FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
AND
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10; rule 13)
TITLE OF THE INVENTION
"LIPOPOLYMERIC NANOCAPSULES FOR DELIVERING HYDROPHOBIC
DRUGS"
APPLICANT
Indian Institute of Technology, Bombay
Powai, Mumbai - 400076
Maharashtra, India
Indian
The following specification particularly describes the invention and the manner in which it is to be performed

FIELD OF INVENTION
The invention deals with a lipopolymeric nanocapsule with a fatty acid core and self crosslinking degradable biopolymers as shells, for improved penetration through ocular and cancerous membranes, delivery of hydrophobic and hydrophilic drugs and a simplified process for its development.
BACKGROUND OF THE INVENTION
There is a need for the development of stable nanocarriers which can penetrate through various barriers for efficient regional and systemic drug delivery for example through the ocular membranes, in drug resistant cancers. The platforms are especially required for delivery of hydrophobic drugs along with a mucoadhesive nature. Clinical administration of many such anticancer drugs requires adjuvants, which may involve numerous side affects and sometimes could be fatal. Similar complications exist in the treatment of ocular diseases For example, topical or regional administration of drugs in the treatment of ocular diseases such as glaucoma, macular degeneration etc. is limited due to the poor penetration of drugs through anatomical barriers and their clearance by physiological mechanisms existing in the corneal tissue.
Prior art
US patent (US 7767219) claims a nanocapsule comprising of a drug containing core and a polyelectrolyte multilayer encapsulating the core. The main application claimed is drug delivery by placing the nanocapsules at a desired location within the body using an implantable or insertable medical device such as pacemaker, heartvalve, stents. The invention involves the embedding of the nanocapsules in a coating layer inside the implantable device. The nanocapsules are not suitable for administration alone in regions of washout or in the presence of harsh conditions like acids. The core of the nanocapsule

comprises of melamine formaldehyde which may have issues of toxicity, and the shell is formed by ionic interaction between polyelectrolytes which alone can result in a relatively weak shell, thereby compromising the stability of the nanocapsule. The nanocapsules are not optimised for increased penetration through anatomical barriers like ocular membranes or drug resistant cancers.
Patent US 7763275 claims a nanocapsule wherein the nanocapsule consists of a template particle which is further coated by polyelectrolyte layers. The template particle is a liposome. This can lead to difficulties in coating the shell on the liposome, poor stability and shear resistance of the particle. The shell of the nanocapsule is formed by polyelectrolyte complexation or a covalent linkage of biopolymers by the addition of an external cross linking agent such as glutaraldehyde. This suffers from poor stability of the ionic interactions or toxic effects of cross linking agents like glutaraldehyde. Further, the proposed formulation cannot withstand harsh conditions like acids and barriers of drug resistant cancers.
Patent US 7723294 claims nanocapsule, where the core consists of either a protein or a drug or a combination thereof. Similarly, the shell also comprises of polypeptide films held together by polyelectrolyte complexation. Protein and polypeptides increase the costs of the formulation and limit the penetration and stability as they are easily degraded by proteolytic enzymes. Concerns of toxicity also arise with foreign peptides.
Patent US 7851189 claims microcapsules (10-100 urn) with a core containing cells and surrounded by a shell made of biodegradable polymer held by polyelectrolyte complexation. The core comprises of biodegradable polymer and self assembling peptides. This formulation again has the disadvantages of a weaker shell held by ionic complexation. The broad size range including 1-100 micron particles is a disadvantage as enhanced cellular uptake and penetration

