[002] The present invention is in the technical field of Titanium dioxide ( TiO ₂ ) Nano-particles (NPs) impregnated Surgical Silk Suture. More particularly, the sutures are biocompatible with antimicrobial, photo-catalytic and self-cleaning properties.

[003] BACKGROUND OF THE INVENTION

[004] The surgical silk is braided and multifilament, which increases the capillarity allowing body fluids and the pathogens getting soaked up and these bacteria prefer to adhere to the silk protein fibroin and hides in the bride gaps, resulting in bacterial colonization, tissue reaction and surgical site infection (SSI), which accounts for 31% of hospital acquired infections as shown in following flowchart.

[005]

[006] At present, there are prior arts available that deals with surgical sutures. However, they have many drawbacks as listed below.

[007] Therefore, there is an urgent need to develop a suture that has less capillarity and not allowing body fluids and the pathogens getting soaked up.

[008] In addition, there is a need for suture not supporting bacterial colonisation, tissue reaction and surgical site infection (SSI).

[009] In summary, there is a need in the art to develop sutures that are biocompatible with antimicrobial, photo-catalytic and self-cleaning properties.

[010] SUMMARY OF THE INVENTION

[011] The primary objective of the present invention is to provide surgical silk sutures and more specifically the sutures coated with titanium dioxide ( TiO ₂ ) nano-particles (NPs), and characterized by non-capillarity, non-brooming, improved knot retention, biocompatible, antimicrobial, photo-catalytic and self-cleaning properties.

[012] In an embodiment, the surgical silk suture, wherein the TiO ₂ substrate is in form of TiO ₂ nano particles, having particle size in range of 40 nanometers to 60 nanometers.

[013] In further embodiment, the TiO ₂ NPs are comprising of Titanium and Oxygen with atomic weight of 24.54% and 41.60% respectively; wherein the hydrodynamic size of TiO ₂ NPs were ranging from 270nm to 400nm; wherein the Zeta potential value of TiO ₂ NPs were -24.7mV; wherein the said of TiO ₂ NPs were stable in the suspension form.

[014] In an embodiment, the support material is a thread with a monofilament or multifilament structure; wherein the suture is a braided, twisted, or covered suture; wherein the support material is a suturing thread joined to a needle; wherein the support material is a staple.

[015] In yet another embodiment, the plurality of plasmonic nanoparticles are evenly dispersed within the silk fibroin matrix; wherein the plurality of nanoparticles are distributed in a gradient within the silk fibroin matrix; wherein the plurality of nanoparticles are distributed in a pattern, said pattern comprises an optical pattern, a micropattem, or a nanopattem; wherein the at least one nanoparticle is selected from the group consisting of a nanosphere, a nanoshell, a nanorod, a nanocage, a nanocrystal, nanopowder, and any combinations thereof.

[016] In yet another embodiment, an automated nanoparticle coating machine, for dipper treatment of sutures to be coated, comprising; a magnetic stirrer; a solution holder; a panel for controlling rate of cycles and time; a chamber for air drying of coated suture and a chamber for sterilizing of coated sutures.

[017] In an embodiment, a method for preparing a surgical silk suture comprising the steps of: [018] adding precursors comprising, Titanium tetra 2-ethylhexyloxide or Titanium tetra oxysulfate or Titanium tetra chloride tetrahydrofiiran or Titanium tetra isopropoxide or Titanium tetra butoxide to solvents comprising, methanol or ethanol or isopropanol at alkaline pH; [019] stirring the solution of step (a) with a magnetic bead at rpm 800 to 1200, and at temperature of 40° to 65°;

[020] forming precipitates by adding distilled water;

[021] cooling of precipitates at the optimum pH & temperature;

[022] centrifuging the precipitates;

[023] forming TiO ₂ Nanoparticles pellets and powdering the said TiO ₂ Nanoparticles;

[024] calcinating TiO ₂ Nanoparticles in a furnace for one to four hours at 400° to 800° C;

[025] cooling down of furnace and collecting Nanoparticles;

[026] impregnating the suture with Nanoparticles; and

[027] drying the coated suture to remove the solvent and collecting the impregnated suture.

[028] According to exemplary aspect of the present invention, the silk is applied in form of a braided thread and structures comprising such substrates for use in various medical devices.

[029] According to a further aspect of the present invention, the essential elements of the disclosed invention are: No capillarity, Antibacterial property (bactericidal), Biocompatible [030] TiO ₂ Nano-particles and Surgical Silk Suture.

[031] In an embodiment, invention also provides kits that are useful for the practice of the methods of the invention.

[032] In summary, the present disclosure relates to surgical silk sutures and more specifically the sutures are biocompatible with antimicrobial, photo-catalytic and self-cleaning properties. Silk is a material widely used in surgery as a non -absorbable suture to sew wounds. Silk is applied in form of a braided thread and structures comprising such substrates for use in various medical devices. The invention relates to surgical silk suture composition that comprises titanium oxide nanoparticles coated sutures, and to a method for producing said surgical silk sutures.

[033] Several aspects of the invention are described below with reference to examples for illustration. However, one skilled in the relevant art will recognize that the invention can be practiced without one or more of the specific details or with other methods, components, materials and so forth. In other instances, well-known structures, materials, or operations are not shown in detail to avoid obscuring the features of the invention. Furthermore, the features/aspects described can be practiced in various combinations, though only some of the combinations are described herein for conciseness.

[034] BRIEF DESCRIPTION OF THE DRAWINGS

[035] Example embodiments of the present invention will be described with reference to the accompanying drawings briefly described below.

[036] FIG. 1 illustrates the titanium oxide nanoparticles treated suture, according to the aspects of the invention.

