Vinblastine-loaded iron oxide (fe3 o4) nanocomposite nanofiber for biomeical application

Abstract

VINBLASTINE—LOADED IRON OXIDE (F6304) NANOCOMPOSITE NANOFIBER FOR BIOMEDICAL APPLICATION A nanofiber for biomedical application is provided. The nanofiber includes [00 milligram (mg) of Iron (II, III) oxide (Fe304) nanoparticles, 500 mg of chitosan, a Poly(vinylpyrrolidone) (PVP) solution and I mg of vinblastine (VBL). The F6304 nanoparticles are sonicaled with 10 milliliters (ml) of Dimethylformamide (DM F) for about | hour to synthesize an uniform F6304 nanoparticles solution. The chilosan is added dropwise to 2 milliliters (ml) of acetic acid and dissolved in double distilled water (DH20), followed by stirring for about 24 hours to synthesize a chitosan solution. The Po|y(vinylpyrrolidone) (PVP) solution is mixed with the chitosan solution and then continuously stirred for about l2 hours to synthesize a PVP-CS solution. The vinblastine is mixed to the F6304 nanopanicles solution to synthesize a Fe304-VBL solution. The Fe;O4-VBL solution is mixed with the PVP-CS solution and then continuously stirred at room temperature for about [2 hours, followed by loading into an electrospinning machine system to synthesize a F6304 nanofiber that has improved drug delivery. FIG. I for Abstract.

Description

BACKGROUNDTechnical Field'

[0001] The present invention generally relates to nanofibers that are suited for biomedicalapplications because of their high surface area, controlled pore size, and drug release, moreparticularly, to a nanofiber and a process of preparing the nanofiber for biomedical application.Description of the Related Art

[0002] Nanotechnology is becoming increasingly important in a variety of disciplines,from the environment to the food industry, and is currently also developing in the biomedical field,which has significant potential in future clinics (Yew et al.2020). But the nanomaterials posesignificant experimental challenges and issues when assessing their toxicity, hence further studieswill be needed to establish nanoparticles’ (NPs') safety and toxicity. A major molecular‘mechanismof toxicity is the induction of oxidative stress through the production of free radicals. Apoptosis,DNA damage, oxidative stress-induced damage, and countless other cellular processes aretriggered by reactive oxygen species (ROS). Biological components such as lipids, proteins, andDNA are oxidized when free radicals accumulate. DNA damage appears to be the main impact ofNPs action. The Fe304 nanofibers have Been used successfully for tumor sensors and as nontoxicmaterials and biocompatible materials to enhance drug delivery.

[0003] Cancer is one of the most troubling public health problems on this planet. Cancertreatment aims to cure the disease and allow the patient to live a normal life, Treatment will reducethe size ofthe tumor or slow its growth when a complete cure is not feasible, thereby prolongingone's survival. The most common cancer therapies include surgery, chemotherapy, bone marrowtransplants, radiotherapy, and immunotherapy. Although these methods are popular today; theyare heavily criticized due to a lack of bioavailability of drugs at the targeted site which leads to avariety of side effects, including death. The leading cause of cancer death is lung cancer, whichaccounts for nearly one in five cancer deaths (Maleti et al. 202l).

[0004] Yet another existing solution (Mownika el al.) reports Acacia coesia (L.) Willd.,which is an ethno medicinal plant used to cure skin, sexual problems, wound, stomach and toothproblems. Different effects of ethanolic extract on Dalton’s Ascitic Lymphoma (DAL) induced tumor inoculation as well as its hematological parameters have been examined for the first time.Further. treatment with test extract- significantly decreased the development of tumor volume,tumor packed cell volume and percentage increase in body weight when compared to DAL inducedtumor control group, also increasing the life span, restoring the total white blood cell count- andhemoglobin. The treatment with test extract at doses of 25 and 50 mg / kg normalized the bodyweight and hematological parameters. The outcome of the present work indicates that Acaciacoesia extract could be used as natural anticancer agent for human health.

[0005] Accordingly, there remains a need for nanofibers that have a great capacity for drugloading and could release therapeutic compounds for a longer period of lime.OBJECTIVES OF THE INVENTION

[0006] The primary objective of the present invention is to develop an Iron oxideNanopanicles (Fe304 NPs) by Co-precipitation method.

[0007] An another objective of the present invention is to develop a Vinblastine-loadediron oxide (F6304) composite by electrospinning method.

[0008] Yet another objective of the present invention is to analyze the [n—vivo cytotoxicityand Anticancer Activity of Vinblastine-loaded iron oxide (F6304) Nanofiber on Daltons AscitesLymphoma in mice.SUMMARY

