A nanoformulation for management of lung cancer and a method of preparation thereof

Abstract

The present invention discloses a nano-formulation comprising of a nanocrystal of a drug, coated with a lipid layer, characterized in a highly stable form of lyophilized dry powder for inhalation that exhibits deep lung deposition potential for pulmonary administration and a method of preparation thereof. Further, the present invention provides a nano-formulation that exhibits properties such as high solubility, high permeability, and high stability owing to the nano range size and increased effective surface area of the lipid-coated nanocrystal. Prepared nano-formulation can be used for pulmonary administration and management of lung cancer owing to good aerosolization performance and high tumor penetration potential. Figure 1 on sheet no. 1 of the drawings may accompany the abstract when published.

Description

FIELD OF THE INVENTIONThe present invention relates to the field of nanotechnology. More particularly,the present invention relates to a nano-formulation of anticancer drug for themanagement of lung cancer that exhibits a higher drug payload and lowexcipient amount as well as releases the drug in a sustained manner along withthe method of preparing the same.BACKGROUND OF THE INVENTIONRecent advancements in combinatorial chemistry and drug design have led todevelopment of drugs that are water-insoluble or hydrophobic, have a highmolecular weight and lipophilicity. Major drawbacks reported in drugdevelopment correspond to the insoluble or hydrophobic nature of the drugsthat result in poor drug bioavailability, non-selective drug distribution andclinical in-efficacy.Nanocrystals are crystalline particles having at least one dimension measuringless than 1000 nm. Due to nano-sizing, nanocrystals have an increased surfacearea to volume ratio resulting in improved dissolution rates. In addition,nanocrystals exhibit properties such as high payload, solubility, permeabilityand stability. Depending on the production technology, nanocrystals ofdifferent shapes and sizes are produced. There are three basic principles usedfor the production of nanocrystals: milling, precipitation methods,homogenization methods and a combination thereof. Nanocrystal formulationis a versatile way of salvaging drugs that are water-insoluble or hydrophobicand equipping them with a carrier free delivery system that improves theiroptimal bioavailability.KR102612705 discloses a cosmetic composition including fucoidan-supportednanoparticles, a manufacturing method thereof wherein the cosmeticcomposition is a drug delivery system after extracting fucoidan that exhibitsincreased antioxidant efficacy through hydrogel nanoparticles (Nano-gel) andpromotes the absorption of fucoidan by meeting the lipid layer of the skin,thereby contributing to skin soothing and increasing efficacy. However, thecited document fails to disclose a nanocrystal exhibiting high dissolution rate,permeability and aerosolization along with the stabilizing property of fucoidan.US20220233576 discloses a method of identifying a subject who is susceptibleto treatment for cancer with lipid-based nanoparticle therapies wherein themethod comprises of determining expression levels of solute carrier family 46member 3 (SLC46A3) in a plurality of cancer cells from a subject having cancerand treating a subject with decreased SLC46A3 expression levels as comparedto historical controls with lipid-based nanoparticles. However, the citeddocument fails to disclose a lipid-based nanoparticle in the form of lyophilizedpowder along with the stabilizing property of fucoidan ensuring sustainedrelease of active ingredient for pulmonary administration.EP3628335B1 discloses a composition for use in a method of treating a diseasein a subject that is due to a deficiency of a pulmonary surfactant-associatedprotein comprising at least one mRNA molecule at least a portion of whichencodes the pulmonary surfactant-associated protein; and a transfer vehiclecomprising a lipid nanoparticle, wherein the lipid nanoparticle comprises oneor more cationic lipids, one or more non-cationic lipids and one or more PEGmodified lipids. However, the cited document fails to disclose fucoidan as acomponent of the nanoparticle that confers stabilizing properties.In view of the above, the existing state of the art provides nanoparticleformulations and the method of preparation thereof. However, there are certainlimitations that the existing prior art fails to address, that includes high load ofexcipients in the formulations, lower dissolution rate, reduced permeability andreduced stability thereby limiting the bioavailability of the drug and thuscompromising their clinical efficacy.Therefore, there is a need of nano-formulations comprising of lipid-coatednanocrystals that have higher drug payload and low excipient amount that isformulated in the form of lyophilized dry powder resulting in improvedstability, higher aqueous solubility, permeability and improved aerosolizationperformance.OBJECT OF THE INVENTIONThe main object of the