[037] FIG. 2. illustrates the FeSEM: (Field Emission Scanning Electron Microscope) of

TiO ₂ NPs. according to the aspects of the invention.

[038] FIG. 3 illustrates energy dispersive spectroscopy showing elemental analysis of the NPs, according to the aspects of the invention.

[039] FIG. 4 illustrates x ray diffraction analysis of TiO ₂ NPs. according to the aspects of the invention.

[040] FIG. 5 illustrates dynamic light scattering of TiO ₂ NPs. according to the aspects of the invention.

[041] FIG. 6 illustrates zeta potential of the NPs, according to the aspects of the invention.

[042] FIG. 7 illustrates automated nanoparticle coating dipper, according to the aspects of the invention.

[043] FIG. 8 illustrates scanning electron microscope of the NPs: 8(A) at 5 micronanometer (μnm) magnification, FIG. 8(B) at 10 μnm magnification, 8(C) at 50 μnm magnification, 8(D) at 100 μnm magnification, according to the aspects of the invention.

[044] FIG. 9 illustrates bacterial growth curve experiments: 9(A) S. aureus, 9(B) E. coli, 9(C) P. aurogenosa, according to the aspects of the invention.

[045] FIG. 10 illustrates measurement of ZOI (zone of inhibition) for S. aureus: 10(A) Control/Uncoated suture and FIG. 10(B) Coated suture with different concentrations of Nano particle impregnated Suture, according to the aspects of the invention.

[046] FIG. 11 illustrates measurement of ZOI (zone of inhibition) for E. coli: 11(A) Control/Uncoated suture and FIG. 11 (B) Coated suture with different concentrations of Nano particle impregnated Suture, according to the aspects of the invention.

[047] FIG. 12 illustrates measurement of ZOI (zone of inhibition) for P. aurogenosa: 12(A) Control/Uncoated suture and FIG. 12 (B) Coated suture with different concentrations of Nano particle impregnated Suture, according to the aspects of the invention.

[048] FIG. 13 illustrates the MTT assay to measure the cell viability and cell proliferation: 13(A)- 24hours and 13(B)- 48hours, according to the aspects of the invention. [049] FIG. 14 illustrates Tensile stress at Maximum Load, compared between (in duplicates): uncoated suture, sample A & B; coated suture & not UV treated, sample C & D; coated suture but UV treated, sample E & F; according to the aspects of the invention.

[050] FIG. 15 illustrates Tensile stress at Breaking Strength, compared between (in duplicates) and compared (in duplicates): uncoated suture, sample A & B; coated suture & not UV treated, sample C & D; coated suture but UV treated, sample E & F; according to the aspects of the invention.

[051] FIG. 16 illustrates tensile stress at yield strength, compared between (in duplicates) and compared (in duplicates): uncoated suture, sample A & B; coated suture & not UV treated, sample C & D; coated suture but UV treated, sample E & F: 16(i) Tensile strain and FIG. 16(ii) Tensile stress, according to the aspects of the invention.

[052] FIG. 17 illustrates tensile stress at Modulous Of Elasticity, compared between (in duplicates) and compared (in duplicates): uncoated suture, sample a & b; coated suture & not uv treated, sample C & D; coated suture but UV treated, sample E & F; according to the aspects of the invention.

[053] FIG. 18 illustrates in vitro bacterial transport assay, according to the aspects of the invention.

[054] FIG. 19 illustrates intradermal injection of the animal, according to the aspects of the invention.

[055] FIG. 20 illustrates administration of test item, according to the aspects of the invention. [056] In the drawings, like reference numbers generally indicate identical, functionally similar, and/or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number.

[057] DETAILED DESCRIPTION OF THE INVENTION

[058] It is to be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The present disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

[059] The use of “including”, “comprising” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. The terms “a” and “an” herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced items. Further, the use of terms “first”, “second”, and “third”, and the like, herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.

[060] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise. By way of example, “a dosage” refers to one or more than one dosage.

[061] The terms “comprising”, “comprises” and “comprised of’ as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non -recited members, elements or method steps.

[062] The term “TiO ₂ ”, means- Titanium dioxide and term “NPs” means nanoparticles.

[063] The term- Zone of inhibition means a circular area around the spot of the antibiotic in which the bacteria colonies do not grow. The zone of inhibition can be used to measure the susceptibility of the bacteria towards the antibiotic.

[064] The term -MTT assay means a colorimetric assay for assessing cell metabolic activity. MTT-assay can be used to determine the viability of nanoparticle-treated cells. 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) is a yellow dye, which is reduced by cellular enzymes to the blue product formazan

[065] The term- tensile stress- can be defined as the magnitude of force applied along the suture material, which is divided by the cross-sectional area of the suture material in a direction perpendicular to the applied force.

[066] The term -breaking strength of suture means Initial tensile strength is a measure of the amount of tension applied in a horizontal plane necessary to break the suturing material.

[067] The term- yield strength is defined as the stress at which a predetermined amount of permanent deformation occurs.

[068] The term- Tensile strain is defined as the deformation or elongation of a solid body due to the application of a tensile force or stress.

[069] The term -Modulous Of Elasticity means a quantity that measures an object or substance's resistance to being deformed elastically when a stress is applied to it.

[070] The term- Automated nanoparticle coating machine means a machine which has programmes installed in it to coat the Nanoparticles.

[071] The term- antimicrobial agent- that kills microorganisms or stops their growth.

[072] The term -skin sensitization is a response of the adaptive immune system, in which there is a delayed T-cell-mediated allergic response to chemically modified skin proteins. [073] The term -intracutaneous reactivity-is a"localized inflammatory response to single, repeated, or continuous application of the test substance, without involvement of an immunological mechanism.

