfate (SS) coated and uncoated nanomagnetic specimens against bacterial pathogens.

Conclusion:
Nanomagnetic spinel ferrites were successfully synthesized by an auto combustion method. The TGA study confirmed the coating of SS on the nanoparticles. TEM images also indicated the coating of drug on nanomagnetic particles. The DLS studies were used to quantify the hydrodynamic diameter of the nanoparticles. These results were in agreement with the particle size measurements in TEM images. The antimicrobial effect on drug-coated and uncoated nanomagnetic specimens is studied on Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus on Mueller Hinton agar plates using disc diffusion assay. There was no effect of uncoated nanoparticles while less effect of streptomycin compared to drug-coated nanomagnetic specimens. This can prove to be useful in controlled drug delivery system. , Claims:CLAIMS:
We claim;
1. A process of preparing streptomycin coated nanomagnetic oxides comprises the steps of,
a) taking metal nitrates and dissolving in distilled water;
b) adding citric acid in step (a) metal nitrate solution;
c) stirring step (b) solution on a magnetic stirrer for 30 min;
d) adding ammonia solution dropwise in step (c) mixture till the pH found 9;
e) stirring step (d) mixture at 80°C constant temperature on a magnetic stirrer;
f) collecting metal ferrite nanomagnetic oxides from step (e) solution and sintering at 400°C for 3 h in a muffle furnace to remove impurities;
g) mixing metal ferrite nanomagnetic oxides from step (f) in distilled water;
h) dissolving streptomycin separately in distilled water;
i) mixing step (g) solution into step (h) solution and centrifuging for 30 min to get final streptomycin coated nanomagnetic functional oxides.
2. The process of preparing streptomycin coated nanomagnetic oxides as claimed in claim 1, wherein said metal nitrates are selected from Mg nitrate, Mn nitrate and Fe nitrate or combinations thereof.
3. The process of preparing streptomycin coated nanomagnetic oxides as claimed in claim 1, wherein the said reaction of step (f) generate magnesium manganese–mixed ferrite nano oxides by using nitrates of Mg (II), Mn (II) and Fe (III).
4. The process of preparing streptomycin coated nanomagnetic oxides as claimed in claim 1, wherein the said reaction of step (f) generate magnesium ferrite nano oxides by using nitrates of Mg (II) and Fe (III).
5. The process of preparing streptomycin coated nanomagnetic oxides as claimed in claim 1, wherein the said reaction of step (f) generate manganese ferrite nano oxides by using nitrates of Mn (II) and Fe (III).
6. The process of preparing streptomycin coated nanomagnetic functional oxides as claimed in claim 1, wherein said streptomycin coated nanomagnetic oxides are having particle size in the range from 10-300 nm.

Dated this 22nd Jul, 2022