through anatomical barriers, are seen only in specific size ranges less than 300 nm. The microcapsules are further encapsulated in a carrier fluid which adheres to the body lumen. This is a complex process and can have several complications inside the body.
US patent 7067153 describes a method for the preparation of microcapsule powder. The method involves heating of the oil component above gel point which can be as high as 90-100oC. Further, the method includes large size microparticles of 10-50 urn which limit the cellular internalisation, membrane penetration and are easily trapped by the reticuloendothelial system causing a short plasma half life.
Similarly, patents US 6979467, 6818296, 6733790 and 6534091 claim large microcapsules (0.1-5mm) for intravenous administration of active principles like perfume, tannins etc. The matrix and membrane, both comprise of biopolymers. This limits the incorporation of hydrophobic drugs. Further, the method includes large size micoparticles of lOmicron -5 mm which limit the cellular internalisation, membrane penetration and are easily trapped by the reticuloendothelial system causing a short plasma half life.
Patent 6958148 claims microparticles, which has surface available transglutaminase substrate reactive groups for targeting/ linking the microcapsules to body tissues using endogenous or exogenous transglutaminase. Unlike the complicated use of transglutaminase substrate, our invention doesnot use any extra ligand on the surface for linking to body tissues. On the other hand, it utilizes the mucoadhesive property of one of its components i.e. chitosan, present on the outer surface, to adhere to the body tissues. Moreover, the large size of the formulation described in US 6958148 devoids it of various features like cellular uptake, penetration through anatomical barriers etc. which are present in our invention.

OBJECTS OF INVENTION
A major objective of this invention is to develop nanocapsules with improved penetration through anatomical barriers like ocular and cancerous membranes for delivery of both hydrophobic and hydrophilic drugs.
Another object is to provide sustained release drug delivery through the invented nanocapsules.
Yet another object is to provide bio-degradable, bio-compatible nanocapsules which withstand dynamic wash out by tear fluids when applied to ocular tissues.
Another object is to provide nanocapsules for drug delivery which show improved gastric stability.
A further object of this invention is in process for preparing lipophilic nanocapsules for delivery hydrophobic and hydrophilic drugs under room temperature.
Advantages
Nanocapsules of the present invention has the following advantages over prior art.
The present invention has the following advantages over the prior art:
- fatty acid core and highly stable self crosslinking shell of biodegradable biopolymers namely alginate dialdehyde and chitosan
- simple room temperature process for preparation -size of 200-300 nm

- shows increased penetration though ocular membranes
- shows high cellular internalization by endocytosis in cancer cells
- bypasses efflux pump in drug resistant cancers
- stable in harsh conditions of gastric acids and enzymes
- withstands tear fluid washout leading to increased ocular residence
- delivers hydrophobic and hydrophilic drugs
BRIEF DESCRIPTION OF INVENTION
This invention relates to lipopolymeric nano capsules with a fatty acid or lipid core and self cross linking degradable biopolymers as shells. Fatty acids may be stearic or palmitic acids but are not limited there to and biodegradable polymers may be alignate dialdehyde and chitosan.
Nano capsules are preferably uniformly sized and has diameter of <300nm. The core consists of a solid matrix of fatty acids or lipids that act as permeating enhancers and are bio compatible GRAS approved (generally regarded as safe) and are easily metabolized in the body. The core is capable of encapsulating a hydrophobic or hydrophilic drug for release under favourable condition. The shell of these nano particles capsules are made of one or more layers of self cross linking degradable bio polymers such as alignate dialdehyde and chitosan which interact covalently and ionically thereby strengthening the shell. Chitosan and dialdehyde may form two separate layers on the core. No additional carriers or fluids are required for drug delivery.
Accordingly this invention relates to lipopolymeric nanocapsules for delivering hydrophobic and hydrophilic drugs each of said nanocapsules comprising a core of one or more bio compatible fatty acids or lipids such as stearic acid and palmitic acid said core capable of containing and releasing drugs, and a shell