[074] The term -acute systemic toxicity is the systemic Toxicity tests evaluate the generalized biological effects to organs and tissues following exposure to a medical device, bio-material, or their extracts.

[075] The term -Cytotoxicity is the quality of being toxic to cells.

[076] The term -Pyrogens are fever-causing agents which contaminate medicines and vaccines as a consequence of the manufacturing process.

[077] All documents cited in the present specification are hereby incorporated by reference in their totality. In particular, the teachings of all documents herein specifically referred to are incorporated by reference.

[078] Example embodiments of the present invention are described with reference to the accompanying figures.

[079] In the drawings, like reference numbers generally indicate identical, functionally similar, and/or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number.

[080] EMBODIMENTS OF THE INVENTION:

[081] The primary objective of the present invention is to provide surgical silk sutures and more specifically the sutures coated with titanium dioxide (TiO ₂ ) nano-particles (NPs), and characterized by non-capillarity, non-brooming, improved knot retention, biocompatible, antimicrobial, photo-catalytic and self-cleaning properties.

[082] In an embodiment, the surgical silk suture, wherein the TiO ₂ substrate is in form of TiO ₂ nano particles, having particle size in range of 40 nanometers to 60 nanometers.

[083] In further embodiment, the TiO ₂ NPs are comprising of Titanium and Oxygen with atomic weight of 24.54% and 41.60% respectively; wherein the hydrodynamic size of TiO ₂ NPs were ranging from 270nm to 400nm; wherein the Zeta potential value of TiO ₂ NPs were -24.7mV; wherein the said of TiO ₂ NPs were stable in the suspension form.

[084] In an embodiment, the support material is a thread with a monofilament or multifilament structure; wherein the suture is a braided, twisted, or covered suture; wherein the support material is a suturing thread joined to a needle; wherein the support material is a staple.

[085] In yet another embodiment, the plurality of plasmonic nanoparticles are evenly dispersed within the silk fibroin matrix; wherein the plurality of nanoparticles are distributed in a gradient within the silk fibroin matrix; wherein the plurality of nanoparticles are distributed in a pattern, said pattern comprises an optical pattern, a micropattem, or a nanopattem; wherein the at least one nanoparticle is selected from the group consisting of a nanosphere, a nanoshell, a nanorod, a nanocage, a nanocrystal, nanopowder, and any combinations thereof.

[086] In an embodiment, the metal is selected from the group consisting of a noble metal, a non- noble metal, an oxide thereof, an alloy thereof, and any combinations thereof.

[087] In an embodiment, the antimicrobial agent is soluble in a solvent which is compatible with both the antimicrobial agent and the suture, wherein the antimicrobial agent is water or alcohol soluble, wherein the antimicrobial agent is anionic, cationic, or nonionic, wherein the antimicrobial agent is selected from the group consisting of tetracycline hydrochloride, neomycin sulfate, chloramphenicol, streptomycin sulfate, potassium penicillin, polymixin B sulfate, oxytetracycline hydrochloride, gentamycin sulfate, sodium cephalothin, nitrofurazone, rifamycin, benzethonium chloride, sodium oxacillin, dihydrostreptomycin sulfate, disodium carbenicillin, and sodium furadantin.

[088] In an embodiment, the multifilament/monofilament suture is a silk, cotton, linen, polyolefin or polyester suture or absorbable or non-absorbable suture.

[089] In an embodiment, the surgical silk sutures have antibiotic property against Gram positive and Gram negative aerobes/anaerobes.

[090] In an embodiment, the surgical silk sutures are biocompatible having tensile strength, breaking strength, yield strength and modulous of elasticity, wherein surgical silk sutures are biocompatible having no skin sensitization or intracutaneous reactivity or acute systemic toxicity or cytotoxicity or pyrogenic activities.

[091] In yet another embodiment, an automated nanoparticle coating machine, for dipper treatment of sutures to be coated, comprising; a magnetic stirrer; a solution holder; a panel for controlling rate of cycles and time; a chamber for air drying of coated suture and a chamber for sterilising of coated sutures.

[092] In an embodiment, a method for preparing a surgical silk suture comprising the steps of: [093] adding precursors comprising, Titanium tetra 2-ethylhexyloxide or Titanium tetra oxysulfate or Titanium tetra chloride tetrahydrofuran or Titanium tetra isopropoxide or Titanium tetra butoxide to solvents comprising, methanol or ethanol or isopropanol at alkaline pH; [094] stirring the solution of step (a) with a magnetic bead at rpm 800 to 1200, and at temperature of 40° to 65°;

[095] forming precipitates by adding distilled water;

[096] cooling of precipitates at the optimum pH & temperature; [097] centrifuging the precipitates;

[098] forming TiO ₂ Nanoparticles pellets and powdering the said TiO ₂ Nanoparticles;

[099] calcinating TiO ₂ Nanoparticles in a furnace for one to four hours at 400° to 800° C;

[0100] cooling down of furnace and collecting Nanoparticles;

[0101] impregnating the suture with Nanoparticles; and

[0102] drying the coated suture to remove the solvent and collecting the impregnated suture. [0103] With the advance in the nanotechnology, titanium dioxide Nanoparticles are prepared and impregnated to suture materials. Being a biocompatible material and having antimicrobial activity can effectively overcome the existing problem associated with the surgical silk (FIG 1).

[0104] EMBODIMENTS OF THE INVENTION:

[0105] Following are the steps followed to prepare and characterize the TiO ₂ NPs.