present invention is to provide a nano-formulation forthe management of lung cancer and a method of preparation thereof.Another object of the present invention is to provide a nano-formulation in theform of lyophilized dry powder for inhalation.Yet another object of the present invention is to provide a nano-formulation forsustained release of the drug in the lungs.Yet another object of the present invention is to provide a nano-formulationexhibiting a higher drug payload and low excipient amount.Still another object of the present invention is to provide a nano-formulationexhibiting higher aqueous solubility, improved permeability, higher flowability,and good aerosolization performance, compared to pure drug (BTB).SUMMARY OF THE INVENTIONThe present invention relates to a nano-formulation for sustained release of adrug in the lungs, exhibiting higher aqueous solubility, improved permeability,high flowability and better aerosolization performance for pulmonaryadministration and effective management of lung cancer and a method ofpreparation thereof.In an embodiment, the present invention provides a nano-formulation for themanagement of lung cancer comprising of a nanocrystal and a plurality of lipidsin a defined ratio. Here, said nanocrystal includes a drug and a stabilizer; saiddrug is a kinase inhibitor, the plurality of lipids include hydrogenated soyphosphatidylcholine (HSPC), cholesterol and stearylamine in a ratio of60:30:15, the nanocrystal is coated with a layer of the plurality of lipids and thedefined ratio of the nanocrystal and the plurality of lipids is in a range of 1:1.In another embodiment, the present invention provides a method ofpreparation of a nano-formulation comprising the steps of: (a) adding 1-2 mLof bosutinib ethanolic solution to 10 mL of aqueous fucoidan solutionin icecold water at a 4-8°C to form a colloidal solution followed by continuousstirring of said colloidal solution at 2000-6000 rpm to form a dispersion; (b)subjecting said dispersion obtained in step (a) to probe sonication for 10minutes at pulse rate of 07 / 03 and amplitude of 60% at 4°C in ice cold water toform a formulation; (c) stirring said formulation obtained in step (b) at 200-600rpm for 15 to 30 minutes at 25°C to form a homogenous formulation followedby centrifuging said homogenous formulation at a temperature of -4°C at 15000rpm for a time duration of 15-30 minutes to form nanocrystals; (d) collectingand lyophilizing said nanocrystals obtained in step (c) to obtain a powderfollowed by desiccating said powder for 24-48 hours to form a dry powder; (e)mixing 60 mg of HSPC, 30 mg of liquid chloesterol (Chol), 15 mg of tocopherolmethoxypolyethylene glycol succinate (TPGS) and 15 mg of stearylamine toform a thin film; (f) adding 100 mg of said dry powder obtained in step (d) to10 mL of water to form a mixture; (g) adding said mixture obtained in step (f)to said thin film obtained in step (e) to form a rehydrated film; (h) subjectingsaid rehydrated film obtained in step (g) to sonication for 15-30 minutes toobtain a suspension followed by passing said suspension through manualextruder to form lipo-nanocrystal; (i) stirring said lipo-nanocrystal obtained instep (h) on ice cold water to obtain uniformly formed lipo-nanocrystal; and (j)centrifuging of said lipo-nanocrystals to obtain sediments followed bylyophilization to obtain said nano-formulation as dry powder.The present invention relates to a nano-formulation for the management of lungcancer and a method of preparation thereof wherein the nano-formulationcomprises of a lipid-coated nanocrystal formulated in the form of lyophilizeddry powder that exhibits high aqueous solubility, permeability, flowability andaerosolization resulting in increased tumor penetration efficacy and deep lungdeposition for pulmonary administration.The above objects and advantages of the present invention will becomeapparent from the hereinafter set forth brief description of the drawings,detailed description of the invention, and claims appended herewith.BRIEF DESCRIPTION OF THE DRAWINGSAn understanding of a nano-formulation for management of lung cancer and amethod of preparation thereof of the present invention may be obtained byreference to the following drawings:Figure 1 is a schematic representation of preparation of lipid coated bosutinibnanocrystals, according to an embodiment of the present invention.Figure 2 is a pictorial representation of preparation of microscopic investigationof fucoidan-stabilized (BTB-NC) and lipid-coated (Lipo / BTB-NC)nanocrystals, according to the present invention.Figure 3 is a pictorial representation of spectroscopic characterization ofprepared fucoidan-stabilized (BTB-NC) and lipid-coated (Lipo / BTB-NC)nanocrystals, according to the present invention.Figure 4 is a pictorial representation of in-vitro drug dissolution andaerosolization performance analysis using Eight-stage Anderson CascadeImpactor, according to the present invention.DETAILED DESCRIPTION OF THE INVENTIONThe present invention will now be described hereinafter with reference to theaccompanying