having one or more layers of self cross-linking degradation biopolymers such as alignate dialdehyde and chitosan.
The process of preparing these nanocapsules involve two separate steps. Alignate dialdehyde is first synthesized by oxidizing alignate. Sodium alignate is dispersed in an alcohol and an aqueous solution of sodium metaperiodate is added under stirring. Excess periodate is then removed by dialize with distilled water and then freeze dried.
Nanocapsules are then prepared with the incorporation of a suitable drug such as paclitaxel / curcumin / Ibuprofen stearic / palmitic acid and the desired drug is dissolved in an alcohol in 2:1 molar ratio and this solution was added to alignate aldehyde solution drop by drop under constant stirring
Sonication was done at 50% amplitude at 20 KHz for 2 minutes or equivalent using a probe sonicator. Following this, the solution was added to 0.1 mg/ml solution of chitosan and stirred for 30 min. Further, the solution was left undisturbed for 12 h to allow the cross linking of chitosan with alginate dialdehyde due to the formation of Schiffs' base as a result of the reaction between the free -NH2 group of chitosan and free -CHO group of alginate dialdehyde. Excess methanol was then evaporated by vacuum evaporation. The solution was finally centrifuged at 25000g, 4oC for 30 minutes. The entire process resulted in the formation of a nanocapsule formulation with a drug containing stearic acid/ palmitic acid core and having a dual coating of alginate dialdehyde and chitosan. Blank nanocapsules (NC-B) were also prepared by similar method without drug.
This invention also resides in a process for preparing lipopolymeric nanocapsules for delivering hydrophobic and hydrophilic drugs comprises the steps of adding to an alcoholic solution of alignate dialdehyde dropwise or

solution of palmitic / stearic acid under constant stirring, sonicating the same and then adding a solution of chitosan under stiring, evaporating the solvent therefrom and then centrifuging the same to produce nanocapsules.
Drug containing core or matrix may result when a mixture of the desired drug and palmitic / stearing acid is stirred in dialdehyde solution.
The invention was evaluated for its anticancer efficacy with paclitaxel and curcumin separately, both encapsulated in the hydrophobic core. The invention was also evaluated with ibuprofen encapsulated in its core for its localized delivery as topical application in several ocular inflammations
The invented formulation (NC-D) had particle sizes less than 300 nm as determined by dynamic light scattering (DLS) using laser particle analyzer and confirmed by transmission electron microscopy. The nanocapsules with such size range can be have specific advantage in cancer chemotherapy as they can result in an increased accumulation of the formulation specifically in the tumor region when given intravenously or orally, due to enhanced permeability and retention (EPR) effect.
The invented nanocapsules had increased penetration through various anatomical barriers like ocular membranes and withstood dynamic washout by tear fluid. Polydispersity index suggested a uniform size distribution of the particles. Zeta potential of the invented formulation was found to be negative (—20 mV). A negative zeta potential ensures the stability of the nanovesicles in suspension which is a crucial parameter for formulation development. As a summary, the physiochemical properties of the formulation are given in table 1.
Present invention had a high encapsulation efficiency of hydrophobic drugs . For example the encapsulation was 83.9 ± 6.4 % (n=3) for paclitaxel and 56 ±

2 % for ibuprofen. Curcumin too showed a high encapsulation of 67.8 ± 5 % (n=3).
The invented formulation showed sustained release of drugs. The invented formulation also showed stability in presence of simulated gastric fluid (can withstand acid and enzyme conditions) In vitro release patterns of paclitaxel, curcumin and ibuprofen were studied. Release studies for paclitaxel and curcumin were done at 37oC temperature and in the presence of 0.1 N HC1 in 25 % methanol (pH 1.2) as the release medium for first two hours followed by release at 37oC temperature in the presence of 25 % methanolic solution of PBS (pH 7.4) as the release medium. 0.1 N HC1 was used to evaluate the gastric stability of the formulation. Release of free curcumin and paclitaxel in the presence of 0.1 N HC1 was also done for 2 h. The present invention showed sustained release profiles of both paclitaxel and curcumin over the entire observation period of 48 h with less than 10 % of drug releasing from the formulation in first 2 h under simulated gastric conditions (figure 2). Free curcumin and paclitaxel on the other hand, showed greater than 70 % drug release in presence of 0.1 N HC1. This suggests that unlike free drugs, present invention exhibits improved gastric stability and shows much sustained release under physiological conditions. The formulation is therefore promising for the oral and intravenous delivery of drugs in diseases such as cancer.
In vitro drug release study of ibuprofen from the invented formulation was done at 37oC in the presence of simulated tear fluid (STF) containing 25 % methanol (pH 7.4). Sustained release profile for ibuprofen was observed with present invention with ~ 62 % of the drug release within 72 hours (figure 2), thereby suggesting the present invention as an effective carrier for ocular drug delivery as topical formulation.