[0009] In view of the foregoing, an embodiment herein provides a nanofiber for biomedicalapplication. The nanofiber includes [00 milligram (mg) of Iron (ll, HI) oxide (F6304)nanopanicles, 500 mg of chitosan, a Poly(vinylpyrrolidone) (PVP) solution and 1 mg ofvinblastine (VBL). The Fe304 nanoparticles are sonicated with 10 milliliters (ml) ofDimethylfomamide (DMF) for about 1 hour to synthesize an uniform F6304 nanoparticlessolution. The chitosan is added dropwise to 2 milliliters (ml) of acetic acid and dissolved in doubledistilled water (DH20), followed by stirring for about 24 hours to synthesize a chitosan solution.The Poly(vinylpyrrolidone) (PVP) solution is mixed with the chitosan solution and thencontinuously stirred for about 12 hours to synthesize a PVP-CS solution. The vinblasline is mixedto the F6304 nanoparticles solution to synthesize a Fe;Oa-V_BL solution. The Fe304-VBL solutionis mixed with the PVP-CS solution and then continuously stirred at room temperature for about I2hours, followed by loading into an electrospinning machine system to synthesize a,Fe;O4 nanofiber that has improved drug delivery.[00l0] In an embodiment, the F6304 nanoparticles are prepared by (i) dissolving 2 g offerric chloride hexahydrate in 25 ml of deionized distilled water (DDW) and stirring the ferricchloride fiexahydrate solution for about 1 hour, (ii) dissolving 2 g of ferrous sulfate heptahydratein 25 ml of distilled water and mixing it with the previously prepared ferric chloride hexahydratesolution to obtain a ferric chloride-ferrous sulfate solution, (iii) continuously stirring the ferricchloride-ferrous sulfate solution for about | hour for maintaining homogeneity, (iv) heating theferric chloride-ferrous sulfate solution to 80 °C and maintaining the temperature for 30 minutes,followed by cooling the ferric chloride-ferrous sulfate solution to room temperature, and (v)gradually adding sodium hydroxide (NaOH) dropwise to the ferric chloride-ferrous sulfate solutionuntil reaching a pH of l I, and stirring the ferric chloride-ferrous sulfate solution for an additional1 hour to generate the F6304 nanoparticles.[001 I] In another embodiment, the ferric chloride-ferrous sulfate solution is washed with’ethanol two times to eliminate any remaining contaminants and dried the resulting ferric chlorideferroussulfate solution by subjecting it to an oven at a temperature range of 70 °C to 80 °C for anovernight period, In yet another embodiment, the F6304 nanofiber is dried at 60 °C for about 12hours afier synthesis. In yet another embodiment, the PVP-CS solution is continuously stirred forabout 12 hours before adding the Fe3O4-VBL solution.[00l2] In yet another embodiment, the F604 nanofiber is synthesized by (i) mixing theFe304 nanopanicles with the PVP-CS solution and (ii) collecting and drying on aluminum foilfrom the mixture of PVP-CS solution and F6304 nanoparticles solution by loading into a 20 mlplastic syringe at a fixed flow rate of l microliler per minute (pl / min), at a voltage of 15 Kilovolt(KV) that is set at a tip of the collector and a collector distance of | 5 centimeter (cm). In yet another.embodiment, the PVP-CS solution is continuously stirred for about l2 hours before adding theF6304 nanoparticles.[OOI3] In one aspect, a process of preparing a nanofiber for biomedical application isprovided. The process includes (a) preparing a F6364 nanoparticles solution by sonicating 100milligram (mg) of Iron (II, III) oxide (F6304) nanopanicles with 10 milliliters (ml') ofDimethylformamide (DMF) for about 1 hour, (b) preparing chitosan solution by adding 500 mg ofchitosan dropwise to 2 milliliters (ml) of acetic acid and dissolving in .double distilled water(DHzO), followed by stirring for about 24 hours, (c) mixing the chitosan solution with aPoly(vinylpyrrolidone) (PVP)' solution and then continuously stirring for about [2 hours tosynthesize a PVP-CS solution, and (d) synthesizing F6304 nanofiber by mixing 1 mg of vinblasline(VBL) to the Fe304 nanopanicles solution to synthesize a F6304-VBL solution, wherein the F6304-VBL solution is mixed with the PVP-CS solution and then continuous|y stirred at roomtemperature for about 12 hours, followed by loading into an electrospinning machine systemItosynthesize a Fe304 nanofiber that has improved drug delivery.

[0014] The Fe304 / vinblastine (VBL) nanofiber has a‘high surface area, a controlled poresize, and a drug release and suited for biomedical applications. The Fe304 nanofiber has a greatcapacity for drug loading>and could release therapeutic compounds for a longer period of time.The (F6304) / vinblasline (VBL) nanofiber is one dimension nanostructures that are ofien madeusing the electrospinning method, which is a quick and affordable way to make ultrafinenanofibers. It is environmentally friendly and can be utilized specifically for cancer applicationsand drug administration when vinblastine is combined with Fe3O4. Among all iron oxides,F6304 has attracted more attention due to its superior magnetic properties and greatbiocompatibility. In-vivo cytotoxicity and anticancer activity of (Fe304) / vinblastine (VBL)nanofiber against Daltons Ascites Lymphoma in mice can be increased by vinblastine (VBL) .integration into F6304 nanopanicles.[00l5] These and other aspects of the embodiments herein will be better appreciated andunderstood when considered in conjunction with the following description and the accompanyingdrawings. It should be understood, however, that the following descriptions, while indicatingpreferred embodiments and numerous specific details there, are given by way of illustration andnot of limitation. Many changes and modifications may be made within the scope of theembodiments herein, and the embodiments herein include all such modifications.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The embodiments herein will be better understood from the following detaileddescription with reference to the drawings, in which[00l7] FIG. 1 illustrates a schematic illustration ofa nanofiber for biomedical applicationaccording to an embodiment herein;[00l8] FIG. 2 illustrates an X-ray powder diffraction (XRDj Analysis of F6304 NPs,F6304 / VBL NPs, F6304 NF and F6304 / VBL NF by using the Rigaku Smart lab X-raydiffractometer (l .5406 A) with Cu Ka radiations according to an embodiment herein;

[0019] FIG. 3 illustrates Field Emission Scanning Electron Microscopy (FESEM) imagesfor F6304 NPs, Fe304 / VBL NPS, F8304 NP and Fe3O4 / VBL NF at 1pm and 500 nm ofmagnifications to obtain morphology images which is performed with Quanta-250-FEG accordingto an embodiment herein;

[0020] FIG. 4 illustrates an Energy Dispersive X-ray (EDX) analysis for Fe304, NPs,F6304 / VBL NPs, Fe304 NF and Feaoa / VBL NF to obtain elemental compound which is performedwith Quanta-ZSO-FEG according to an embodiment herein;

[0021] FIG. 5 illustrates a Fourier transform infrared (FTIR) spectrum for F6304 NPs,FC3OA / VBL NPs, Fe304 NF and F6304 / VBL NF by using the Bruker Tensor 27 according to anembodiment herein;

[0022] FIG. 6 illustrates an‘ Ultra Violet (UV) Spectrum Analysis for F6304 NPs,FegO4 / VBL NPs, Fe304 NF and Fe304 / VBL NF by using a UV-Visible spectrophotometer and aJoscoV-650, Japan is used to evaluate the absorption (200-800nm) spectra of the samplesaccording to an embodiment herein;

[0023] FIG; 7 illustrates a body weight of control and all treated animal (Fe304 / VBL NF)group according to an embodiment herein;4

[0024] FIG. 8 illustrates a decreased level of tumor in treated group compared to controlaccording to an embodiment herein;

[0025] FIG. 9 illustrates 3 showed Red blood cells (RBC), White blood cells (WBC) andHemoglobin (HB) level of control, only DAL, DAL+STD and DAL+Fe304 / VBL NF (250mg / Kgand 500mg / Kg) according to an embodiment herein;

[0026] FIG, I0 illustrates an effect of Fe304 / VBL NF on packed cell volume according toan embodiment herein;

[0027] FIG. ll illustrates an effect of Fegoa / VBL NF on serum biochemical parametersaccording to an embodiment herein;’

[0028] FIG. [2 illustrates an effect of Fe304 / VBL NF on antioxidant enzymes accordingto an embodiment herein.