[0106] 1 TiO ₂ NPs PREPARATION (sol-gel method)

[0107] 2.TiO ₂ NPs CHARECTERISATION

[0108] 3. COATED SUTURES PREPARATION & CHARECTERISATION

[0109] 4. ANTIBIOTIC PROPERTY TESTING

[0110] 5. BIOCOMP ATABILITY TESTS

[0111] 6. PHYSICAL PROPERTIES ANALYSIS

[0112] 7. CAPILLARITY EXPERIMENT

[0113] 8. QUANTIFICATION OF NPs IN SUTURE

[0114] 9. RELEASE KINETIC EXPERIMENT

[0115] 10. ANTIFUNGAL TEST\

[0116] 11. PRECLINIC AL ANIMAL SAFETY STUDY [0117] 1. TiO2 NPs PREPARATION (sol-gel method)

[0118] Various precursors (Titanium tetra 2-ethylhexyloxide / Titanium tetra oxysulfate / Titanium tetra chloride tetrahydrofuran / Titanium tetra isopropoxide / Titanium tetra butoxide), solvents (methanol / ethanol / isopropranolol), and distilled water mixed at alkaline pH in a conical flask placed over a stirring hot plate and with the magnetic bead, stirring done with the rpm 800 to 1200 with a temperature of 40° to 65°. The said preparation method is described in the following flowchart.

[0119] Under cold condition mixture of precursor and solvent was prepared by adding them slowly in stirring condition [250-500 rpm] at 60°C to 90°C on a hot plate for 5-10hours. Then the distilled water [pH-8] was added. Yellow precipitates start to form. After an incubation period of 1 - 2 hr let cool down the setup, collect the precipitates and wash with distilled water by centrifugation method at 5000 - 7000 rpm for 5- 10 minutes at least for 2- 3 times.

[0120] The supernatant was thrown and collected the yellowish precipitates in MCT tubes or petri plates, further stand them for an incubation of 8-10hrs under 70°C to 80°C. Precipitates are further collected in dried condition crushed in a small mortar pestle collect the powder form. Then the powder form was kept in a crucible inside a furnace at 400°C to 800°C for 4hrs. Let cool down the furnace [6- 8 hrs] and collect the formed TiO ₂ powders.

[0121] General Observation: Advantages of this method includes, the method is highly versatile, high degree of purity of the obtained NPs, possibility to obtain a fine control of microstructure of final product by modulation of synthesis parameters, and also prepare NPs using less temperature.

[0122] 2. MORPHOLOGICAL CHARACTERISATION OF TITANIUM DIOXIDE (TiO ₂ ) NANOPARTICLES

[0123] Titanium dioxide (TiO ₂ ) nanoparticles are characterized by following methods.

A) FIELD EMISSION SCANNING ELECTRON MICROSCOPE

[0124] FIG 2 depicts FeSEM: Field Emission Scanning Electron Microscope. General Observation : FeSEM was done to examine minor area contamination spots at electron accelerating voltages well-matched with EDS. This revealed the size, shape, aggregation of NPs and the elements present. Scientific Observation: FeSEM showed the spherical, dispersed, Nanocrystals with a size range of 38nm to 45nm in a magnification of 250kx.

[0125] B) ENERGY DISPERSIVE SPECTROSCOPY

[0126] FIG 3 shows energy dispersive spectroscopy. General Observation: Energy Dispersive

Spectroscopy was done to get the Elemental analysis of the NPs.

[0127] Scientific Observation: EDS showed the presence of elements Titanium and oxygen with its atomic weight of 24.54% & 41.60% respectively.

[0128] C) X RAY DIFFRACTION ANALYSIS:

[0129] FIG 4 shows the X ray diffraction analysis. General Observation: X ray diffraction analysis was done for phase identification, and sample purity. Scientific Observation: the crystalline phases can be confirmed by the presence of (101), (004), (105) and (200) diffraction peaks, for the case of anatase phase and (110), (111), (211) and (002) forthe rutile crystalline phase.

[0130] D) DYNAMIC LIGHT SCATTERING

[0131] FIG 5 shows the dynamic light scattering. General Observation: Dynamic light scattering was done for phase identification, and sample purity. Scientific Observation: The hydrodynamic size of TiO ₂ NPs were ranging from 270nm to 400nm.

[0132] E) ZETA POTENTIAL

[0133] FIG 6 illustrates the zeta potential. General Observation: It measures effective electric charge on the nanoparticle surface. The magnitude of the zeta potential provides information about particle stability, with particles with higher magnitude zeta potentials exhibiting increased stability due to a larger electrostatic repulsion between particles. When the nanoparticles have zeta potential value of <-30mv or >30mv, that indicates the stabile form of particles. Scientific Observation: Zeta potential value of -24.7mV shows that TiO ₂ particles are stable in the suspension form.

[0134] 3. COATED SUTURES PREPARATION & CHARECTERISATION [0135] Following are the steps of preparation & characterization of coated sutures.

[0136] Treatment of sutures to be coated ->‘3D dipping machine’ with the magnetic stirrer on top kept ready for dipping->Solution of desired concentration of Nanoparticles prepared and kept onto the stirrer->Surgical suture is being dipped at fixed rate of cycles for a fixed period->Air drying of coated suture-> Sterilization of ‘Coated sutures.

[0137] FIG 7 depicts the picture of Automated nanoparticle coating dipper, according to the embodiment of the invention.