Since the present invention is also intended for regional drug delivery by topical administration in various ocular disorders, we evaluated the biocompatibility of blank nanocapsules (NC-B) at different concentrations with SIRC cell line (rabbit corneal epithelial cell line) over an exposure period of 24 h. As can be observed from figure 3, the formulation showed greater than 80 % cellular viability at concentrations as high as 0.5 mg/ml suggesting it to be biocompatible with corneal epithelial cells, thereby its suitability as topical formulation for ocular diseases.
In vitro anticancer efficacy of the invented formulation was evaluated on A549 (human non small cell lung carcinoma) and MDAMB-231 (drug resistant human breast carcinoma) cell lines. Inhibitory activity of nanocapsules with paclitaxel and curcumin were evaluated separately. Blank nanocapsules were also evaluated in order to determine if the material itself is toxic to the cancer cells. IC50 of the formulations as obtained as a result of their 72 hours exposure are given in table 2.
As can be seen, nanocapsule formulations of both paclitaxel and curcumin showed significantly less IC50 as compared to that of free drugs (p<0.05), indicating a clear advantage of the invented formulation. Blank nanocapsules (NC-B) did not show any cytotoxicity even at a concentration as high as 10 uM which confirmed that blank nanocapsules do not contribute to the cytotoxic effects.
The invented formulation was effective in bypassing efflux pumps in drug resistant cancers. It showed lower IC50 in MDAMB-231, a drug resistant breast carcinoma cell line clearly suggests that present formulation bypasses P-glycoprotein pump, which is overexpressed in such drug resistant cell lines and efflux the drug out of the cell. The invented formulation was taken up by cancerous cells and ocular cells actively. Cellular uptake study for present

invention was done on A549 (human non small cell lung carcinoma) and SIRC (rabbit corneal epithelia) cell lines in order to study its uptake and internalization. The formulation was prepared as per the protocol mentioned earlier with calcein and rhodamine 6G loaded into the nanocapsules instead of drug. As shown in figure 4, cells incubated in the presence of rhodamine-6G or calcein loaded formulation showed bright fluorescence, colocalized uniformly inside the cells. In contrast to this, cells incubated with free rhodamine-6G or free calcein showed relatively less/no fluorescence. This finding implies the role of present invention in mediating and facilitating the cellular uptake and interaction of the encapsulated materials. Increased cellular uptake of the nanocapsules can be attributed to their unique size of 200-300 nm and their structure.
The. invented formulation was actively taken up by ATP dependant endocytosis. Mechanism of cellular uptake and interaction of rhodamine-6G loaded nanocapsules with A549 cells and SIRC cells was studied by incubating the cells with rhodamine-6G loaded nanocapsules in normal and ATP depleted conditions. ATP depleted conditions were obtained by either pre incubation of cells in the presence of metabolic inhibitor i.e. 0.1 % sodium azide or incubation at 4oC temperature. As shown in figure 5; cells pretreated with 0.1 % sodium azide and cells incubated at 4oC showed significantly less (p<0.05) intracellular rhodamine-6G content at all time points as compared to those incubated under normal conditions, i.e. 37oC without azide. Sodium azide being a metabolic inhibitor depletes the cell of ATP and hence no active process is possible thereafter. Similar effect is also caused by the incubation of cells at 4oC rather than 37oC. This suggests that the cellular uptake of nanocapsules described in present invention, is a highly energy dependent or active process and therefore a statistically significant reduction (p<0.05) in the intracellular rhodamine-6G content was observed in the case of cells pretreated with 0.01 % sodium azide and cells incubated at 4oC. Moreover, uptake of