[0029] FIG. l3 illustrates an effect of Fegoa / VBL NF on mean survival time (MST)according to an embodiment herein;

[0030] FIG; I4 illustration effect of Fe304 / VBL NF on a percentage of lifespan groupaccording to an embodiment herein;[003l] FIG. 15 illustrates a histopathological examination of control mice liver samplesaccording to an embodiment herein;

[0032] FIG. l6 illustrates a hislopalhological examination of Only DAL group accordingto an embodiment herein;

[0033] FIG. 17 illustrates a histbpathological examination of DAL+STD group accordingto an embodiment herein;4

[0034] FIG; 18 illustrates a histopathological examination of F6304 / VBL NF (250 mg / kg)group according to an embodiment herein;

[0035] FIG. 19 illustrates a histopathological examination of Fe3O4 / VBL NF (500 mg / kg)group according to an embodiment herein; and

[0036] FIG. 20 illustrates a process of preparing a nanofiber for biomedical ‘applicationaccording to an embodiment herein.DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0037] The embodiments herein and the various features and advantageous details thereofare explained more fully with reference to the non-limiting embodiments that are illustrated in theaccompanying drawings and detailed in the following description. Descriptions of well-knowncomponents and processing techniques are omitted so as to not unnecessarily obscure theembodiments herein. The examples used herein are intended merely to facilitate an understandingof ways in which the embodiments herein may be practiced and to further enable those of skill inthe art to practice the embodiments herein. Accordingly, the examples should not be construed aslimiting the scope of the embodiments herein.

[0038] As mentioned, there remains a need for a nanofiber and a process of preparing the'nanofiber for biomedical application. Referring now to the drawings, and more particularly toFIGS. 1 through 20, where similar reference characters denote corresponding features consistentlythroughout the figures, there are shown preferred embodiments.

[0039] FIG. I illustrates a schematic illustration of a nanofiber for biomedical application according to an embodiment herein. The nanofiber includes IOO milligram (mg) of Iron (II, ”Doxide (Fe304) nanoparticles, 500 mg ocilosan, a Poly(vinylpyrrolidone) (PVP) solution and 1mg of vinblasline (VBL). The F6304 nanopanicles are sonicated with |0 milliliters (ml) ofDimethylformamide (DMF) for about I hour to synthesize an uniform F6304 nanoparticlessolution. The chitosan is added dropwise to 2 milliliters (ml) of acetic acid and dissolved in doubledistilled water (DHzO), followed by stirring for about 24 hours to synthesize a chitosan solution.The Poly(vinylpyrrolidone) (PVP) solution is mixed with the chitosan solution and thencontinuously stirred for about l2 hours to synthesize a PVP-CS solution. The vinblastine is mixedto the F6304 nanoparticles solution to synthesize a Fe;O4-VBL solution. The F6304-VBL solutionis mixed with the PVP-CS solution and then continuously stirred at room temperature for about 12hours, followed by loading into an electrospinning machine system to synthesize a Fe304 nanofiberthat has improved drug delivery.

[0040] In an embodiment, the F6304 rganopanicles are prepared by (i) dissolving 2 g offerric chloride hekahydrate in 25 ml of deionized distilled water (DDW) and stirring the ferricchloride hexahydrate solution for about 1 hour, (ii) dissolving 2 g of ferrous sulfate heptahydratein 25 ml of distilled water and mixing it with the previously prepared ferric chloride hexahydratesolution to obtain a ferric chloride-ferious sulfate solution, (iii) continuously stirring the ferricchloride-ferrous sulfate solution for about 1 hour for maintaining homogeneity, (iv) heating theferric chloride-ferrous sulfide solution to 80 °C and maintaining the temperature for 30 minutes,followed by cooling the ferric chloride-ferrous sulfate solution to room temperature, and (v)gradually adding sodium hydroxide (NaOH) dropwise to the ferric chloride-ferrous sulfate solutionuntil reaching a pH of l l, and stirring the ferric chloride-ferrous sulfate solution for an additional'l hour to generate'the Fe304 nanoparticles.

[0041] In another embodiment, the ferric chloride-ferrous sulfate solution is washed withethanol two times to eliminate any remaining contaminants and dried the resulting ferric chlorideferroussulfate solution by subjecting it to an oven at a temperature range of 70 °C to 80 °C for an'overnight period. In yet another embodiment, the F6304‘nanoflber is dried at 60 °C for about 12hours afier synthesis. In yet another embodiment, the PVP-CS solution is continuously stirred forabout I2 hours before adding the Fe3Oa-VBL solution. In yetlanother’ embodiment, the F6304nanofiber is synthesized ‘by (i) mixing the F6304 nanoparticles with the PVP-CS solution and (ii)collecting and drying on aluminum foil from the mixture of PVP-CS solution and F6304nanoparticles solution by loading into a 20 ml plastic syringe at a fixed flow rate of l microliterper minute (pl / min), at a voltage of 15 Kilovolt (KV) that is set at a tip of the collector and acollector distance of 15 centimeter (cm). In yet another embodiment, the PVP-CS solution iscontinuously stirred for about [2 hours before adding the F6304 nanoparticles.

[0042] Superparamagnetic iron oxide nanoparticles (SPIONS) have unique magnetizationproperties, such as high magnetization values and superparamagnetism, which makes them idealas hypenhennia agents. In such a case, the efficacy of the treatment may be improved byconcurrently delivering anticancer drugs or biologically active molecules. One ofthe advantagesof using SPIONs as pulmonary drug delivery carriers is that the nanoparticles can be guided toaccumulate in the affected regions of the lungs by using an external magnetic field. SPIONs aremost commonly combined with polymers to enhance their efficacy in biomedical applications.Due to its higher solubility in acidic environments and non-toxic, hydrophilic, biocompatible, andbiodegradable properties, chitosan has been an important component of drug delivery systems(DDSs). Among the many beneficial properties of chilosan and its derivatives can be ascribed tothe presence of reactive functional groups, namely hydroxyl (-OH) and aminé (-NH2) groups. Achitosan composite is magnetically guided and conducted pulsatile VBL drug release fromchitosan composites using an external magnet.