[0138] CHARACTERISATION OF COATED SUTURES WITH SCANNING ELECTRON MICROSCOPE General Observation : Scanning electron microscope assess the surface morphology with qualitative analysis of the ‘TiO ₂ Coated Surgical Silk’. FIG. 8 illustrates scanning electron microscope of the NPs at different magnification: 8(A) 5 micronanometer (μnm) magnification, 8(B) 10 micronanometer (μnm) magnification, 8(C) 50 micronanometer (μnm) magnification, 8(D) 100 micronanometer (μnm) magnification, according to the aspects of the invention. Scientific Observation: At 5 μnm FIG 8(A), 10 μnm FIG 8 (B), 50 μnm FIG 8 (C), and 100 μnm FIG 8 (D), magnification showed the deposition of TiO ₂ Nanoparticles onto the surface of Surgical Silk.

[0139] 4. ANTIBIOTIC PROPERTY TESTING

[0140] Antibiotic property testing was carried out by a) bacterial growth curve experiment and b) diffusion method, three organisms are tested for bacterial growth curve experiment; S. aureus, E. coli and P. aurogenosa.

[0141] BACTERIAL GROWTH CURVE EXPERIMENT

[0142] Following is the flowchart for bacterial growth curve experiment.

[0143] 2 sets of flasks with broth (1-control, 2-study)->Treatment of Study flask with TiO ₂ NPs & bacterial culture, Control flask treatment only with bacterial culture->Ovemight Incubation- >Sample from the flasks collected in 1ml aliquots every day for 5 days and plated on agar plates- >Incubation of agar plates & counting of bacterial colonies->Graph plotting

[0144] Steps:

1) Take 2 sets of conical flasks consisting of 100mL autoclaved broth in 250ml conical flask.

2) Inoculate 5ml of bacterial culture in both the sets. Inoculums of bacteria are maintained to OD-0.1 at 600nm. 3) Keep one set for control experiment for showing growth of bacteria in absence of antibacterial agent; another set is treated with 1ml of titanium Nanoparticles with concentration of 8mg/ml.

4) The setup was kept for 5 days in incubation at 37°C, 1ml aliquots were collected every 24 hours.

5) Collected aliquots were further diluted to 10 ^-6 ; dilutions 10 ^-5 , 10 ^-6 were further plated on agar plates of both set control and Nanoparticle inoculated.

6) Plates of each interval are incubated for 18hours and then the colonies are counted. The colony count is further directly multiplied to dilution factor [10 ^-5 , 10 ^-6 ].

7) At the end of the experiment, plot graph of time intervals on x-axis vs OD at 600nm on y-axis to obtain a growth curve of bacteria.

[0145] FIG. 9 illustrates bacterial growth curve experiment: 9(A) S. aureus, 9(B) E. coli, 9(C) P. aurogenosa, according to the aspects of the invention.

[0146] General Observation: This curve represents the increase in numbers or bacterial mass can be measured as a function of time. Several distinct growth phases can be observed within a growth curve. These include the lag phase, the exponential or log phase, the stationary phase, and the death phase. Each of these phases represents a distinct period of growth that is associated with typical physiological changes in the cell culture. Scientific Observation: The control suture (uncoated) showed the ideal bacterial growth curve, whereas ‘TiO ₂ Coated Surgical Silk’ at 4 different concentrations inhibited the bacterial growth. The Minimal Inhibitory Concentrations of the TiO ₂ against these organisms are 2 mg/ml, 4 mg/ml, 6 mg/ml, and 8 mg/ml. The antibacterial activity of different concentration solution (200, 100, 50 and 25 μM) of titanium oxide nanoparticles coated sutures were evaluated by agar diffusion method which was tested against gram-positive (Staphylococcus aureus) bacteria. The bacterial strains were cultured on nutrient agar and plates were incubated for 24h at 37°C.

[0147] The clear inhibition area of bacterial growth around the titanium oxide nanoparticles treated suture indicates a good antibacterial. No inhibition zone was observed for the untreated suture.

[0148] B) DIFFUSION METHOD

[0149] Luria-Bertani agar plates (2 plates; 1 for control suture and other for coated one)- >Addition of bacterial culture to the plates->‘TiO ₂ coated Surgical Silk sutures’ of particular concentrations placed in the spread plate->Ovemight Incubation->Measurement of ZOI (Zone Of Inhibition)

[0150] FIG 10 illustrates measurement of ZOI (zone of inhibition) for S. aureus: 10(A) Control/Uncoated suture, FIG. 10 (B) Coated suture with different concentrations of Nano particle impregnated Suture, according to the aspects of the invention.

[0151] FIG. 11 illustrates measurement of ZOI (zone of inhibition) for E. coli: 11(A) Control/Uncoated suture, FIG. 11(B) Coated suture with different concentrations of Nano particle impregnated Suture, according to the aspects of the invention.

[0152] FIG. 12 illustrates measurement of ZOI (zone of inhibition) for P, aurogenosa : 12(A) Control/Uncoated suture, FIG. 12 (B) Coated suture with different concentrations of Nano particle impregnated Suture, according to the aspects of the invention.

[0153] General Observation : Agar diffusion test is the primary method to determine the antimicrobial activity of the nanofibrous scaffolds. It is only suitable for the diffusive test materials. The test is qualitative, easy to perform, and simple .Scientific Observation: The presence of antimicrobial activity is indicated by the absence of bacterial growth, which is indicated by the ZOI.

[0154] 5. BIOCOMPAT ABILITY TESTS

[01561 MTT ASSAY

MTT Assay was done by following method.

[0157] Plate cells (RAW CELLS) at 1,000 to 100,000 per well->Incubate for 6 to 24 hours->Add 10 μL MTT Reagent->Incubate for 2 to 4 hours until purple precipitate is visible->Add 100 μL Detergent Reagent->Leave at room temperature in the dark for 2 hours-> Record the absorbance at 570 nm

[0158] Procedure:

[0159] Harvest suspension cells by centrifugation. Adherent cells should be released from their substrate by trypsinization or scraping. Trypsinised cells are counted by using haemocytometer. Maintained the preferred cell density according to the plates used. Control and samples are considered in triplicates. Incubated for 24-48 hrs for cell confluence.