drawings in which a preferred embodiment of the invention isshown. This invention may, however, be embodied in many different formsand should not be construed as being limited to the embodiment set forthherein. Rather, the embodiment is provided so that this disclosure will bethorough, and will fully convey the scope of the invention to those skilled inthe art.The present invention now will be described hereinafter with reference to thedetailed description, in which some, but not all embodiments of the inventionare indicated. Indeed, the invention may be embodied in many different formsand should not be construed as limited to the embodiments set forth herein;rather, these embodiments are provided so that this disclosure will satisfyapplicable legal requirements. Like numbers refer to like elements throughout.The present invention is described fully herein with non-limiting embodimentsand exemplary experimentation.The present invention provides a nano-formulation for sustained release of adrug in the lungs, characterized in high dissolution rate, improved permeabilityresulting in improved tumor penetration efficacy, better flow and aerosolizationperformance, wherein the nano-formulation is in the form of lyophilized drypowder for pulmonary administration and effective management of lungcancer.In a preferred embodiment, the present invention provides a nano-formulationcomprising of a nanocrystal; and a plurality of lipids in a defined ratio. Here,said nanocrystal includes a drug and a stabilizer; said drug is a kinase inhibitor;said plurality of lipids include hydrogenated soy phosphatidylcholine (HSPC),cholesterol and stearylamine in a ratio of 60:30:15; said nanocrystal is coatedwith a layer of said plurality of lipids; and said defined ratio of said nanocrystaland said plurality of lipids is in a range of 1:1.Here, the drug is bosutinib in a concentration range of 10 to 20 mg / ml inorganic phase for nanocrystal preparation, the stabilizer is fucoidan in aconcentration range of 0.5 - 1% (w / v) for nanocrystal preparation, the nanoformulation is in the form of a liquid nanocrystals and as lyophilized drypowder. Further, the nano-formulation is a cube to spherical with size rangingfrom 250 to 300 nm, the nano-formulation has a drug content of 20-25%, ispermeable in a range of 80 to 95%, exhibits a flowability in a range of 23 to 29°,disperses in a range of 54 to 60%. Moreover, the drug is released in a sustainedmanner at a rate of 60 to 90% in 12 to 48 hours.In another preferred embodiment, the present invention provides a method ofpreparation of a nano-formulation comprising the steps of: (a) adding 1-2 mLof bosutinib ethanolic solution to 10 mL of aqueous fucoidan solutionin icecold water at a 4-8°C to form a colloidal solution followed by continuousstirring of said colloidal solution at 2000-6000 rpm to form a dispersion; (b)subjecting said dispersion obtained in step (a) to probe sonication for 10minutes at pulse rate of 07 / 03 and amplitude of 60% at 4°C in ice cold water toform a formulation; (c) stirring said formulation obtained in step (b) at 200-600rpm for 15 to 30 minutes at 25°C to form a homogenous formulation followedby centrifuging said homogenous formulation at a temperature of -4°C at 15000rpm for a time duration of 15-30 minutes to form nanocrystals; (d) collectingand lyophilizing said nanocrystals obtained in step (c) to obtain a powderfollowed by desiccating said powder for 24-48 hours to form a dry powder; (e)mixing 60 mg of HSPC, 30 mg of liquid chloesterol (Chol), 15 mg of tocopherolmethoxypolyethylene glycol succinate (TPGS) and 15 mg of stearylamine toform a thin film; (f) adding 100 mg of said dry powder obtained in step (d) to10 mL of water to form a mixture; (g) adding said mixture obtained in step (f)to said thin film obtained in step (e) to form a rehydrated film; (h) subjectingsaid rehydrated film obtained in step (g) to sonication for 15-30 minutes toobtain a suspension followed by passing said suspension through manualextruder to form lipo-nanocrystal; (i) stirring said lipo-nanocrystal obtained instep (h) on ice cold water to obtain uniformly formed lipo-nanocrystal; and (j)centrifuging of said lipo-nanocrystals to obtain sediments followed bylyophilization to obtain said nano-formulation as dry powder.Further, said dry powder in step (d) was stored in a sealed container at atemperature range of 2-8°C and said nano-formulation was stored in a sealedcontainer at a temperature range of -8 to -20°C.Referring to Figure 1, a schematic representation of preparation of lipid coatedbosutinib nanocrystals is depicted.EXAMPLE 1For Experimentation DataMaterialsBosutinib was received from MSN laboratories Private Limited Unit II,Telangana - 502, 300, India. Fucoidan (Undaria pinnatifida) of medicine gradewas received from Nutra Green Biotechnology, Co. Ltd. Shanghai-200, 129,China. Hydrogenated soy phosphatidylcholine was gifted by Lipoid, GmBH,Germany. Vitamin E TPGS NF Grade was gifted by Antares Health Products,St. Charles, USA. Cholesterol extrapure AR, 99% and Stearylamine AR, 99%were procured