rhodamine-6G loaded nanocapsules was found to be a time dependent process as increase in intracellular rhodamine-6G content was observed with increasing time points.
The invented formulation showed superior penetration through ocular membranes even in the present of simulated tear fluid washout. Present invention was evaluated for its potential to be used as a an ocular drug delivery system for topical administration in various ocular disorders, including those involving the posterior segments of the eye such as vitreous, retina etc. For this, we did an ex vivo penetration study of sodium fluorescein containing nanocapsules in goat's eyeball. Briefly, sodium fluorescein containing nanocapsules were applied on the corneal surface of the eye ball. Equivalent amount of free dye was used as the control. To simulate the wash out and blinking reflex of the living animal's eye on the application of formulations, STF was flowed on the surface of eyeballs at the rate of 33 μL/min till 3 mins. After 12 hrs the eyeballs were dissected to separate cornea, aqueous humor, iris, vitreous humor and retina. Figure 6 shows the percentage of sodium fluorescein accumulated in different tissues of eye as a result of application of nanocapsule formulation and free dye. As can be seen, dye accumulation reduced drastically from corneal tissues to retinal tissues in the case of free dye administration. Unlike this, when nanocapsule based formulation was administered topically, almost 3-4 folds increase in dye content was observed in all the tissues as compared to what was observed with free dye. This finding clearly suggests the ability of the invented formulation to penetrate through several tissue barriers such as corneal epithelial cells and accumulate in posterior segments of the eye in significant amounts.
Physical properties of the nanocapsules encapsulating drugs like paclitaxel, Curcumin and Ibuprofen are shown in the table below

TABLE I

TABLE II below show IC 50 and combination index or different cancer cell line

The following examples illustrate the preparation of the nanocapsules and the enhanced efficacy, of the drug released there through, sustained release efficacy and other advantages noticed.
Example 1
The invented formulation was prepared as a two step process. The first step involved the synthesis of alginate dialdehyde by oxidation of alginate. Briefly, sodium alginate was dispersed in ethanol, to which sodium metaperiodate in distilled water was added and the reaction mixture was stirred in dark at room temperature for 6 h. The weight ratio of sodium alginate and sodium metaperiodate was kept as 1.85:1. Subsequently, solution was dialyzed against distilled water for 48 h with several changes of water till the solution is free of periodate. The dialyzate was then freeze dried. The second step involved the preparation of nanocapsules. For the preparation of nanocapsules, stearic acid/

palmitic acid and curcumin/ paclitaxel/ ibuprofren in 2:1 molar ratio were dissolved in methanol and the solution was added drop by drop to alginate dialdehyde solution (0.6 mg/ml) with continuous stirring. Sonication was done at 50% amplitude at 20 KHz for 2 minutes or equivalent using a probe sonicator. Following this, the solution was added to 0.1 mg/ml solution of chitosan and stirred for 30 min. Further, the solution was left undisturbed for 12 h to allow the cross linking of chitosan with alginate dialdehyde due to the formation of Schiffs' base as a result of the reaction between the free -NH2 group of chitosan and free -CHO group of alginate dialdehyde. Excess methanol was then evaporated by vacuum evaporation. The solution was finally centrifuged at 25000g, 4oC for 30 minutes. The entire process resulted in the formation of a nanocapsule formulation with a drug containing stearic acid/ palmitic acid core and having a dual coating of
alginate dialdehyde and chitosan. Blank nanocapsules (NC-B) were also prepared by similar method without drug.
Example 2
The invented formulation showed particle sizes of 200-300 ran. This is seen by dynamic light scattering (DLS) using laser particle analyzer (BI 200SM, Brookhaven Instruments Corporation).
The zeta potential is -20 mV and the invented formulation has a low polydispersity indicating uniform sized particles (Table 1) Transmission electron microscopy (TEM) as shown in figure 1 further confirmed the size of the nanocapsules. TEM imaging was done by negative staining method using CM200 (Philips) transmission electron microscope operating at 120 kV.
Example 3
The invented formulation showed high encapsulation of hydrophobic drugs. Present invention was found to have a high encapsulation efficiency of 83.9 ± 6.4 % (n=3) for paclitaxel and 56 ± 2 % for ibuprofen as determined by UV-