[0160] After 90% cell confluence, the cells are treated with the samples for 24hrs. After the incubation period, the samples were replaced with the MTT reagent (tetrazolium) of specific concentration and quantity into each well. Return plate to cell culture incubator for 2 to 4 hours. Periodically view the cells under an inverted microscope for presence of intracellular punctate purple precipitate. When the purple precipitate is clearly visible under the microscope add Detergent Reagent (DMSO) to all wells, including controls. FIG. 13 illustrates the MTT assay to measure the cell viability and cell proliferation: 13(A)- 24hours, 13(B)- 48hours, according to the aspects of the invention.

[0161] Leave plate with cover in the dark for 2 to 4 hours. Remove plate cover and measure the absorbance in each well, including the blanks, at 570 nm in a microtiter plate reader. It should yield an absorbance of 0.75 - 1.25. Determine the average values from triplicate readings. FIG 13 (A and B).

[0162] General Observation: MTT Assay measures the cell viability and cell proliferation. The reduction of tetrazolium salts is now widely accepted as a reliable way to examine cell proliferation. The yellow tetrazolium MTT (3 -(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) is reduced by metabolically active cells, in part by the action of dehydrogenase enzymes, to generate reducing equivalents such as NADH and NADPH. The resulting intracellular purple formazan can be solubilized and quantified by spectrophotometric means.

[0163] Scientific Observation: All 4 concentrations of ‘ TiO2 Coated Silk Suture’ showed 80% to 90% of cell viability in both the samples of 24 hrs and 48hrs condition.

[0164] B) LIVE & DEAD STAINING ASSAY [0165] Live & dead staining assay was done by following method.

[0166] Cell seeding in the culture flask-> Cell Incubation under specific condition - >Trypsinisation of cells ->Addition of media with serum->Separation of cell clumps & their resuspension->Addition of dye (Propidium Iodide / Syto 9)->Cell Incubation->Analyzing the percentage of viable cells using ‘flow cytometer’.

[0167] Observation: Percentage of viable cells stained were more as compared to that of dead cells

[0168] Cl EARLY APOPTOSIS

[0169] Early apoptosis assay was done by following method.

[0170] Cell seeding in the culture flask (three T25 culture flask for control/unstained, Annexin only, and propidium iodide only) in triplicates->Cell Incubation under specific condition- >Trypsinisation of cells->Centrifugation of cells & resuspension of pellets->Incubation of study group cells with the dye->Cell analysing using ‘Flow Cytometer’.

[0171] Observation: the annexin V binding assay provides an excellent indicator for the early stages of apoptosis

[0172] D) IN VITRO SCRATCH ASSAY

[0173] In vitro scratch assay was done by following method.

[0174] RAW cells are grown to monolayer confluently and then serum is deprived for 24 h->A scratch wound with a p20 pipette tip OR anything with sharp tip is used to inflict a straight line across the confluent cells->Cell wash & Incubation with the Media treated with NPs->Scratch wound closure is photographed after 6 and 24 h and monitored under a light microscope.

[0175] Using these photographs, the acellular area will be calculated as the number of pixels by Image

[0176] Observation: Acellular area is covered slowly by the cells which migrated towards the scratch area. [0177] 6. PHYSICAL PROPERTIES OF COATED SUTURE

[0178] Following four parameters were analyzed to assess the physical properties of coated sutures.

[0179] There were 6 different samples were taken, and compared between (in duplicates) and compared (in duplicates): uncoated suture, sample A & B; coated suture & not UV treated, sample C & D; coated suture but UV treated sample E & F.

[0181] FIG. 14 illustrates tensile stress at Maximum Load, compared between (in duplicates) and compared (in duplicates): uncoated suture, sample A & B; coated suture & not UV treated, sample C & D; coated suture but UV treated sample E & F; according to the aspects of the invention. [0182] FIG. 15 illustrates tensile stress at Breaking Strength, compared between (in duplicates) and compared (in duplicates): uncoated suture, sample A & B; coated suture & not UV treated, sample C & D; coated suture but UV treated sample E & F; according to the aspects of the invention.

[0183] FIG. 16 illustrates tensile stress at yield strength, compared between (in duplicates) and compared (in duplicates): uncoated suture, sample A & B; coated suture & not UV treated, sample C & D; coated suture but UV treated sample E & F: 16(i) Tensile strain and FIG. 16(ii) Tensile stress, according to the aspects of the invention.

[0184] FIG. 17 illustrates tensile stress at Modulous Of Elasticity, compared between (in duplicates) and compared (in duplicates): uncoated suture, sample a & b; coated suture & not uv treated, sample C & D; coated suture but UV treated sample E & F; according to the aspects of the invention.

[0185] 7. CAPILLARITY EXPERIMENT

[0186] 2 test tubes with the rubber fork, containing broth was connected with a aglass tube through which suture material inserted. 2 sets were made one for uncoated suture and the other for the coated suture. The rubber stoppers prevented the propagation of bacteria on the surface of the thread. The broth in one of the test tubes was inoculated with Staphylococcus aureus, which was immobile. Incubation followed at 37° C, and the other, non -inoculated broth was inspected daily for bacterial growth. FIG. 18 illustrates invitro bacterial transport assay, according to the aspects of the invention. General Observation: This is an invitro Bacterial transport assay, where bacteria transports from one test tube to the other through the capillarity of the suture material. Scientific Observation: Non inoculated broth with the Uncoated suture showed bacterial growth due to the capillarity nature of the suture material. Whereas there was no bacterial growth in the broth associated with the Coated suture.