from SRL Pvt. Ltd., India. Acetonitrile and water of HPLCgrade were procured from Finar chemicals India. All other chemicals used wereof analytical grade.Method of preparationA method of preparation of a nano-formulation comprising the steps of: (a)adding 1-2 mL of bosutinib ethanolic solution (20 mg in 1-2 mL) with a 2 mLsyringe with 24G needle dropwise to 10 mL of aqueous fucoidan solutionmaintained at a temperature range of 4-8°C on ice-cold water to form a colloidalsolution under continuous stirring of said colloidal solution at 2000-6000 rpmto form a dispersion; (b) subjecting said dispersion obtained in step (a) to probesonication for 10 minutes at pulse rate of 07 / 03 and amplitude of 60% whilemaintaining at 4°C using ice cold water to form a formulation; (c) stirring saidformulation obtained in step (b) at 200-600 rpm for 15-30 min at 25°C to forma homogenous solution followed by centrifuging said homogenous formulationat temperature range of -4°C at 15000 rpm for time duration of 15-30 minutesto form nanocrystals; (d) collecting and lyophilizing said nanocrystals obtainedin step (c) to obtain a powder followed by desiccating said powder for a timeduration of 24-48 hours to form a dry powder; (e) mixing 60 mg ofhydrogenated soy phosphatidylcholine (HSPC), 30 mg of Liquid Chloesterol(Chol), 15 mg of Tocopherol methoxypolyethylene glycol succinate (TPGS)and 15 mg of stearylamine to form a thin film; (f) adding 100 mg of said drypowder obtained in step (d) to 10 mL of water to form a mixture; (g) addingsaid mixture obtained in step (f) to said thin film obtained in step (e) to form arehydrated film; (h) subjecting said rehydrated film obtained in step (g) tosonication for 15-30 minutes to obtain a suspension followed by passing saidsuspension through manual extruder to form lipo-nanocrystal; and (i) stirringsaid lipo-nanocrystal as obtained in step (h) on ice cold water to obtainuniformly formed lipo-nanocrystal; and (j) centrifuging of said liponanocryatals to obtain sediments followed by lyophilization to obtain saidnano-formulation as dry powder. Further, said dry powder in step (d) wasstored in a sealed container at a temperature range of 2-8°C and said nanoformulation was stored in a sealed container at a temperature range of -8 to -20°C.EXAMPLE 2For characterizationPhysical characterizationParticle size, polydispersity index (PDI) and zeta potential of FNBC weredetermined at 25°C using Zetasizer (Nano ZS, Malvern instrument, MalvernUK). Particles size was measured based on dynamic light scattering (DLS)technique. The zeta potential or surface charge was achieved by laser Dopplervelocimetry and phase analysis light scattering technique. For analysis,formulation was diluted 10-times prior to measurement. The sample wasloaded in zen size cell or zen potential cell (folded capillary with parallel Pdelectrode pairs). The measurements were taken in triplicate and analyzed usingZetasizer software. The nanocrystals exhibited hydrodynamic diameter ofabout 223.27±73.83 for BTB-NCs and 267.12±58.38 nm for Lipo / BTB-NCs.Prepared nanocrystals were homogeneously dispersed with polydispersityindex (PDI) of 0.24.37±0.23 and 0.134±0.73. The nanocrystals exhibited zetapotential of -12.37±2.24 mV (BTB-NCs) and 9.329±1.38 mV (Lipo / BTBNCs).The drug content (DC) of nanocrystals was measured by HPLC. A reversephase HPLC column (C18) was used. The flow rate of the mobile phase ofACN-Buffer pH 5.8 (48:52 v / v) was set at 1.0 ml / min. The column effluentwas detected with a UV / VIS detector at 267 nm. The calibration curve waslinear in the range of 10-100,000 ng.ml, with a correlation coefficient of R2 =0.999. For analysis, 100 mg of lyophilized powder was added to 5 mL ofethanol and vortexed for 10 minutes for stabilizer membrane disruption andobtain free-drug in solution. The resultant solution was then centrifuged at15,000 rpm for 10 min to separate polymer followed by collection ofsupernatant and filtration through a 0.20 μM syringe filter. The DC was definedas the ratio of the drug in nanocrystals to the weight of nanocrystals used.Prepared nanocrystals exhibited the drug content of about 51.73 ± 2.37 % forBTB-NCs and approximately 26.37 ± 3.02 % for Lipo / BTB-NCs.Robustness to dilution was checked to determine the effect of dilution onnanocrystal size. For this, 1 mg / ml of nanocrystals were diluted to 10, 20 and50-fold and bath sonicated for 30s to redisperse the particles homogeneously.Then, the particle size was measured using Zetasizer at various time points.The nanocrystals observed to robust the dilution, showing no significant (p <0.001) change in particle size, PDI and zeta potential during their dilution.For storage stability study, lyophilized samples of nanocrystals were stored ina sealed container at 2-8°C, and lipid coated nanocrystals at a temperaturerange of -8 to -20°C. The storage stability was determined by comparing thechanges in particle size, zeta potential and drug content with time (0, 15, 30, 90and 180 days). Prepared nanocrystals were stable during the storage periodshowing no significant (p < 0.001) changes in particle size, zeta potential anddrug content.Microscopic investigationsThe nanocrystals were evaluated for the microscopic characteristics like shapeand area equivalent diameter using EVO scanning electron microscope (SEM)(MA15 / 18, CARL ZEISS Microscopy Ltd.). For the sample preparation forSEM analysis, 40-fold dilution was done and drop casted on 1 cm2 glass slide.The sample was air dried and stored in a desiccator to completely dry thesample. The dried sample was then gold coated using sputter coater (DSR1) for120 sec and loaded in the instrument. For the analysis, working distance wasset to 5 mm and accelerating voltage at 20kV. Contrast and brightness wereadjusted to obtain clear particles images easily distinguishable frombackground. The image magnification was set to 50K X and images werecaptured. The size of prepared nanocrystals was determined using ImageJSoftware. The nanocrystals exhibited cubic-rectangle shape of about205.56±99.34 nm for BTB-NCs. Lipid coating resulted in crystals of about240.83±86.07 nm for Lipo-BTB-NCs with rounded edges and towardsspherical morphology.The nanocrystals surface characteristics like smoothness and shape. The shapewas determined using scanning probe microscope (SPM) (NTEGRA Prima,NT-MDT Service, and Logistics Ltd). For analysis, sample was prepared bycasting a drop of 40-fold diluted sample on glass slide dried at roomtemperature. The SPM results confirmed the smooth topography for preparednanocrystals, showing non-crystalline nature of prepared polymer or lipidcoated nanocrystals.The size and crystallinity of prepared nanocrystals were also evaluated usingHigh-Resolution Transmission Electron Microscope (HR-TEM, Tecnai G2 20TWIN, FEI Corporation of USA (S.E.A.) PTE, LTD.). For sample analysis, asingle drop of 40-fold diluted sample was casted on TEM grid and air dried atroom temperature. The TEM images revealed nanocrystals of about213.45±85.75 nm and 245.28 ±73.82 for BTB-NCs and Lipo-BTB-NCs,respectively.EXAMPLE 3Spectroscopic analysis, wettability and saturation solubilitySolid-state characterization of Coarse drug (BTB), fucoidan (FP), bosutinibnanocrystals (BTB-NCs) and lipid-coated nanocrystals (Lipo / BTB-NCs) wasdone using fourier transform infrared (FTIR), powder X-ray diffraction (XRD),differential scanning calorimetry (DSC), and Brunner-Emmett-Teller (BET).Fourier Transform Infrared (FTIR) SpectroscopyFor FTIR analysis, the sample was mixed with KBr in a ratio of 1:5 and pressedto form the pellets using hydraulic press. The pellets were then loaded in theinstrument (SHIMADZU 8400 S, Tokyo, Japan), and percentagetransmittance corresponding to characteristic peaks were determined inwavelength range of 600-4000 cm-1 at a resolution of 4 cm-1 and 64accumulation per min as rate. The IR Peaks of bosutinib (BTB) were nitrilestretching at 2210 cm-1, N-H stretching at 3530 cm-1; Aromatic C-H Stretchingat 3238 cm-1; Alkyl C-H stretching 2944 and 2828 cm-1; N-H bending at 1599cm-1; C-C stretching (in ring) at 1506 cm-1; Methylene group C-H bending at1466 cm-1; Methyl group C-H bending at 1427 cm-1; Aromatic amine C-Nstretching at 1372 cm-1; Alkyl aryl ether C-O stretching at 1247 and 1220 cm-1;C-N stretching at 1051 cm-1; C-Cl stretching at 815 cm-1 and N-H wag at 727cm-1. Fucoidan (FP) showed characteristic peaks corresponding to broad O-Hstretching at 3500-3200 cm-1 (3451 cm-1) of monomeric monosaccharides,alkane C-H stretching at 2943 cm-1, C=O stretching at 1640 cm-1, S=Ostretching at 1252 cm-1, alcohol O-H bending at 1430-1330 cm-1; S=O stretchingat 1165 cm-1, ether C-O stretching 1132 cm-1; ester C-O stretching at 1054 cm-1;and C-O-S stretching at 853 cm-1. For fucoidan-stabilized nanocrystals (BTBNCs), the intensity of characteristic drug peaks (especially 2210 cm-1) decreasedand additional peaks (mainly 3450 cm-1 and 2947 cm-1) were present similar tofucoidan spectra. This was attributed to molecular dispersion of crystalline drugin hydrophilic fucoidan core to give stable hydrophilic nanocrystals. Thepresence of fucoidan on nanocrystals surface marked the peaks of pure drug.Furthermore, the characteristic peak of bosutinib was also absent in lipidcoated nanocrystals (Lipo / BTB-NCs) revealing no drug was leached out duringthe lipid coating. Also, the trend followed by Lipo-NCs was different fromFPNC and was of lipid mixture used for coating of nanocrystals surface.Lipo / BTB-NCs exhibited peaks at 3306 cm-1, 2917 cm-1, 2851 cm-1, 2218 cm-1,1736 cm-1, 1464 cm-1, 1372 cm-1, 1223 cm-1, 1089 cm-1, 1058 cm-1, and 966 cm-1.X-Ray Diffraction (XRD) AnalysisFor XRD analysis, the sample powder was used to determine the diffractionpattern using Rigaku Miniflex 600 powder x-ray diffractometer equipped witha D / teX Ultra detector. The measurements were done at a step size of 0.02°and scan rate of 5° per min over 2θ of 5-50°. X-Ray Powder Diffraction studieswere conducted to determine the stabilizer's effect on the drug's crystalline stateafter formulation as nanocrystals. Coarse drug (BTB) exhibited characteristicspeak at 2θ= 9.136 [°], 11.410 [°], 14.296 [°], 18.332 [°], 19.385 [°], 20.401 [°],21.224 [°], 22.279 [°], 23.929 [°], 24.90 [°], 25.22 [°], 25.778 [°], 27.556 [°],28.346 [°], 29.514 [°], 30.227 [°] and 32.292 [°]. The stabilizer was observed tohave no characteristic peaks and showed a broader diffraction pattern, probablydue to the amorphous nature of fucoidan. No sharp peaks similar to BTB wereobserved in BTB-NCs and in contrast were of very low intensity and slightlyright-shifted probably due to formation of fucoidan stabilized nanocrystals witha new material structure. The shifting in peak is due to a decrease in particlesize while reduction in intensity was attributed to conversion of crystallinecoarse drug to amorphous structure. For Lipo / BTB-NCs, a major peakobserved was at 21.47 [°] as a broad high intensity peak corresponding to HSPCsuggesting the successful coating on nanocrystals surface.Diffraction Scanning Calorimetry (DSC) AnalysisFor DSC analysis, the sample powder was subjected to heating at a rate of20°C / min over temperature range - 25 to 300°C, using Shimadzu DSC-60 Plus.The DSC thermogram obtained were studied for endothermic and exothermicevents. Herein, BTB showed a sharp endothermic peak at 82°C and anexothermic peak at 283°C, corresponding to the crystalline drug. The polymerfucoidan had an exothermic peak at 258°C owing to amorphous fucoidandecomposition at a higher temperature. BTB-NCs lack endothermic peak butexhibited an exothermic peak of polymer. This is due to the stabilizer fucoidandriven amorphous phase surrounding the drug's crystalline phase.Additionally, absence of weak transition or endothermic peak near 0°Csuggested the absence of water traces in prepared nanocrystals. In addition,Lipo-NCs exhibited distinct peaks at 38°C and 200°C attributed to lipid coating(TPGS and HSPC).Brunauer-Emmett-Teller (BET) AnalysisFor Brunauer-Emmett-Teller (BET) analysis, the lyophilized powder was usedand analysis was made using BELLSORP MAX II & BELCAT-II,MicrotracBEL Corp. The BET study was conducted to determine the effectivesurface area that increases as a result of their nanocrystal preparation. The puredrug exhibited a surface area of about 2.52 m2g-1 while both, BTB-NCs andLipo / BTB-NCs exhibited a surface area of about 30 m2g-1. This suggestedsignificant increase in the effective surface area of drug due to the nano size ofNCs. As a result, gas adsorption significantly increased to about 300 cm3g-1 fornanocrystals as compared to BTB having a low nitrogen adsorption capacity ofonly 25 cm3g-1.Wettability AnalysisThe wettability analysis was conducted using static contact angle measurementinstrument (KRUSS GmbH Germany, DSA 10). For analysis, the sampleswere punched into tablets (100 mg, pressure 5 kg). The tablet was placed at thestage of instrument followed by casting a drop of simulated lung fluid (SLF)(~4 μL) on the plain surface of the tablet through a microinjector and theimages were captured. The measurements were done in triplicate and contactangle values were noted. The water contact angle of nanocrystals as comparedto bosutinib was also determined. Bosutinib belonging to BCS Class IV drug ishydrophobic in nature. The hydrophobic drugs on pulmonary administrationare generally cleared from the lungs by ciliary clearance or macrophage uptake.Therefore, hydrophobic drugs must be converted to a highly soluble form usinga hydrophilic polymer to increase their wettability in lung fluid and avoid theirclearance. Herein, hydrophobic BTB and fabricated nanocrystals (BTB-NCsand Lipo / BTB-NCs) were evaluated for their affinity toward water using a dropof SLF. The results revealed a lower contact angle of about 19° and 27° forBTB-NCs and Lipo / BTB-NCs, respectively. Compared to this, coarse drugexhibited a higher contact angle of 96°. Lower contact angle of nanocrystalswas attributed to hydrophilic fucoidan used for nanocrystal stabilization thatform a layer around drug nanocrystals responsible for improving the drugwettability. The results confirmed the conversion of the hydrophobic drug to ahydrophilic amorphous particle, which significantly enhances the solubility ordissolution of the drug in lung fluid. The hydrophilic surface and increasedeffective surface area together may further improve the saturation solubility ofdrug.Saturation SolubilityThe saturation solubility of drug compared to nanocrystals was studied insimulated lung fluid (SLF, 0.02% w / v DPPC maintained at 35°C) to determinethe effect of nanocrystal's size-dependent surface area on drugs solubility. Forthis, equilibrium solubility of the BTB and BTB-NCs in SLF was measured bySaturation shake-flask method. The sample in excess was added to 10 ml ofmedia shaken at 37 ± 0.5°C in a water-bath shaker for 48 h to achieve theequilibrium. The equilibrated liquid was centrifuged for 15 min at 15,000 rpmand filtered through a 0.2 μm syringe filter (AXIVA) to remove the surplusinsoluble excipients. The filtrate was then analyzed at 267 nm using HPLCmethod. Both nanocrystals were found to significantly increase the saturationsolubility of the drug by about ten-folds compared