[0043] Polyvinylpyrrolidone (PVP) possesses physicochemical features that make itacceptable for use in a variety of industries, including pharmaceutical, biomedical, cosmetic, andfood. PVP and chitosan interact through hydrogen bonds formed between the pyrrolidine rings ofPVP and [He amino and hydroxyl groups of chitosan, resulting in high material miscibility andimproved characteristics. When PVP is present, the miscibility of chitosan with hydrophilicpolymers like PVP is examined, and mechanical / physical and thermal properties are improved.The Polyvinylpyrrolidone (PVP) enhances the controlled drug releasement.

[0044] Vinblastine (VBL), a naturally occurring alkaloid extracted from Vinca rosea Linn,acts as an antioxidant and is also used in cancer therapies. Vinblastine is one oFthe most commonlyused vinca alkaloids (VBL). The VBL can prevent cell division by inhibiting mitosis, purine, andRNA synthesis. These properties cause the dividing cells to be destroyed quickly. VBL isincorporated with magnetic NPs into cationic liposomes based on the above hypotheses to reducetumour nodules and target the tumour vasculature.

[0045] 'Dalton‘ lymphoma is frequently used as an intriguing model for cancer researchbecause it functions as a preclinical system for assessing brand-new or established medications inthe treatment of various cancers. Ovarian, uterine, Cervical, colon, gastric, pancreatic, breast, andliver cancers are the most common cancers in which Dalton lymphoma (DL) ascites develops.Ascites may develop as a result of liver-related cancer spread (Koiri et al. 2017).

[0046] Nanofibers are exquisite materials used for enhancing biomedical applications liketarget drug transport, scaffolds in tissue engineering, and enzyme immobilization. Nanofibers havea high surface area and porosity in addition to their small fiber size, wound healing function andbiosensor. The delivery of chemotherapeutic herbal products and pills through nanofibers hasdemonstrated increased anticancer efficacy and decreased systemic toxicity in the treatment ofcancer.

[0047] Preparation of Fe304 / VBL NF: I00 mg F6304 NP is sonicated with DMF for Ihour to prepare a uniform solution. Then, 500 mg of chitosan is added dropwise to 2 ml of acelicacjd it is dissolved in double DHzO, and the mixture is stirred for 24 hours to obtain'a uniformsolution of chitosan. Then PVP is added in CS solution and the mixture is continuously stirfed for12 hours. Finally, Fe304 solution is added in PVP / CS solution and the mixture is continuouslystirred at room temperature for 12 hours. After stirring for 24 hours, 1 mg of VBL drug is alsoadded to the solution. Finally, F6304 / VBL solution is added to the PVP / CS solution and themixture is continuously stirred at room temperature for [2 hours. The mixture is loaded into anelectrospinning machine system and dried at 60°C for 12 hours.

[0048] The Fe304 / VBL nanofiber (NF) demonstrated in viva anticancer efficacy againstDaitons Ascites Lymphoma in mice, The lifeSpan of mice is increased to 70% of F6304 / VBL NFwhen compare to control by reducing the tumor cell and packed cell volume, normalizing the bodyweight, haematological profile, and serum biochemical parameters, and in-vivo cytotoxicity andanticancer activity of Fe3O4 / VBL NF on Daltons Ascites Lymphoma in mice.

[0049] The enzymatic and non-enzymatic antioxidant systems are improved in the 'groupgiven treatments with Fe304 / VBL NF. Increased levels oftotal protein, GSH and LPO showedtheir potential as an inhibitor of DLA-induced intracellular oxidative stress in mice treated withFesO4 / VBL NF. No significant changes observed in SOD, CAT and GPX showed their potential as an inhibitor of DLA-induced intracellular oxidative stress in mice treated with FCJOA / VBL NFas a result, mice treated with F6304 / YBL NF showed remarkable in viva antitumor activity againstDAL; Therefore, the F6301a / VBL NF safer fomulations of these materials improvebiocompatibility and can be used for multifunctional applications in future devices.

[0050] In viva antitumor activities in Swiss inbred female mice: Method-l: Themethod-l is used to determine mean survival time and weight ana|ysis. The specifications of theexperimental groups for F6304 / VBL are as follows:[005 I] Method-2: The method-2 is used to determine peritoneal tumor volume, tumorpacked cell volume, haematological, biochemical and histopathological studies for F6304 / VBLNF and F6304Ninblastine NF.

[0052] FIG. 2 illustrates an X-ray powder diffraction (XRD) Analysis of F6304 NPs,F6304 / VBL NPs, F6304 NF and FeaO4 / VBL NF by using the Rigaku Sman lab X-raydiffractometer (1.5406 A) with Cu Ku radiations according to an embodiment herein. FIG. 2shows the XRD patterns ofF6304 NPs, Fe3O4 / VBL NPs, Fe304 NF and Fe304 / VBL NF. The Fe304NPs diffraction patterns have six main characteristic peaks at 29 value is 30.l°, 355°, 425°,52.7°,57.|°,62.7° corresponding to crystal plane of (220), (3| I), (400), (422), (5| I), (440)respectively. The major peak at 26 value is 35.5" are indexed to (3| 1) plane that perfectly matcheswith magnetite plane of standard (JCPDS Card No. 89-0950). To verify the crystalline magnetitestructure is cubic phase to confirm the F6304 NPS. The XRD diffraction peak of Fe304 / VBL NPs,Fe3O4 NF and Fe3O4 / VBL NF shows the no change for phase structure and position of the allpeaks. However, the incorporated by polymer Fe304 / VBL NF does not have any effect on thecrystalline plane of Fe304 NPs. The F6304 / VBL NF is a lower peak intensity and crystallinitycompare to the F6304 NPs. To calculated the average particles size by Debye-Scherrer equation:D=Kcose. Where, D is the average particles size, K is a constant, A is the X-ray radiationwavelength in angstroms, B is the full width at half maximum (FWHM) and 9 is the angle of thepeak. The approximated average crystalline particles size for F6304 Nanoparticles is 12.4 nmrespectively. Therefore, this confirms the formation of Fe304 NPs, FC3O4 / VBL NPs, Fe304 NF andFe304 / VBL NF in XRD pattern._ [0053} FIG. 3 illustrates Field Emission Scanning Electron Microscopy (FESEM) imagesfor R304 NPs, F6304 / VBL NPs, Fe304 NF and F6304 / VBL NF at lpm and 500 nm ofmagnifications to obtain morphology images which is performed with Quanta-ZSO—FEG accordingto an embodiment herein. FIG. 3a and FIG. 3b shows the cubic and few fine grains shape for theF6304 nanoparticles. FIG. 3c and FIG. 3d shows the both cubic and spherical in the shape of-Fe304 / VBL nanopanicles. As shown in FIG. 3e-f, the surface of nanofiber is smooth withoutvisible beads, indicating all nanoparticles are wrapped inside the nanofibers. The homogeneousmorphology is also maintained with the addition of F6304 nanoparticles in incorporation bypolymer (F6304 NF) and FegOa / VBL NF. Therefore, this confirms the formation of nanofibers inboth F6304 NF and Fe304 / VBL NF by the electrospinning techniques.