[0187] 8. QUANTIFICATION OF NPs IN SUTURE [0188] Following method was followed.

[0189] Nanoparticle coated suture ->Dipped in the 5%-10% of Sulphuric acid for 8-12 Hrs->Only suture dissolves->Undissolved NPs collected->NPs Quantification by ‘Inductive Coupled Plasma- Mass Spectroscopy’ Analysis.

[0190] 9. RELEASE KINETIC EXPERIMENT [0191] Following method was followed.

[0192] Coated suture of particular length dipped in specific quantity of artificial saliva->Seven sets of suture and artificial saliva prepared->Artificial saliva collected per day from day 1 to day 7->From the collected samples of each day the suture was removed by sterile forceps->Samples examined in ‘Inductive Coupled Plasma- Mass Spectroscopy’ (ICP-MS) and the concentration of the nanoparticles released was measured.

[0193] 10. ANTIFUNGAL TEST [0194] Following method was followed.

[0195] Required quantity of test item kept for incubation at 50±2°C for 72±2 hours based on the surface area ->Candida albicans tester strain is adjusted to 1 x 10 ⁸ cells/mL and is streaked using swabs onto preincubated and prelabelled Sabouraud Dextrose Agar plates. ->Then well is made using cork borer and to the well post incubated 1 mL of test item extract is added->Triplicates prepared and plates incubated for 24 to 72 hours at 25±l°C->Blank media plate kept as control and Media plate streaked with Candida albicans kept as positive control->Post incubation plates observed for Zone of inhibition

[0196] 11. PRECLINICAL ANIMAL SAFETY STUDY

[0197] A] SKIN SENSITIZATION STUDY OF NANOSUTRA IN GUINEA PIGS BY

MAXIMIZATION TEST

[0198] [As per ISO 10993-10: 2010 (E)]

[0199] Study Design: l

[0200] Magnusson and Kligman Grading Sca e for the Evaluation of Challenge Patch Test

Reactions (FIG 19)

Patch Test Reaction Grading Scale

No visible change 0 Discrete or patchy erythema 1 Moderate and confluent erythema 2 Intense erythema and/or swelling 3 [0201] EVALUATION OF RESULTS

[0202] A Magnusson and Kligman grade of 1 or greater in the treatment group indicates sensitization, provided grades of less than 1 are seen in solvent control. If grades of 1 or greater are observed in solvent control animals, then the reactions of test animals which exceed the most severe reaction in solvent control animals are presumed to be due to sensitization. [0203] Observation: No visible changes revealed over injection site

[0204] B] INTRACUTANEOUS REACTIVITY TESTOF COATED SUTURE IN NEW ZEALAND WHITE RABBITS [0205] [As per ISO 10993-10: 2010 (E)]

[0206] Study Design:

[0207] Grading System for Intracutaneous (Intradermal) Reactions

[0208] INTERPRETATION OF RESULTS

[0209] The numerical values corresponding to each injection site, at each time interval will be recorded in all the animals. The erAythema grades and oedema grades recorded at 24 (±2 hr), 48 (±2 hr) and 72 (±2 hr) hours will be totaled separately for each test item extract and solvent control for each animal. The total will be divided by 15 (3 time points x 5 injection sites). The mean score of each test item extract and each corresponding solvent control will be calculated. The final test item reaction score will be obtained by subtracting the mean score of solvent control from the mean score of test item. The requirements of the experiment will be meet if the final test sample score is 1.0 or less. If at any observation period the average reaction to the test item is questionably greater than the average reaction to the solvent control, the amendment will be made and test will be repeated using three additional rabbits. (FIG 20)

[0210] Observation: No intracutaneous reactivity is seen.

[0211] C] ACUTE SYSTEMIC TOXICITY STUDY OF COATED SUTURE IN SWISS ALBINO MICE

[0212] [As per ISO 10993-11: 2017 (E)]

[0213] Study Design

[0214] Clinical Signs Observation

[0215] All the animals will be observed for clinical signs of toxicity and mortality at 30 to 40 min, 1 hr (±10 mins), 2 hrs (±10 mins) and 4 hrs (±10 mins) post dosing on Day 1. On day 2, 3 and 4 all the animals will be observed once daily for clinical signs of toxicity and twice daily for mortality. The observation will be terminated, if the animals do not develop any clinical signs up to three days after dosing. The observation period may extend up to 14 days after dosing based on the toxic reactions, rate of onset and length of recovery period. The appearance, change and disappearance of the toxicity signs will be recorded.

[0216] Observation: No acute systemic toxicity is seen.

[0217] C] MATERIAL MEDIATED PYROGEN TEST OF NANOSUTRA IN NEW ZEALAND WHITE RABBITS [0218] [As per USP General Chapter <151>]

[0219] Sham Test

[0220] Within 7 days prior to the main test, the rabbits will be subjected to sham test for selecting for main test. This test will include all steps in the main test except the injection.

[0221] Animals showing control temperature variation of not more than 1°C from each other and having temperature not exceeding 39.8°C will be selected for main test.

[0222] Main Test [0223] Procedure: [0224] Body weight of the animals will be taken at least one hour before recording the temperature, animals will be kept in the restrainer; temperature sensing probes will be inserted to the rectum of rabbits to a depth of not less than 7.5 cm and left for stabilization.

[0225] Clinical Signs of Toxicity and Morbidity/Mortality

[0226] Rabbits will be observed once daily for clinical signs of toxicity and twice daily for morbidity/mortality throughout the experimental period.