to the coarse drug. TheOstwald-Freundlich equation explains increased solubility of drug that was dueto reduction in particle size that increased surface area exposed to mediaresponsible for larger dissolution pressure and, thus, saturation solubility.EXAMPLE 4In vitro studiesFor in-vitro drug release study, modified dialysis bag diffusion technique wasused. A falcon tube of 50 ml with an open upper end and a lower end assembledwith a dialysis bag (12 kD) was used for the study. The sample was redispersedin 2 ml SLF and transferred to falcon tube (donor compartment) immersed in48 ml of SLF (acceptor compartment) in a beaker. The system was maintainedat 37 ± 0.5°C under continuous stirring at 100 rpm / min. At predetermined timeintervals, aliquots of 2 ml were collected from the acceptor compartment andreplaced with the same volume of fresh medium. Since, BTB exhibits pooraqueous solubility in phosphate-buffered saline, 0.1% (w / v) Tween 80 wasadded in the release medium to maintain the sink condition. The samples werefiltered through a 0.20 μm syringe filter before analysis by HPLC. The coarsedrug exhibited drug release at a very slow rate as compared to nanocrystals.The drug release was only 12% in 48 hours attributed to its hydrophobic nature.Fucoidan-stabilized nanocrystals (BTB-NCs) exhibited drug release at a fasterrate showing almost complete drug release in 48 hours due to the hydrophilicproperty rendered by fucoidan. Contrary to this, lipid-coated nanocrystals(Lipo / BTB-NCs) demonstrated drug release at a slower rate than BTB-NCs buthigher than pure BTB. The higher rate of release by BTB-NCs was due to thehydrophilic stabilizer used. While slow drug release in a controlled manner byLipo / BTB-NCs was due to lipid layer on the nanocrystals surface that mighthave increased the drug diffusion layer.Flow PropertyTraditional angle of repose method was used for determining the flow propertyof prepared nanocrystals. For this, lyophilized nanocrystals were converted toa fine dry powder of nanocrystals by rotating slowly clockwise in mortar pestle,without applying any pressure. Then, nanocrystals powder was added to afunnel and passed through it to from a static heap of powder. The height andradius of pile were noted and the angle of repose was calculated. Thenanocrystals observed to have a high flow property compared to coarse drug.Pure drug exhibited an angle of repose of 47°. While, nanocrystals (BTB-NCsand Lipo / BTB-NCs) exhibited an angle of repose of 19-21° and 25-29°. Thissuggested higher flowability of nanocrystals as compared to nanocrystalsdemonstrating their suitability for pulmonary delivery.Aerosolization PerformanceThe aerosolization performance of nanocrystals was determined using an eightstage Anderson cascade impactor (ACI, Westech Instruments, UK). TheRotahaler inhaler device (D) was used as an actuator for the evaluation of theprepared nanoformulation. The device was loaded with size-3 HPMC capsules(C) filled with lyophilized nanoformulations and connected to mouthpieceadaptor (MP) of ACI. The formulation (powder) was then actuated for about3-sec at a flow rate of 60 L / min into the ACI. In total, ten capsules weresubjected to ACI for the study. Post aerosolization, the quantitativedetermination of the drug content in each part of ACI including D, MP,induction port (I), and pre-separator (PS) along with all eight stages (Stage 0:8600 nm, 1: 6500 nm, 2: 4400 nm, 3: 3200 nm, 4: 1900 nm. 5: 1200 nm, 6: 600nm, and 7: 300 nm) was done using HPLC method. Emitted dose (ED) wasdetermined as particles deposited at each stage of ACI. Fine particle fraction(FPF) was calculated as percentage particles deposited at stages 2-7 of ACI.FPF is particle fraction less than 5 μm size and is suitable for inhalation. Herein,prepared nanocrystals distributed to all the stages with maximum deposition atlower stages. The FPF value for BTB-NCs and Lipo / BTB-NCs was 54.23%and 57.39%, respectively. In contrast to nanocrystals, BTB had FPF of only44%. As FPF over 50% is suitable for pulmonary administration, preparednanocrystals are used for drug deposition to the lungs and pulmonaryapplications.Therefore, the present invention provides a nano-formulation in the form oflyophilized dry powder for sustained release of the anti-cancer drug in order tomanage the lung cancer. Further, the nano-formulation of the present inventionexhibits a higher drug payload and low excipient amount as well as goodaerosolization performance.Many modifications and other embodiments of the invention set forth hereinwill readily occur to one skilled in the art to which the invention pertain havingthe benefit of the teachings presented in the foregoing descriptions and theassociated drawings. Therefore, it is to be understood that the invention is notto be limited to the specific embodiments disclosed and that modifications andother embodiments are intended to be included within the scope of theappended claims. Although specific terms are employed herein, they are usedin a generic and descriptive sense only and not for purposes of limitation.