[0054] FIG. 4 illustrates an Energy Dispersive X-ray (EDX) analysis for F6304, NPs,Fe304 / VBL NPs, Fe304 NF and Fegoa / VBL NF to obtain elemental compound which is performedwith Quanta-250-FEG according to an embodiment herein. In .FIG. 4a, F6304 NPs show thecharacteristic element both Fe and 0 present at 6 l .7 l % and 38.29% respectively. In FIG. 4b, showsthe F6304 / VBL element both Fe and 0 present at 61.06% and 38.94%. Comparatively, FIGS. 4aand 4b show the F6304 NPs and Fe304 / VBL NPs lo evaluated the elemental éomposition is afierloaded the VBL béth Fe is decreases and O is increases in the FezO4 / VBL NPs. The EDX spectrumof (F6. 4c) F6304 NF and (FIG. 4d) Fezoa / VBL NF shows corresponding Fe, C and O atoms ofR304. But the C% of F6304 NF and Fe304 / VBL NF is lower abofit 3.2.2|%, [8.79% and 49% respectively. This can be due to the presence of the VBL drug used in the NF fortification. Hence,this confirms the atomic weight percentage ofthe elements present in the samples.

[0055] FIG. 5 illustrates a Fourier transform infrared (FTIR) spectrum for Fe304 NPs,F6304 / VBL NPs, Fe304 NF and Fe304 / VBL NF by using the Bruker’ Tensor 27 according to Anembodiment herein. FIG. 5 depicts the FTIR spectra of Fe304 NPs, Fe3O4NBL NPs, Fe304 NFand Fe;O4 / VBL NF are in the 400-4000 cm" region. The absorption spectrum for Fe304 has peaksat 583 cm" (the Fe-O stretching vibration mode), 622 cm" (the C-Br stretching vibration mode),I I IS cm'l (the C- O stretching vibration mode) and 3637 cm'l (the O-H stretching vibration mode)..The peak at 1672 cm" and 2689 cm" corresponds to the stretching C=0 and OH stretchingvibration for overlapping of methyl, methylene and -CH groups in Fe304 loaded by Vinblastinedrug inside the nanopanicles respectively. The bands at l 125 cm’l and |559 cm'I are related to theC-0 stretching vibration mode of C-OH and the bending vibration of—NHZ groups of chitosan inF6304 NF. The C-N bending stretching of polymer and C-H overlapping stretching groups ofVinblastine in Fe304 / VBL NF. The FTIR spectrum for F6304 / VBL NF shows no shift inwavenumber or appearance of new peaks are presence, indicating tha! chemical and druginteraction does not occur between the adsorbent.

[0056] FIG; 6 illustrates an Ultra Violet (UV) Spectrum Analysis for F6304 NPs,FCJO4 / VBL NPs, F6104 NF and F6304 / VBL NF by using a UV-Visible spectrophotometer and a,JoscoV-650, Japan is used to evaluate the .absorption (200-800nm) spectra of the samplesaccording to an embodiment herein. As shown in FIG. 6, the UV spectra obtained for FégOa NPs,Fe304 / VBL NPs, F6304 NF and Fe;O4 / VBL NF is visible at regions from 200 to 800 nm. As shownin FIG. 6, the absorption peak at 270 nm is due to the clue of F6304 NPs may be formed. Theabsorption peak is shifted at 250 nm and the absorption intensity is decreased because of iron oxideloaded by VBL drug in the Fe304 / VBL NPs. The position and absorption peak are shifted From230 nm may be due to the colloidal solution of nanofibers in F940: incorporated by polymer inF6304 NF. The peak of F6304 / VBL NF shifted at 260 nm and the absorption intensity wasincreased compared to other saniples because of iron oxide nanoparticles incorporated by polymerloaded with VBL drug to the colloidal solution of nanofibers to confirms the formation of F6304Nanofibers.

[0057] FIG. 7 illustrates a body weight ofcontrol and all treated animal (F6304 / VBL NF)group according to an embodiment herein. As shown in FIG. 7, the values represented as mean i SD of initial body weight and final body weight. The experimental data are performed by the OnewayAnalysis of Variance (ANOVA) followed by Dunnett comparison. lnitial body weight ofDAL+ Fegoa / VBL NF (500mg / kg) group compared to final body weight of DAL+ Fé304 / VBLNF (500mg / kg) group (FIG. 7). Results showed more are less similar. High body weight showedin final body weight DAL+ F6304 / VBL NF (250mg / kg) group compared with Initial body weightof DAL+ F6304 / VBL NF (250mg / kg) group. DAL+ Fe304 / VBL NF (250mg / kg and SOOmg / kg)group compared with initial and final body weight of control, only DAL, DAL+STD group. In this.group no significant change of body weight observed. Only DLA group showed high level of finalbody weight (32.8il0.4). Its levels compared to DAL+ Fe3O4 / VBL NF (250mg / kg and500mg / kg). The composite reacts with the camper induced animal (only DAL), afier 14 daystreatment body weight to be reduced, respectively (232t7.33, 2 | .72k4.4). The result to be consideredstatistically significant when the "P<0.0 / .‘

[0058] FIG. 8 illustrates a decreased level of tumor in treated group compared to controlaccording to an embodiment herein. FIG. 8 shows that the antitumor activity of the (DAL+Fe304 / VBL NF 250mg / Kg and DAL+S| L.D 500mg / Kg) compared with only DAL group andDAL+STD group, The results showed decreased amount of tumor in DAL+ Fe304 / VBL N_F(250mg / Kg and 500mg / Kg) respectively (5.7i2.2 and 4.8:tl.3|) compared to only DAL(l3.7i4.42). The result to be considered statistically significant when the **P<0.0I.,