spectrophotometry (Perkin Elmer Lambda 25) at 228 nm and 273 nm respectively. Curcumin too showed a high encapsulation of 67.8 ± 5 % (n=3) as determined by
fluorescence spectrophotometry using a Hitachi 250098, F2500 spectrophotometer at 420 nm excitation and 552 nm emission wavelengths.
Example 4
In vitro anticancer efficacy of the invented formulation was evaluated in A549 (human non small cell lung carcinoma) and MDAMB-231 (drug resistant human breast carcinoma) cell lines. Inhibitory activity of nanocapsules with paclitaxel and curcumin were evaluated separately. Blank nanocapsules were also evaluated in order to determine if the material itself is toxic to the cancer cells. IC50 of the formulations as obtained as a result of their 72 hours exposure are given in Table 2. As can be seen, nanocapsule formulations of both paclitaxel and curcumin showed significantly less IC50 as compared to that of free drugs (P<0.05), indicating a clear advantage of the invented formulation. Blank nanocapsules (NC-B) did not show any cytotoxicity even at a concentration as high as 10 μM which confirmed that blank nanocapsules do not contribute to the cytotoxic effects.
Example 5
The invented formulation showed sustained release of drugs. The invented formulation also showed stability in presence of simulated gastric fluid (can withstand acid and enzyme conditions) In vitro release patterns of paclitaxel, curcumin and ibuprofen were studied. Release studies for paclitaxel and curcumin were done at 37oC temperature and in the presence of 0.1 N HC1 in 25 % methanol (pH 1.2) as the release medium for first two hours followed by release at 37oC temperature in the presence of 25 % methanolic solution of PBS (pH 7.4) as the release medium. 0.1 N HC1 was used to evaluate the gastric stability of the formulation. Release of free curcumin and paclitaxel in

the presence of 0.1 N HC1 was also done for 2 h. The present invention showed sustained release profiles of both paclitaxel and curcumin over the entire observation period of 48 h with less than 10 % of drug releasing from the formulation in first 2 h under simulated gastric conditions (figure 2). Free curcumin and paclitaxel on the other hand, showed greater than 70 % drug release in presence of 0.1 N HC1. This suggests that unlike free drugs, present invention exhibits improved gastric stability and shows much sustained release under physiological conditions. The formulation is
therefore promising for the oral and intravenous delivery of drugs in diseases such as cancer.
Example 6
In vitro drug release study of ibuprofen from the invented formulation was done at 37oC in the presence of simulated tear fluid (STF) containing 25 % methanol (pH 7.4). Sustained release profile for ibuprofen was observed with present invention with ~ 62 % of the drug release within 72 hours (figure 3), thereby suggesting the present invention as an effective carrier for ocular drug delivery as topical formulation.
Example 7
The invented formulation was biocompatible at different concentrations with SIRC cell line (rabbit corneal epithelial cell line) over an exposure period of 24 h. As can be observed from figure 4, the formulation showed greater than 80 % cellular viability at concentrations as high as 0.5 mg/ml suggesting it to be biocompatible with corneal epithelial cells, thereby its suitability as topical formulation for ocular diseases.
Example 8
Cellular uptake study for the invented formulation was done in A549 (human non small cell lung carcinoma) cell lines in order to study its uptake and

internalization. The formulation was prepared as per the protocol mentioned earlier with calcein and rhodamine 6G loaded into the nanocapsules instead of drug. Cells were incubated with either free rhodamine-6G or free calcein or rhodamine-6G loaded or calcein loaded nanocapsules at final rhodamine-6G concentration of 1.6 μM and final calcein concentration of 2 μM. Following an incubation of 1 h, slides were prepared and observed by a confocal laser scanning microscope (CLSM) (Olympus Fluoview, FV500, Tokyo, Japan) using an excitation wavelength of 570 nm and emission wavelength of 590 nm for rhodamine 6G and an excitation wavelength of 503 nm and emission wavelength of 525 nm for calcein. Images were acquired and analyzed with 60X water immersion objective using the Fluoview software (Olympus, Tokyo, Japan). As shown in figure 5, cells incubated in the presence of rhodamine-6G or calcein loaded formulation showed bright fluorescence, colocalized uniformly inside the cells. In contrast to this, cells incubated with free rhodamine-6G or free calcein showed relatively less/no fluorescence. This finding implies the role of present invention in mediating and facilitating the cellular uptake and interaction of the encapsulated materials. This is due to the nanosize range of 100-300 nm. (Figure 5)
Example 9
Cellular uptake study for the invented formulation was done in SIRC (rabbit corneal epithelial cells) cell lines in order to study its uptake and internalization (Figure 6). The formulation was prepared with calcein and rhodamine 6G loaded into the nanocapsules instead of drug. Cells were incubated with either free rhodamine-6G or free calcein or rhodamine-6G loaded or calcein loaded nanocapsules at final rhodamine-6G concentration of 1.6 μM and final calcein concentration of 2 μM. Following an incubation of 1 h, slides were prepared and observed by a confocal laser scanning microscope (CLSM) (Olympus Fluoview, FV500, Tokyo, Japan) using an excitation wavelength of 570 nm