Claims

1. A nano-formulation for management of lung cancer comprising of: a nanocrystal and a plurality of lipids in a defined ratio; wherein, said nanocrystal includes a drug and a stabilizer; said drug is a kinase inhibitor; said plurality of lipids include hydrogenated soy phosphatidylcholine (HSPC), cholesterol and stearylamine in a ratio of 60:30:15; said nanocrystal is coated with a layer of said plurality of lipids; and said defined ratio of said nanocrystal and said plurality of lipids is in a range of 1:1.

2. The nano-formulation as claimed in claim 1, wherein said drug is bosutinib in a concentration range of 10 to 20 mg / ml in organic phase for nanocrystal preparation.

3. The nano-formulation as claimed in claim 1, wherein said stabilizer is fucoidan in a concentration range of 0.5 - 1% (w / v) for nanocrystal preparation.

4. The nano-formulation as claimed in claim 1, wherein said nanoformulation is in the form of a liquid nanocrystals and as lyophilized dry powder.

5. The nano-formulation as claimed in claim 1, wherein said nanoformulation is a cube to spherical with size ranging from 250 to 300 nm.

6. The nano-formulation as claimed in claim 1, wherein said nanoformulation has a drug content of 20-25%.

7. The nano-formulation as claimed in claim 1, wherein said nanoformulation is permeable in a range of 80 to 95%.

8. The nano-formulation as claimed in claim 1, wherein said nanoformulation exhibits a flowability in a range of 23 to 29°.

9. The nano-formulation as claimed in claim 1, wherein said nanoformulation disperses in a range of 54 to 60%.

10. The nano-formulation as claimed in claim 1, wherein said drug is released in a sustained manner at a rate of 60 to 90% in 12 to 48 hours.

11. A method of preparation of a nano-formulation for management of lung cancer comprising the steps of: (a) adding 1-2 mL of bosutinib ethanolic solution to 10 mL of aqueous fucoidan solution by a 2 mL syringe with 24G needle dropwise in ice-cold water at 4-8°C to form a colloidal solution followed by continuous stirring of said colloidal solution at 2000-6000 rpm to form a dispersion; (b) subjecting said dispersion obtained in step (a) to probe sonication for 10 minutes at pulse rate of 07 / 03 and amplitude of 60% at 4°C in ice cold water to form a formulation; (c) stirring said formulation obtained in step (b) at 200-600 rpm for 15 to 30 minutes at 25°C to form a homogenous formulation followed by centrifuging said homogenous formulation at a temperature of - 4°C at 15000 rpm for a time duration of 15-30 minutes to form nanocrystals; (d) collecting and lyophilizing said nanocrystals obtained in step (c) to obtain a powder followed by desiccating said powder for 24-48 hours to form a dry powder;(e) mixing 60 mg of HSPC, 30 mg of liquid chloesterol (Chol), 15 mg of tocopherol methoxypolyethylene glycol succinate (TPGS) and 15 mg of stearylamine to form a thin film; (f) adding 100 mg of said dry powder obtained in step (d) to 10 mL of water to form a mixture; (g) adding said mixture obtained in step (f) to said thin film obtained in step (e) to form a rehydrated film; (h) subjecting said rehydrated film obtained in step (g) to sonication for 15-30 minutes to obtain a suspension followed by passing said suspension through manual extruder to form lipo-nanocrystal; (i) stirring said lipo-nanocrystal obtained in step (h) on ice cold water to obtain uniformly formed lipo-nanocrystal; and (j) centrifuging of said lipo-nanocrystals to obtain sediments followed by lyophilization to obtain said nano-formulation as dry powder.

12. The method as claimed in claim 11, wherein said dry powder in step (d) was stored at a temperature range of 2 to 8°C.

13. The method as claimed in claim 11, wherein said nano-formulation in step (j) was stored at a temperature range of -8 to -20°C.