[0059] FIG. 9 illustrates a showed Red blood cells (RBC), White blood cells (WBC) andHemoglobin (HB) level of control, only DAL, DAL+STD and DAL+FegOa / VBL NF (250mg / Kgand 500mg / Kg) according to an embodiment herein. The collected blood is stored in 4°C.Followed by blood parameter checked in haemocjtometer (RBC, WBC and HB‘ level). DAL+Fe304 / VBL NF (250mg / Kg and 500mg / Kg) (9.37:t0.26 and 9.33i0.667) and DAL+STD(9.8i0524) group showed count ofWBC, more are less similar to Control, only DAL respectivelylO.|i0.896, 8.471057. DAL+ Fe304 / VBL NF (250mg / Kg and 500mg / Kg) (4.25i0.073l and4.54i'0.l8) and DAL+STD (4.84t0.l7l) group showed count of RBC, more are less similar toControl, on|y DAL respectively 5.0lztO.l07, 4.5i0.243. DAL+ Fe304 / VBL NF (250mg / Kg am;500mg / Kg) (I l.9t0.296 and 12.4:t0.4|6) and DAL+STD (l2.6:!:0.33) group showed count ofHB,more are less similar to Control, only DAL respectively 12.710.669, l|.9i0.458. In these; resultsindicates no changes observed the all groups. The result to be considered statistically significantwhen the **P<0.0I.

[0060] FIG. 10 illustrates an effect ofF6304 / VBL NF on packed cell volume according toan embodiment herein. The values represented as mean 1- SD of packed cell volume. Theexperimental data are performed by the One—way Analysis of Variance (ANOVA) followed byDunnett comparison. FIG. IO shows the result of packed cell volume compared between only DAL}and Fe304 / VBL NF (250mg / kg and 500mg / kg) concentratibn. Only DAL group having highpacked cell_ volume (3.03i0.203) compared to FezO4 / VBL NF V(250mg / kg and 500mg / kg)concentration respectively (2.63i0.066 and 2.07d:0.3l8). Packed cell volume compared betweenonly DAL group and DAL+STD drug (FU). Only DAL group having high packed cell volume(3.03i0.203) compared to DAL + STD (2.5:i:0.08). Packed cell volume of DAL+STD and DAL+Fe;O4 / VBL (250mg / kg and 500mg / kg) concentration group have fiimilar results. The result to beconsidered statistically significant when the **P<0.01,‘[006l] FIG. ll illustrates an effect of F6304 / VBL NF.on serum biocherfiical parametersaccording to an embodiment herein. The serum biochemical parameters such as (liver markerenzymes), Serum Glutamate Pyruvate Transaminase (SGPT), Glutamate Oxalo AcetateTransaminase (SGOT) and Alkaline Phosphate (ALP) activities observed in treated mice. As~ shown in FIG. ll, SGPT value of DAL+ FegOMVBL NF (250mg / Kg: 36.9i0.981) compared toonly DAL and control respectively (66.3il.5, 46.91:].01). Reduced SGPT value of DAL+Fe;O4 / VBL NF (250mg / K'g) observed in treated mice. SGPT value of DAL+ Fegoa / VBL NF(500mg / Kg: 1848i0.549) compared to only DAL and control respectively (66.3115, 46.9:tl.0l).‘Reduced SGPT value of DAL+ Fe304 / IVBL NF (500mg / Kg) observed in treated mice; In500mg / kg concentration have been very low level of SGPT value compared to 250mg / kgconcentration. SGOT value of DAL+ F6304 / VBL NF (250mg / Kg: |38i0.635) compared to onlyDAL and control respectively (l73zt2.l7, [21i0.346). Reduced SGOT range observed in250mg / Kg concentration compared to only DAL. Elevated level of SGOT range observed incontrol compared with 250mg / kg concentration. SGOT value of DAL+ Fe304 / VBL NF(500mg / Kg: 95.2:t0.l89) compared to only DAL and control respectively (l73:t2. l7, 121i0.346).Reduced SGOT range observed in 500mg / Kg concentration compared to only DAL and control.250mg / kg concentration have very high SGOT range compared to 500mg / kg. ALP results showed DAL+ F6304 / VBL NF (250mg / Kg: I29il‘96) compared to control and only DAL respectively(l24i0.907, 22lil.18). 250mg / kg concentration have low ALP value compared with only DALand thus value higher in compared to control. ALP results showed DAL+ Fe304 / VBL NF (500mg / Kg: |80i0.953) compared to control and only DAL respectively (l24:t0.907, 22li|.18).500mg / kg concentration have low ALP value compared with only DAL and thus value higher incompared to control. 500mg / kg concentration have very high ALP range compared to 250mg / kg.Approximately level of SGPT, SGOT and ALP are indicating of impaired liver functions due tocancer. The significantly almost level of SGPT, SGOT and ALP in the serum of tumor -inoculatedanimals indicated liver damage and loss of functional integrity of the cell membrane. Treatmentwith F6304 / VBL NF (250mg / Kg and 500mg / Kg) body weight restored the above - mentionedparameters to a normal level. The result to be considered statistically significant when the**P<0.0l.I