and emission wavelength of 590 nm for rhodamine 6G and an excitation wavelength of 503nm and emission wavelength of 525 nm for calcein. Images were acquired and analyzed with 60X water immersion objective using the Fluoview software (Olympus, Tokyo, Japan). As shown in figure 6, cells incubated in the presence of rhodamine-6G or calcein loaded formulation showed bright fluorescence, colocalized uniformly inside the cells. In contrast to this, cells incubated with free rhodamine-6G or free calcein showed relatively less/no fluorescence. This finding implies the role of present invention in mediating and facilitating the cellular uptake and interaction of the encapsulated materials. This is due to the nanosize range of 100-300 nm.
Example 10
Mechanism of cellular uptake and interaction of rhodamine-6G loaded nanocapsules with A549 cells and SIRC cells was studied by incubating the cells with rhodamine-6G loaded nanocapsules in normal and ATP depleted conditions. ATP depleted conditions were obtained by either pre incubation of cells in the presence of metabolic inhibitor i.e. 0.1 % sodium azide or incubation at 4oC temperature. As shown in figure 7; cells pretreated with 0.1 % sodium azide and cells incubated at 4oC showed significantly less (p<0.05) intracellular rhodamine-6G content at all time points as compared to those incubated under normal conditions, i.e. 37oC without azide. Sodium azide being a metabolic inhibitor depletes the cell of ATP and hence no active process is possible thereafter. Similar effect is also caused by the incubation of cells at 4oC rather than 37oC. This suggests that the cellular uptake of nanocapsules described in present invention, is a highly energy dependent or active process and therefore a statistically significant reduction (p<0.05) in the intracellular rhodamine-6G content was observed in the case of cells pretreated with 0.01 % sodium azide and cells incubated at 4oC. Moreover, uptake of rhodamine-6G loaded nanocapsules was found to be a time dependent process

as increase in intracellular rhodamine-6G content was observed with increasing time points.
Example 11
Present invention was also evaluated for its potential to be used as a an ocular drug delivery system for topical administration in various ocular disorders, including those involving the posterior segments of the eye such as vitreous, retina etc. For this, we did an ex vivo penetration study of sodium fluorescein containing nanocapsules in goat's eyeball. Briefly, sodium fluorescein containing nanocapsules were applied on the corneal surface of the eye ball. Equivalent amount of free dye was used as the control. To simulate the wash out and blinking reflex of the living animal's eye on the application of formulations, STF was flowed on the surface of eyeballs at the rate of 33 μL/min till 3 mins. Tear turnover effect of the eye was stimulated by flowing STF at the rate 2.2 uL/min for 3 hrs on the eye balls [5]. After 12 hrs the eyeballs were dissected to separate cornea, aqueous humor, iris, vitreous humor and retina. Each tissue was suspended in the 5 ml of milli Q water for 3 hrs so that the dye will be released from the tissue into the solution and then the fluorescence was observed with the help of spectrofluorometer (Hitachi 250098, F2500) at an excitation wavelength of 421 nm and emission wavelength of 520 nm. Figure 8 shows the percentage of sodium fluorescein accumulated in different tissues of eye as a result of application of nanocapsule formulation and free dye. As can be seen, dye accumulation reduced drastically from corneal tissues to retinal tissues in the case of free dye administration. Unlike this, when nanocapsule based formulation was administered topically, almost 3-4 folds increase in dye content was observed in all the tissues as compared to what was observed with free dye. This finding clearly suggests the ability of our invention to penetrate through several tissue barriers such as corneal epithelial cells and accumulate in posterior segments of the eye in