[0227] Observation: No material mediated pyrogenecity is seen.

[0228] D] 28 DAYSINTRAMUSCULAR IMPLANTATION TEST OF COATED SUTURE IN NEW ZEALAND WHITE RABBITS [0229] [As Per ISO 10993 PART 6:2016(E)]

[0230] Study Design-The study will be performed in 3 rabbits as per the following study design.

[0232] The evaluation will be semi-quantitative as per ISO 10993-6:2016(E)Table E.3 depending upon the type of reaction shown by the implant.

[0233] Depending on the scoring of implantation sites, the test sample will be graded as follows when compared to the reference item samples:

[0234] Minimal or non-reaction (0.0 up to 2.9)

[0235] Slight reaction (3.0 up to 8.9)

[0236] Moderate reaction (9.0 up to 15.0)

[0237] Severe reaction (>15)

[0238] STUDY REPORT PREPARATION AND RESULTS

[0239] Individual animal data will be presented as appendices and summarized and presented as tables. All findings will be presented in the report as per the standard reporting procedure of the test facility. [0240] Observation: On intaramuscular implanatation, non-reactive was observed.

[0241] E] IN VITRO CYTOTOXICITY STUDY OF COATED SUTURE BY ELUTION METHOD

[0242] [As per ISO 10993-5: 2009(E)]

[0243] Extraction Procedure

[0244] *Total surface area of test item is 1.8856 cm ² per 30 cm.

[0245] Scoring for cytotoxicity will be done based on the following criteria:

[0246] treatment date.

[0247] Cells will be incubated at 37±1°C in the presence of 5±1% CO ₂ for 26±2 hours. Post incubation of 26±2 hours, the cultures will be examined microscopically to verify any changes in cell morphology after treatment with the test item.

[0248] Scoring for cytotoxicity will be done based on the following criteria:

[0249] ACCEPTANCE CRITERIA

[0250] If the numerical grading is greater than 2, the test item will be considered as cytotoxic. [0251] If there are evidences of difference in test results for replicates (grade <2 or grade >2), the test will be considered invalid and the study will be repeated.

[0252] If the negative and positive control does not exhibit the expected response (Grade 0 and 3 or 4 respectively) in the test system, the test will be repeated.

[0253] Observation: Non cytotoxic is observed.

[0254] F] BACTERIAL REVERSE MUTATION TEST OFCOATED SUTURE USING SALMONELLA TYPHIMURIUM AND ESCHERICHIA COLI TESTER STRAINS [0255] (As per ISO 10993-3:2014, ISO 10993-33:2015)

[0256] Test Concentrations [0257] Plate Incorporation Method:

[0258] The first trial will be conducted using direct plate incorporation method.

[0259] Preincubation Method:

[0260] EVALUATION AND INTERPRETATION OF RESULTS [0261] Effect on Bacterial Background Lawn

[0262] The condition of the bacterial background lawn will be evaluated for evidence of the test item toxicity.

[0263] Controls

[0264] Extraction Control

[0265] Polar solvent (normal saline) and non-polar solvent (dimethyl sulphoxide) will be used as a solvent.

[0266] Positive Controls [0267] Dimethyl Sulphoxide will be used to prepare the stock and/or dilutions of the following positive controls:

[0268]

[0269] Test Concentrations

[0270] Limit test will be performed using 100% test item extraction/plate test sample. If any cytotoxicity or mutation observed further study will be repeated using five different lower concentrations including cytotoxicity test by raising an amendment to study plan.

[0271] Performance of the Assay

[0272] Labeling: The petri-dishes will be labeled to indicate the study number, strain number, treatment group, metabolic activation, experiment date and replicate number.

[0273] Triplicates will be maintained for solvent control, negative control, positive control and one treatment level will be maintained in the experiment

[0274] Plate Incorporation Method:

[0275] The first trial will be conducted using direct plate incorporation method.

[0276] Preincubation Method:

[0277] Observation: No bacterial mutation is observed.

[0278] ADVANTAGES OF THE INVENTION:

[0279] The technology and product developed herein is novel in terms of its application and usage. [0280] Cost-effective and highly reliable and user friendly compared to the existing technologies. [0281] A 3D printing machine for coating the thread/suture and a method of coating the suture using the said machine.

[0282] Sustained antibiotic property of the suture.

[0283] Sustained release of NPs.

[0284] BENEFITS OF THE INVENTION

[0285] The said sutures are, anti-bacterial, photo -catalytic, self-cleansing, uv protective, cost effective and better tensile strength.

[0286] BEST MODE TO PRACTICE

[0287] Best mode to practice the invention is through commercialization of this invention through the distributors, e-commerce sites, and marketing to reach private dental clinics and hospitals. [0288] According to aspects of the present invention, the invention also provides kits that are useful for the practice of the methods of the invention.

[0289] Merely for illustration, only representative number/type of graph, chart, block, and sub- block diagrams were shown. Many environments often contain many more block and sub-block diagrams or systems and sub-systems, both in number and type, depending on the purpose for which the environment is designed.

[0290] While specific embodiments of the invention have been shown and described in detail to illustrate the inventive principles, it will be understood that the invention may be embodied otherwise without departing from such principles.

[0291] Reference throughout this specification to “one embodiment”, “an embodiment”, or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment”, “in an embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

[0292] It should be understood that the figures and/or screen shots illustrated in the attachments highlighting the functionality and advantages of the present invention are presented for example purposes only. The present invention is sufficiently flexible and configurable, such that it may be utilized in ways other than that shown in the accompanying figures.

[0293] It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.