[0062] FIG. [2 illustrates an effect of F6304 / VBL NF on antioxidant enzymes accordingto an embodiment herein. The antioxidant enzymes activities of liver marker such as Total Protein(TP), Superoxide Dismulase (SOD), Calalase (CAT), Glutathione Peroxidises (GPX), ReducedGlutathione (GSH) and Lipid Peroxidation (LPO) seen in oxidative stress induced treated mice.Only DAL (l.|7i0.106) group having high level of total protein compared to Control(0.69i0.0242), DAL+STD (0.72:0.0219), F6304 / VBL NF (250mg / kg:0.952i0.0208 and500mg / kg:0.718:|:0.0l73). No significant changes observed in SOD values t6 all groups such asControl (0.]52i0.00346), Only DAL (0.402zh0.00549), DAL+STD (0.197i0.0052), Fe304 / VBLNF (250mg / kg:0.369:t0.0075| and 500mg / kg:0.24i0,0023l). No significant changes observed incatalysis 'values to all groups such as Control (0.497i0.00866), Only DAL (0.24i0.00346),DAL+STD (0.36i0.0098 l ), F6304 / VBL NF (250mg / kg:0.227i0.00664 andSOOmg / kg:0.336:t0.0277). No significant changes observed in GPX values to all groups such asControl (0.048i0.00346), Only DAL (0.086zt0.001 I5), DAL+STD (0.04H:0.00l73), F6304 / VBLNF (250mg / kg:0.0903i0.00433 and 500mg / kg:0.0583:l:0.00l45). Only DAL (0.]08i0.00577),Fe304 / VBL NF (250mg / kg:0. l49i0.00404) groups having high level ofGSH compared i0 Control(0.0263i0.00l45), DAL+STD (0.075i0.0|9|), Fe304 / VBL NF (500mg / kg:0.067:t0.0225). OnlyDAL (0.329t0‘00751) group having high level of LOP compared to COntrol (0.]04t0.00664),DAL+STD (0.| l9:|:0.02 I 4), F6304 / VBL NF (250mg / kg:0.52i0.00808 and500mg / kg:0£l39:h0.00404). As a result, Fe;O4 / VBL NF treatment improved both the enzymaticand non-enzymatic antioxidant systems. The result to be considered statistically significant when the *P<0.05.

[0063] FIG. 13 illustrates an effect of Fe304 / VBL NF on mean survival time (MST)according to an embodiment herein. The values represented as mean survived time (MST) ofinitialbody weight and final body weight. Initial body weight of DAL+ Fe3O4NBL NF (500mg / kg:23.8i0.307) group compared to final body weight of DAL+ Fe3O4 / VBL ~NF (500mg / kg:23.2:h4.66) group. After treatment measurement of body weight results showed to be equal. Highbody weight showed ih final body weight DAL+ Fe304 / VBL NF (250mg / kg: 26.8i5.46) groupcompared with Initial body weight ofDAL+ Fe3O4NBL NF (250mg / kg:24.3:t0.33) group. DAL+Fe;O4 / VBL NF (250mg / kg and 509mg / kg) group compared with mean survived time of final bodyweight of only DAL, DAL+STD group. Changes of body weight observed in this group. OnlyDLA group showed high level of final body weight (33.3i10.7). Its levels compared to DAL+STDand DAL+ Fe304 / VBL NF (250mg / kg and 500mg / kg). Our composite reacts with the cancerinduced animal, afier l4days treatment body weight to be reduced, respectively (2|.7dz4.34,26.8:t5,46, 23.2i4.66). The result to be considered statistically significant when the **P<0.01.

[0064] FIG. l4 illustrates an effect of Fegoa / VBL NF on a percentage of lifespan groupaccording to an embodiment herein. Only DAL group having very low (l7.7:b0.88) mean survivaltime (MST) compared to DAL+-STD, DAL+ FegO4 / VBL NF (250mg / kg and 500mg / kg)respectively 21.711.45, 43.3i0.882***, 3|.3il.76. Highest level of MST showed in group [II'(DAL+ Fe3O4 / VBL NF: 250mg / kg) concentration. After 2| days treatment MST increased in alltreated and STD group compared to only DAL. The result to be considered statistically significantwhen the **l’<0.0 / ,

[0065] FIG. IS illustrates a histopathological examination of control mice liver samplesaccording to an embodiment herein. It is viewed under the microscope IOX and 40Xmagnifications. The histopathological studies ofthe tissues ofthe antitumor activity area treatedwith the Fe3O4 / VBL NF (250mg / kg and 500mg / kg) treated groups, control, only DAL and DAL+5Fluro Uracil was used as a standard drug treated groups were performed on the liver measuring2.6xl.4x0.8cms and‘histopalhological examinations of animal tissue; Histopathology is themicroscopic examination of tissues. FIGS. lS(a-i) of control animals with IOX and 40Xmagnification show the various histopathological chahges that occurred in the control groups.Individual hepatocytes show no significant pathology, the portal triad shows bile duct hyperplasia,and the central vein and sinusoids show no significant pathology.

[0066] FIG. l6 illustrates a histopathological examination of Only DAL group accordingto an embodiment herein. It is viewed under the microscope IOX and 40X magnifications.Individual hepatocyles in FIGS. 16(a-j) of only QAL animals with 10X and 40X magnificationexhibited with the altered Iobular architecture show moderate eosinophilic cytoplasm, moderate tomarked pleomorphic hyperchromatic nuclei, and inconspicuous nucleoli. Parenchyma alsoexhibits diffuse inflammatory infiltrates, portal triad exhibits normal morphology, Central veinexhibits congestion, and sinusoids exhibit mild;

[0067] FIG. l7 illustrates a histopathological examination of DAL+STD group accordingto an embodiment herein. It is viewed under the microscope IOX and 40X magnifications. FIGS.|7(a-j) of DAL+STD animals with lOX and 40X magnification showed mild loés of architecture,individual hepatocytes showed interface hepatitis and mild reactive atypia, portal triad showed nosignificant pathology, central vein showed congestion and dilatation, and sinusoids showed milddilatation.

[0068] FIG. [8 illustrates a histopathological examination of F6304 / VBL NF (250 mg / kg)group according to an embodiment herein. It is viewed under the microscope IOX and 40Xmagnifications. FIGS. l8(a-i) shows that animals exposed to Fegoa / VBL NF (250 mg / kg) hadaltered Iobular architecture and interface hepatitis. Individual hepatocytes exhibit focalparenchymal necrosis, cytoplasmic vacuolation, and no discernible pathology is visible in theportal triad. Sinusoids and the central vein both exhibit mild dilatation.

[0069] FIG. [9 illustrates a histopathological examination of Fe304 / VBL NF (500 mg / kg)group according to an embodiment herein. It is viewed under the microscope IOX and 40Xmagnifications. FIGS. l9(a-j) demonstrates that animals exposed to F6304NBL NF (500 mg / kg)had mild architecture loss and interface hepatitis. Binucleation is visible in individual hepatocytes,the portal triad is normal, the central vein is dilated and congested, and the sinusoids are dilated.