significant amounts. The increased accumulation of dye as a result of administration of nanocapsule based formulation can be due to two main reasons. Firstly, due to a unique size range of 200-300 nm, these nanocapsules can easily penetrate through various ocular barriers. Secondly, due to its unique composition which involves mucoadhesive crosslinked biopolymers on the outer side, these nanocapsules can adhere to the various ocular tissues thereby preventing their loss by tear fluid mechanism and hence increasing their residence time.
Graphs and figures shown in the accompanying drawings illustrate the efficacy of drugs encapsulated in the nanocapsules of this invention.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1(a) and (b): show the in vitro release of paclitaxel and curcumin respectively from the invented nanocapsule formulation (NC-D). Release studies were done at 37oC temperature and in the presence of 0.1 N HC1 in 25 % methanol (pH 1.2) as the release medium for first two hours followed by release at 37oC temperature in the presence of 25 % methanolic solution of PBS (pH 7.4) as the release medium. Release of free paclitaxel and curcumin in the presence of 0.1 N HC1 was also done for 2 h as shown in (a) and (b) respectively.
Figure 2: shows the release profile of ibuprofen from the invented nanocapsules as studied at 37oC in the presence of simulated tear fluid (STF) containing 25 % methanol (pH 7.4).
Figure 3(a) and (b): show the CLSM images of SIRC cells incubated with calcein loaded nanocapsules and free calcein respectively. Incubation time was kept as 1 h.

Figure 4: Cellular levels of rhodamine-6G after incubation of (a) A549 cells and (b) SIRC cells with rhodamine-6G loaded nanocapsules under normal and ATP depleted conditions. (n=3).
Figure 5: Percentage penetration of the tagged nanocapsule and free sodium fluorescein dye in goat's eye ball as done ex vivo. (n=3)
Obvious modifications and alterations known to person skilled in the art are within the scope and ambit of the appended claims.

WE CLAIM
1. Lipopolymeric nanocapsules for delivering hydrophobic and hydrophilic drugs, each of said nanocapsules comprising a core of one or more biocompatible fatty acids or lipids such as stearic acid and palmitic acid, said core capable of containing and releasing drugs and a shell having one or more layers of self cross linking degradable biopolymers such as alignate dialdehyde and chitosan.
2. Nanocapsules as claimed in claim 1 wherein said core contains drugs such as curcumin, paclitaxel, ibubrofen or metoprolol in about 2:1 ratio of fatty acid to drug for stable and sustained release therefrom.
3. Nanocapsules as claimed in claims 1 & 2 containing tracking substances like fluorescent dyes, iron oxide, gold nano particles and the like encapsulated in said core.
4. Nanocapsules as claimed in claims 1-3 having a diameter of <300nm.
5. A process for preparing lipopolymeric nanocapsules for delivery hydrophilic and hydrophobic drugs comprising the steps of:
adding to an alcoholic solution of alignate dialdehyde dropwise, a solution palmitic / stearic acid under constant stirring, sonicating the same and then adding a solution of chitosan under stirring, evaporating the solvent therefrom and then centrifuging the same to produce lipopolymeric nanocapsules.
6. The process as claimed in claim 5 wherein drugs like curcumin,
paclitaxel, ibuprofen, or metoprolol in about 2:1 ratio with the fatty acid drug is
added to produce drug encapsulated nano capsules.

7. The process as claimed in claims 5 & 6 wherein the solution is centrifuged at 25000g for 30 mts at 4°C.
8. Lipopolymeric nanocapsules substantially as herein described and exemplified
9. A process for producing lipopolymerics nano capsules substantially as herein described and exemplified.