[0070] FIG. 20 illustrates a process of preparing a nanofiber for biomedical applicationaccording to an embodiment herein. At a step 2002, a Fe304 nanoparticles solution is prepared by adding 500 mg of chitogan dropwise to 2 milliliters (ml) of acetic acid and dissolving in doubledistilled water (DHzO), followed by stirring for about 24 hours. At a step 2006, the chitosansolution is mixed with a Poly(vinylpyrrolidone) (PVP) solution and then continuously stirring forabout 12 hours to synthesize a PVP-CS solution. At a step 2008, Fe304 nanofiber is synthesizedby mixing I mg of vinblastine (VBL) to the F6304 nanoparticles solution to synthesize a Fe304-VBL solution. The Fegoa-VBL solution is mixed with the PVP-CS solution and then continuouslystirred. at room temperature for about 12 hours, followed by loading into an electrospinningmachine system to synthesize a F6304 nanofiber that has improved drug delivery.ADVANTAGES OF THE PRESENT INVENTION[007l] Vinbiastine-loaded iron oxide (Fe304) can be an eco-friendly and can be usedspecifically- for drug delivery and cancer applications. Therefore, nanoparliqles / nanofibercomposites with nanoparticles bonded with nanoflbers offer a brand-new concept for destinynanopanicle utility and a likely answer for side-results resulting from nalural nanuparliclcs.Nanofibers is an excessive level region and porosity, in addition to small fiber sjze, nanofibers areexquisite substances used for boosting biomedical program's like goal drug transport, scaffolds intissue engineering, enzyme immobilization. wound restoration activity, and biosensor. Nanofibers / Nanoparticles composites mediated transport of chemotherapeutic herbal merchandise and pillshas verified improved anticancer efficacy and decreased systematic toxicity in most cancersremedy.

[0072] The foregoing description of the specific embodiments will so fully reveal thegeneral nature oft embodiments herein that others can, by applying current knowledge, readily- modify and / or adapt for various applications such Specific embodiments without departing fromthe generic concept, and, therefore, such adapt'ations and modifications should and are intended tobe comprehended within the meaning and range of equivalents oflhe disclosed embodiments. Itis to be understood that the phraseology or terminology employed herein is for the purpose ofdescription and not oflimitation. Therefore, while the embodiments herein‘have been described interms of preferred embodiments, those skilled in the art will recognize that the embodiments hereincan be practiced with modification within the scope of the appended claims.

Claims

CLAIMSI / We claim:I. A nanofiber for biomedical application, comprising:100 milligram (mg) of Iron (II, III) oxide (F6304) nanoparticles, wherein the F6304nanoparticles are sohicated with IO milliliters (ml) of Dimethylformamide (DMF) for about 1 hourto synthesize an uniform F6304 nanopanicles solution;500 mg of chitosan, wherein the chitosan is added dropwise to 2 milliliters (ml) of aceticacid and dissolved in double distilled water (DHzO), followed by stirring for about 24 hours tosynthesize a chilosan solution;a Poly(vihylpyrrolidone) (PVP) solution, wherein the PVP solutiqn is mixed with thechitosan solution and then continuously stirred for about l2 hours to synthesize a PVP-CS solution;characterized in that, 1 mg of vinblasline (VBL), wherein the vinblastine is mixed to theF6304 nanoparticles solution to synthesize a F6304-VBL solution, wherein the F6304-VBL solutionis mixed with the PVP-CS solution and then continuously stirred at room temperature for about 12hours, followed by loading into an electrospinning machine system to synthesize a F6304 nanofiberthat has improved drug delivery.

2. The nanofiber as claimed in claim I, wherein the F6304 nanoparticles are prepared by (i)dissolving 2 g of ferric chloride hexahydrate in 25 ml of deionized distilled water (DDW) andstirring the ferric chloride hexahydrate solution fdr about I hour, (ii) dissolving 2 g of ferroussulfate heptahydrate in 25 ml of distilled water and mixing it with the previously prepared ferricchloride hexahydrate solution to obtain a ferric chloride-ferrous sulfate solution, (iii) continuouslystirring the ferric chloride-ferrous sulfate solution for about | hour for maintaining homogeneity,(iv) heating the ferric chloride-ferrous sulfate solution to 80 °C and maintaining the temperaturefor 30 minutes,» Followed by cooling the ferric chloride—ferrous sulfate solution to roomtemperature, and (v) gradually adding sodium hydroxide (NaOH) dropwise to the ferric chlorideferroussulfate solution until reaching a pH of l I, and stirring the ferric chloride-ferrous sulfatesolution for an additional 1 hour to generate the F6304 nanoparticles.

3. The nanofiber as claimed in claim I, wherein the ferric chloride-ferrous sulfate solution iswashed with ethanol two times to eliminate any remaining contaminants and dried the resultingferric chloride-ferrous sulfate solution by subjecting it to an oven at a temperature range of70 °Cto 80 °C for an overnight period.

4. The nanofiber as claimed in claim I, wherein the F6304 nanofiber is dried at 60 °C for about [2hours after synthesis.

5. The nanofiber as claimed in claim I, wherein the PVP-CS solution is continuously stirred forabout 12 hours before adding the Fe304-VBL solution.‘6.The nanofiber as claimed in claim 1, wherein the F6304 nanofiber is synthesized by (i) mixingthe Fe304 nanopanicles with the PVP-CS solution and (ii) collecting and drying on aluminum foilfrom the mixture of PVP-CS solution and F6304 nanopanicles solution by loading into a 20 mlplastic syringe at a fixed flow rate of | microliter per minute (pl / min), at a voltage of IS Kilovolt(KV) that is set at a tip of‘the collector and a collector distance of 15 centimeter (cm).74 The nanofiber as claimed in claim 6, wherein the PVP-CS‘solution is continuously stirred forabout l2 hours before adding the F6304 nanoparticles.

8. A process of preparing a nanofiber for biomedical application, comprising:preparing 31:6304 nanoparticles solution by sonicating |00 milligram (mg) of Iron (II, III)oxide (F6304) nanoparticles with IO milliliters (ml) of Dimethylformamide (DMF) for about Ihour;preparing chitosan solution by adding 500 mg of chitosan dropwise to 2 milliliters (ml) ofacetic acid and dissolving in double distilled water (DHzO), followed by stirring for about 24hours;mixing the chitosan solution With a Poly(vinylpyrrolidone) (PVP) solution and thencontinuously stirring for about 12 hours to synthesize a PVP-CS solution; and synthesizing F6304 nanofiber by mixing l mg of vinblastine (VBL) to the F6304nanopanicles solution to synthesize a F6304-VBL solution, wherein the Fe304-VBL solution is mixed with the PVP-CS solution and then continuously stirred at room temperature for about |2